WO2015048577A2 - Crispr-related methods and compositions - Google Patents

Crispr-related methods and compositions Download PDF

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Publication number
WO2015048577A2
WO2015048577A2 PCT/US2014/057905 US2014057905W WO2015048577A2 WO 2015048577 A2 WO2015048577 A2 WO 2015048577A2 US 2014057905 W US2014057905 W US 2014057905W WO 2015048577 A2 WO2015048577 A2 WO 2015048577A2
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vii
molecule
nucleic acid
composition
grna
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PCT/US2014/057905
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French (fr)
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WO2015048577A3 (en
Inventor
Alexandra Glucksmann
Deborah PALESTRANT
Louis Anthony Tartaglia
Jordi MATA-FINK
Agnieszka Dorotz CZECHOWICZ
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Editas Medicine, Inc.
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Priority to US15/025,222 priority Critical patent/US20160237455A1/en
Publication of WO2015048577A2 publication Critical patent/WO2015048577A2/en
Publication of WO2015048577A3 publication Critical patent/WO2015048577A3/en
Priority to US16/938,661 priority patent/US20210040506A1/en
Priority to US18/450,549 priority patent/US20240067992A1/en

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    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to CRISPR-related methods and components for editing of, or delivery of a payload to, a target nucleic acid sequence.
  • CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats
  • RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. That RNA, which contains sequence complimentary to the viral genome, mediates targeting of a Cas9 protein to the sequence in the viral genome. The Cas9 protein cleaves and thereby silences the viral target.
  • the CRISPR/Cas system has been adapted for genome editing in eukaryotic cells.
  • the introduction of site-specific double strand breaks (DSBs) allows for target sequence alteration through one of two endogenous DNA repair mechanisms— either non-homologous end-joining (NHEJ) or homology-directed repair (HDR).
  • NHEJ non-homologous end-joining
  • HDR homology-directed repair
  • the CRISPR/Cas system has also been used for gene regulation including transcription repression and activation without altering the target sequence.
  • Targeted gene regulation based on the CRISPR/Cas system uses an enzymatically inactive Cas9 (also known as a catalytically dead Cas9).
  • Methods and compositions disclosed herein e.g., a Cas9 molecule complexed with a gRNA molecule, can be used to target a specific location in a target DNA.
  • a Cas9 molecule/gRNA molecule complex used specific editing or the delivery of a payload can be effected.
  • the disclosure features a gRNA molecule comprising a targeting domain which is complementary with a target sequence from a target nucleic acid disclosed herein, e.g., a sequence from: a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, 1X-3, XIV- 1 , or Section VIII.
  • a target sequence from a target nucleic acid disclosed herein e.g., a sequence from: a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1
  • the disclosure features a composition, e.g., pharmaceutical
  • composition comprising a gRNA molecule described herein.
  • the composition further comprises a Cas9 molecule, e.g., an eaCas9 or an eiCas9 molecule.
  • a Cas9 molecule e.g., an eaCas9 or an eiCas9 molecule.
  • said Cas9 molecule is an eaCas9 molecule.
  • said Cas9 molecule is an eiCas9 molecule.
  • said composition comprises a payload, e.g., a payload described herein, e.g., in Section VI, e.g., in Table VI-1, VI-2, VI-3, VI-4, VI-5, VI-6, or VI-7.
  • a payload e.g., a payload described herein, e.g., in Section VI, e.g., in Table VI-1, VI-2, VI-3, VI-4, VI-5, VI-6, or VI-7.
  • the payload comprises: an epigenetic modifier, e.g., a molecule that modifies DNA or chromatin; component, e.g., a molecule that modifies a histone, e.g., an epigenetic modifier described herein, e.g., in Section VI; a transcription factor, e.g., a
  • transcription factor described herein e.g., in Section VI; a transcriptional activator domain; an inhibitor of a transcription factor, e.g., an anti-transcription factor antibody, or other inhibitors; a small molecule; an antibody; an enzyme; an enzyme that interacts with DNA, e.g., a helicase, restriction enzyme, ligase, or polymerase; and/or a nucleic acid, e.g., an enzymatically active nucleic acid, e.g., a ribozyme, or an mRNA, siRNA, of antisense oligonucleotide.
  • a transcriptional activator domain an inhibitor of a transcription factor, e.g., an anti-transcription factor antibody, or other inhibitors
  • a small molecule an antibody
  • an enzyme an enzyme that interacts with DNA, e.g., a helicase, restriction enzyme, ligase, or polymerase
  • a nucleic acid e.g., an
  • the composition further comprises a Cas9 molecule, e.g., an eiCas9, molecule.
  • said payload is coupled, e.g., covalently or noncovalently, to a Cas9 molecule, e.g., an eiCas9 molecule.
  • said payload is coupled to said Cas9 molecule by a linker.
  • said linker is or comprises a bond that is cleavable under physiological, e.g., nuclear, conditions.
  • said linker is, or comprises, a bond described herein, e.g., in Section XI.
  • said linker is, or comp ises, an ester bond.
  • said payload comprises a fusion partner fused to a Cas9 molecule, e.g., an eaCas9 molecule or an eiCas9 molecule.
  • said payload is coupled, e.g., covalently or noncovalently, to the gRNA molecule.
  • said payload is coupled to said gRNA molecule by a linker.
  • said linker is or comprises a bond that is cleavable under physiological, e.g., nuclear, conditions.
  • said linker is, or comprises, a bond described herein, e.g., in Section XI.
  • said linker is, or comprises, an ester bond.
  • the composition comprises an eaCas9 molecule. In some embodiments, the composition comprises an eaCas9 molecule which forms a double stranded break in the target nucleic acid.
  • the composition comprises an eaCas9 molecule which forms a single stranded break in the target nucleic acid.
  • said single stranded break is formed in the complementary strand of the target nucleic acid.
  • said single stranded break is formed in the strand which is not the complementary strand of the target nucleic acid.
  • the composition comprises HNH-like domain cleavage activity but having no, or no significant, N-terminal RuvC-like domain cleavage activity. In some embodiments, the composition comprises N-terminal RuvC-like domain cleavage activity but having no, or no significant, HNH-like domain cleavage activity.
  • said double stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position. In some embodiments, said single stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
  • the composition further comprises a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
  • said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, V1I- 16, VII- 17, VII-18, VII-19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- IA, IX-2, IX-3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1 , IX-IA, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • Section VIIB e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1 , IX-IA, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, IX-2, IX-3, XIV- 1 , or Section VIII.
  • the composition further comprises a second gRNA molecule, e.g., a second gRNA molecule described herein.
  • said gRNA molecule and said second gRNA molecule mediate breaks at different sites in the target nucleic acid, e.g., flanking a target position.
  • said gRNA molecule and said second gRNA molecule are complementary to the same strand of the target.
  • said gRNA molecule and said second gRNA molecule are complementary to the different strands of the target.
  • said Cas9 molecule mediates a double stranded break.
  • said gRNA molecule and said second gRNA molecule are configured such that first and second break made by the Cas9 molecule flank a target position.
  • said double stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
  • the composition further comprises a template nucleic acid.
  • the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
  • said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of a target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VI1B, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- IA, IX-2, IX-3, XIV- 1 , or Section VIII.
  • the template nucleic acid is a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to ' 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g.', in Section V11B, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- IA, IX-2, IX-3, XIV- 1 , or Section VIII.
  • the template nucleic acid is a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX-I A, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • said Cas9 molecule mediates a single stranded break.
  • said gRNA molecule and said second gRNA molecule are configured such that a first and second break are formed in the same strand of the nucleic acid target, e.g., in the case of transcribed sequence, the template strand or the non-template strand.
  • said first and second break flank a target position.
  • one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
  • the composition further comprises a template nucleic acid.
  • the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
  • said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , ⁇ - 1 ⁇ , rX-2, IX-3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX-1 , IX- I A, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XTV- 1 , or Section VIII.
  • said gRNA molecule and said second gRNA molecule are configured such that a first and a second breaks are formed in different strands of the target.
  • said first and second break flank a target position.
  • one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
  • the composition further comprises a template nucleic acid.
  • the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
  • said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII- 14, VII- 15, VII-16, VII-17, VII- 18, VII- 19, VII-20, VII-21 , VIl-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, IX-2, LX-3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1 , IX-IA, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII-19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1, LX- 1 A, IX-2, lX-3, XIV- 1 , or Section VIII.
  • the composition comprises a second Cas9 molecule.
  • one or both of said Cas9 molecule and said second Cas9 molecule are eiCas9 molecules.
  • said eiCas9 molecule is coupled to a payload by a linker and said second eiCas9 molecules is coupled to a second payload by a second linker.
  • said payload and said second payload are the same. In some embodiments, said payload and said second payload are different. In some embodiments, said linker and said second linker are the same. In some embodiments, said linker and said second linker are different, e.g., have different release properties, e.g., different release rates.
  • said payload and said second payload are each described herein, e.g., in Section VI, e.g., in Table VI- 1, VI-2, VI-3, VI-4, VI-5, VI-6, or VI-7.
  • said payload and said second payload can interact, e.g., they are subunits of a protein.
  • one of both of said Cas9 molecule and said second Cas9 molecule are eaCas9 molecules.
  • said eaCas9 molecule comprises a first cleavage activity and said second eaCas9 molecule comprises a second cleavage activity.
  • said cleavage activity and said second cleavage activity are the same, e.g., both are N-terminal RuvC- like domain activity or are both HNH-like domain activity.
  • said cleavage activity and said second cleavage activity are different, e.g., one is N-terminal RuvC-like domain activity and one is HNH-like domain activity.
  • said Cas9 molecule and said second Cas9 molecule both mediate double stranded breaks.
  • said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein.
  • said gRNA molecule and said second gRNA molecule are configured such that first and second break flank a target position.
  • one of said first and second double stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
  • the composition further comprises a template nucleic acid.
  • the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
  • said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- IA, IX-2, IX-3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • Section VIIB e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, rX-2, IX-3, XIV- 1 , or Section VIII.
  • one of said Cas9 molecule and said second Cas9 molecule mediates a double stranded break and the other mediates a single stranded break.
  • said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein.
  • said gRNA molecule and said second gRNA molecule are configured such that a first and second break flank a target position. In some embodiments, said first and second break flank a target position. In some embodiments, one of said first and second breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
  • the composition further comprises a template nucleic acid.
  • the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
  • said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII-19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VTT-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • Section VIIB e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VTT-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XIV- 1 , or Section VIII.
  • said Cas9 molecule and said second Cas9 molecule both mediate single stranded breaks.
  • said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein.
  • said first and second break flank a target position.
  • one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
  • the composition further comprises a template nucleic acid.
  • the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
  • said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, ' VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- IA, IX-2, IX-3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, IX-2, IX- XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII-18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XIV- 1 , or Section VIII.
  • said gRNA molecule, said second gRNA molecule are configured such that a first and second break are in the same strand.
  • said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein.
  • said gRNA molecule, said second gRNA molecule are configured such that a first and second break flank a target position.
  • one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
  • the composition further comprises a template nucleic acid.
  • the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
  • said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% 6f its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , ⁇ - 1 ⁇ , IX-2, IX-3, XIV- 1 , or Section VIII.
  • said first and second break are on the different strands.
  • said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another Pam described herein.
  • said gRNA molecule, said second gRNA molecule are configured such that a first and second break are on different strands.
  • said gRNA molecule, said second gRNA molecule are configured such that a first and second break flank a target position. In some embodiments, said first and second break flank a target position.
  • one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
  • the composition further comprises a template nucleic acid.
  • the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
  • said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XIV- 1 , or Section VIII. -
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII-18, VII- 19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1 , IX-1A, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • Section VIIB e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII-18, VII- 19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1 , IX-1A, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XIV- 1 , or Section VIII.
  • the disclosure features a composition, e.g., a pharmaceutical composition, comprising a gRNA molecule and a second gRNA molecule described herein.
  • the composition further comprises a nucleic acid, e.g., a DNA or mRNA, that encodes a Cas9 molecule described herein. In some embodiments, the composition further comprises a nucleic acid, e.g., a DNA or RNA, that encodes a second Cas9 molecule described herein. In some embodiments, the composition further comprises a template nucleic acid described herein.
  • the disclosure features a composition, e.g., a pharmaceutical composition, comprising, nucleic acid sequence, e.g., a DNA, that encodes one or more gRNA molecules described herein.
  • a composition e.g., a pharmaceutical composition, comprising, nucleic acid sequence, e.g., a DNA, that encodes one or more gRNA molecules described herein.
  • said nucleic acid comprises a promoter operably linked to the sequence that encodes a gRNA molecule, e.g., a promoter described herein.
  • said nucleic acid comprises a second promoter operably linked to the sequence that encodes a second gRNA molecule, e.g., a promoter described herein.
  • the promoter and second promoter are different promoters. In some embodiments, the promoter and second promoter are the same.
  • the nucleic acid further encodes a Cas9 molecule described herein. In some embodiments, the nucleic acid further encodes a second Cas9 molecule described herein.
  • said nucleic acid comprises a promoter operably linked to the sequence that encodes a Cas9 molecule, e.g., a promoter described herein.
  • said nucleic acid comprises a second promoter operably linked to the sequence that encodes a second Cas9 molecule, e.g., a promoter described herein.
  • the promoter and second promoter are different promoters. In some embodiments, the promoter and second promoter are the same.
  • the composition further comprises a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.
  • a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.
  • the disclosure features a composition, e.g., a pharmaceutical composition, comprising nucleic acid sequence that encodes one or more of: a) a Cas9 molecule, b) a second Cas9 molecule, c) a gRNA molecule, and d) a second gRNA molecule.
  • a composition e.g., a pharmaceutical composition, comprising nucleic acid sequence that encodes one or more of: a) a Cas9 molecule, b) a second Cas9 molecule, c) a gRNA molecule, and d) a second gRNA molecule.
  • each of a), b) v c) and d) present are encoded on the same duplex molecule.
  • a first sequence selected from of a), b), c) and d) is encoded on a first duplex molecule and a second sequence selected from a), b), c), and d) is encoded on a second duplex molecule.
  • said nucleic acid encodes: a) and c); a), c), and d); or a), b), c), and d).
  • the composition further comprises a Cas9 molecule, e.g., comprising one or more of the Cas9 molecules wherein said nucleic acid does not encode a Cas9 molecule.
  • the composition further comprises an mRNA encoding Cas9 molecule, e.g., comprising one or more mRNAs encoding one or more of the Cas9 molecules wherein said nucleic acid does not encode a Cas9 molecule.
  • the composition further comprises a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.
  • a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.
  • the disclosure features a nucleic acid described herein.
  • the disclosure features a composition
  • a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and c) optionally, a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.
  • the disclosure features a composition
  • a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.
  • a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.
  • the disclosure features a composition
  • a nucleic acid e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.
  • the disclosure features a composition
  • a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g.
  • the disclosure features a method of altering a cell, e.g., altering the structure, e.g., sequence, of a target nucleic acid of a cell, comprising contacting said cell with:
  • composition comprising:
  • gRNA molecule a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
  • an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and
  • a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV;
  • a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
  • a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV;
  • a nucleic acid e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
  • an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule);
  • a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV; or
  • composition comprising:
  • nucleic acid e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
  • nucleic acid e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
  • a template nucleic acid e.g., a template nucleic acid described herein, e.g.. in Section IV.
  • a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule are delivered in or by, one dosage form, mode of delivery, or formulation.
  • a) a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by, a first dosage form, a first mode of delivery, or a first formulation; and b) an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.
  • the cell is an animal or plant cell .
  • the cell is a mammalian, primate, or human cell.
  • the cell is a human cell, e.g., a cell from described herein, e.g., in Section VIIA.
  • the cell is: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blastocyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.
  • the cell is a human cell, e.g., a cancer cell or other cell characterized by a disease or disorder.
  • the target nucleic acid is a chromosomal nucleic acid. In some embodiments, the target nucleic acid is an organellar nucleic acid. In some embodiments, the target nucleic acid is a mitochondrial nucleic acid. In some embodiments, the target nucleic acid is a chloroplast nucleic acid.
  • the cell is a cell of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
  • a disease causing organism e.g., a virus, bacterium, fungus, protozoan, or parasite.
  • the target nucleic acid is the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
  • a disease causing organism e.g., a virus, bacterium, fungus, protozoan, or parasite.
  • said method comprises: modulating the expression of a gene or inactivating a disease organism.
  • said cell is a cell characterized by unwanted proliferation, e.g., a cancer cell.
  • said cell is a cell characterized by an unwanted genomic component, e.g., a viral genomic component.
  • the cell is a cell described herein, e.g., in Section ⁇ .
  • a control or structural sequence of at least, 2 3, 4, or 5 genes is altered.
  • the target nucleic acid is a rearrangement, a kinase, a
  • the method comprises cleaving a target nucleic acid within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
  • said composition comprises a template nucleic acid.
  • the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
  • said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, V1I-20, VII, 21 , VIl-22, Vl -23, VII-24, VII-25, IX- 1 , IX- IA, 1X-2, IX-3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VlI- 17, Vll-18, VII- 19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1 , IX- I A, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a con-esponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, IX-2, IX-3, XIV- 1 , or Section VIII.
  • a control region e.g., a cis-acting or tans-acting control region
  • the sequence of a control region e.g., a cis-acting or tans-acting control region, of a gene is altered, e.g., by an alteration that modulates, e.g., increases or decreases, expression a gene under control of the control region, e.g., a control sequence is disrupted or a new control sequence is inserted;
  • a transcribed region e.g., a coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that increases expression of or activity of the gene product is effected, e.g., a mutation is corrected;
  • the sequence of a transcribed region e.g., the coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that decreases expression of or activity of the gene product is effected, e.g., a mutation is inserted, e.g., the sequence of one or more nucleotides is altered so as to insert a stop codon.
  • a control region or transcribed region e.g., a coding sequence, of at least 2, 3, 4, 5, or 6 genes are altered.
  • the disclosure features a method of treating a subject, e.g., by altering the structure, e.g., altering the sequence, of a target nucleic acid, comprising administering to the subject, an effective amount of:
  • composition comprising:
  • gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule) ;
  • an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and
  • a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV;
  • gRNA molecule a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
  • a nucleic acid e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
  • a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV;
  • a nucleic acid e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
  • an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule);
  • a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV; and/or
  • composition comprising:
  • nucleic acid e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
  • a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.
  • a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule are delivered in or by one dosage form, mode of delivery, or formulation.
  • a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage form, in a first mode of delivery, or first formulation; and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.
  • the subject is an animal or plant. In some embodiments, the subject is a mammalian, primate, or human.
  • the target nucleic acid is the nucleic acid of a human cell, e.g., a cell described herein, e.g., in Section VIIA.
  • the target nucleic acid is the nucleic acid of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.
  • the target nucleic acid is a chromosomal nucleic acid. In some embodiments, the target nucleic acid is an organellar nucleic acid. In some embodiments, the nucleic acid is a mitochondrial nucleic acid. In some embodiments, the nucleic acid is a chloroplast nucleic acid.
  • the target nucleic acid is the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
  • said method comprises modulating expression of a gene or inactivating a disease organism.
  • the target nucleic acid is the nucleic acid of a cell characterized by unwanted proliferation, e.g., a cancer cell.
  • said target nucleic acid comprises an unwanted genomic component, e.g., a viral genomic component.
  • a control or structural sequence of at least, 2 3, 4, or 5 genes is altered.
  • the target nucleic acid is a rearrangement, a kinase, a rearrangement that comprises a kinase, or a rumor suppressor.
  • the method comprises cleaving a target nucleic acid within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
  • said composition comprises a template nucleic acid.
  • the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
  • said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18. VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, IX-2, IX-3, XIV- 1 , or Section Vlll.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table Vll- 13, VII- 14, VII- 15, VI1- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1, IX-I A, IX-2, IX- 3, XIV- 1 , or Section VIII.
  • the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 1 0 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:
  • a control region e.g., a cis-acting or trans-acting control region
  • the sequence of a control region, e.g., a cis-acting or trans-acting control region, of a gene is altered, e.g., by an alteration that modulates, e.g., increases or decreases, expression a gene under control of the control region, e.g., a control sequence is disrupted or a new control sequence is inserted;
  • c) the coding sequence of a gene is cleaved; d) the sequence of a transcribed region, e.g., a coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that increases expression of or activity of the gene product is effected, e.g., a mutation is corrected;
  • a transcribed region e.g., the coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that decreases expression of or activity of the gene product is effected, e.g., a mutation is inserted, e.g., the sequence of one or more nucleotides is altered so as to insert a stop codon.
  • a control region or transcribed region e.g., a coding sequence, of at least 2, 3, 4, 5, or 6 genes are altered.
  • the disclosure features a composition
  • a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload coupled, covalently or non- covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule or the gRNA molecule.
  • the disclosure features a composition
  • a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is: coupled, covalently or non-covalently, the gRNA molecule; or a fusion partner with the Cas9 molecule.
  • the disclosure features a composition
  • a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is coupled, covalently or no -covalently, to the Cas9 molecule.
  • a nucleic acid e.g., a DNA
  • a Cas9 molecule e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule
  • the disclosure features a composition
  • a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g.
  • a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) a payload which is a fusion partner with the Cas9 molecule.
  • the disclosure features a method of delivering a payload to a cell, e.g., by targeting a payload to target nucleic acid, comprising contacting said cell with:
  • composition comprising:
  • gRNA molecule a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
  • a Cas9 molecule e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
  • a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule or the gRNA molecule;
  • gRNA molecule a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
  • a nucleic acid e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
  • a payload which is: coupled, covalently or non-covalently, the gRNA molecule; or a fusion partner with the Cas9 molecule;
  • a nucleic acid e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
  • a Cas9 molecule e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
  • composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g.
  • a DNA or mRNA .encoding a Cas9 molecule e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
  • a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule are delivered in or by one dosage form, mode of delivery, or formulation.
  • a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage form, first mode of delivery, or first formulation; and a Cas9 molecule, or nucleic acid encoding a Cas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.
  • the cell is an animal or plant cell.
  • the cell is a mammalian, primate, or human cell.
  • the cell is a human cell, e.g., a human cell described herein, e.g., in Section VIIA.
  • the cell is: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.
  • the cell is a human cell, e.g., a cancer cell, a cell comprising an unwanted genetic element, e.g., all or part of a viral genome.
  • the gRNA mediates targeting of a chromosomal nucleic acid. In some embodiments, the gRNA mediates targeting of a selected genomic signature. In some embodiments, the gRNA mediates targeting of an organellar nucleic acid. In some
  • the gRNA mediates targeting of a mitochondrial nucleic acid. In some embodiments, the gRNA mediates targeting of a chloroplast nucleic acid.
  • the cell is a cell of a disease causing organism, e.g., a vims, bacterium, fungus, protozoan, or parasite.
  • the gRNA mediates targeting of the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
  • the payload comprises a payload described herein, e.g., in Section VI.
  • said cell is a cell characterized by unwanted proliferation, e.g., a cancer cell.
  • said cell is characterized by an unwanted genomic component, e.g., a viral genomic component.
  • a control or structural sequence of at least, 2 3, 4, or 5 genes is altered.
  • the gRNA targets a selected genomic signature, e.g., a mutation, e.g., a germline or acquired somatic mutation.
  • the gRNA targets a rearrangement, a kinase, a rearrangement that comprises a kinase, or tumor suppressor.
  • the gRNA targets a cancer cell, e.g., a cancer cell disclosed herein, e.g., in Section VIIA.
  • the gRNA targets a cell which has been infected with a virus.
  • the disclosure features a method of treating a subject, e.g., by targeting a payload to target nucleic acid, comprising administering to the subject, an effective amount of:
  • gRNA molecule a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
  • a Cas9 molecule e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
  • a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule;
  • a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
  • the gRNA molecule is coupled, covalently or non-covalently, the gRNA molecule; or is a fusion partner with the Cas9 molecule;
  • a nucleic acid e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
  • a Cas9 molecule e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
  • composition comprising:
  • a nucleic acid e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g.
  • a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) a payload which is a fusion partner with the Cas9 molecule.
  • a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule are delivered in or by one dosage form, mode of delivery, or formulation.
  • a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage, mode of delivery form or formulation; and a Cas9 molecule, or nucleic acid encoding a Cas9 molecule, is delivered in or by a second dosage form, mode of delivery, or formulation.
  • the subject is an animal or plant cell. In some embodiments, the subject is a mammalian, primate, or human cell.
  • the gRNA mediates targeting of a human cell, e.g., a human cell described herein, e.g., in Section VIIA. In some embodiments, the gRNA mediates targeting of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.
  • the gRNA mediates targeting of a cancer cell or a cell comprising an unwanted genomic element, e.g., all or part of a viral genome. In some embodiments, the gRNA mediates targeting of a chromosomal nucleic acid. In some embodiments, the gRNA mediates targeting of a selected genomic signature. In some embodiments, the gRNA mediates targeting of an organellar nucleic acid. In some embodiments, the gRNA mediates targeting of a mitochondrial nucleic acid. In some embodiments, the gRNA mediates targeting of a chloroplast nucleic acid.
  • the gRNA mediates targeting of the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
  • the gRNA targets a cell characterized by unwanted proliferation, e.g., a cancer cell, e.g., a cancer cell from Section VIIA, e.g., from Table VII- 1 1 .
  • a cancer cell e.g., a cancer cell from Section VIIA, e.g., from Table VII- 1 1 .
  • the gRNA targets a cell characterized by an unwanted genomic component, e.g., a viral genomic component.
  • a control element e.g., a promoter or enhancer
  • the gRNA targets a rearrangement, a kinase, a rearrangement that comprises a kinase, or a tumor suppressor.
  • the gRNA targets a selected genomic signature, e.g., a mutation, e.g., a germline or acquired somatic mutation.
  • the gRNA targets a cancer cell. In some embodiments, the gRNA targets a cell which has been infected with a virus.
  • At least one eaCas9 molecule and a payload are administered.
  • the payload comprises a payload described herein, e.g., in Section VI.
  • the disclosure features a reaction mixture comprising a composition described herein and a cell.
  • Headings including numeric and alphabetical headings and subheadings, are for organization and presentation and are not intended to be limiting.
  • FIG. 1 A-G are representations of several exemplary gRNAs.
  • FIG. 1 A depicts a modular gRNA molecule derived in part (or modeled on a sequence in part) from Streptococcus pyogenes ⁇ S. pyogenes) as a duplexed structure (SEQ ID NOS 42 and 43, respectively, in order of appearance);
  • FIG. IB depicts a unimolecular (or chimeric) gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 44);
  • FIG. IC depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 45);
  • FIG. I D depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 46);
  • FIG. I E depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 47);
  • FIG. I F depicts a modular gRNA molecule derived in part from Streptococcus
  • Ihermophilus as a duplexed structure (SEQ ID NOS 48 and 49, respectively, in order of appearance);
  • FIG. 1G depicts an alignment of modular gRNA molecules of S. pyogenes and S.
  • FIG. 2 depicts an alignment of Cas9 sequences from Chylinski et al. , RNA BIOL. 2013; 10(5): 726-737.
  • the N-terminal RuvC-like domain is boxed and indicated with a "Y”.
  • the other two RuvC-like domains are boxed and indicated with a "B”.
  • the HNH-like domain is boxed and indicated by a "G”.
  • Sm S. mutatis (SEQ ID NO: 1 ); Sp: S. pyogenes (SEQ ID NO: 2); St: S. thermophil s (SEQ ID NO: 3); Li: L.
  • innocua SEQ ID NO: 4
  • FIG. 3A shows an alignment of the N-terminal RuvC-like domain from the Cas9 molecules disclosed in Chylinski et al. (SEQ ID NOS 54-103, respectively, in order of appearance). The last line of FIG. 3A identifies 3 highly conserved residues.
  • FIG. 3B shows an alignment of the N-terminal RuvC-like domain from the Cas9 molecules disclosed in Chylinski et al. with sequence outliers removed (SEQ ID NOS 104-177, respectively, in order of appearance).
  • SEQ ID NOS 104-177 sequence outliers removed.
  • the last line of FIG. 3B identifies 4 highly conserved residues.
  • FIG. 4A shows an alignment of the HNH-like domain from the Cas9 molecules disclosed in Chylinski et al. (SEQ ID NOS 178-252, respectively, in order of appearance). The last line of FIG. 4A identifies conserved residues.
  • FIG. 4B shows an alignment of the HNH-like domain from the Cas9 molecules disclosed in Chylinski et al. with sequence outliers removed (SEQ ID NOS 253-302, respectively, in order of appearance).
  • SEQ ID NOS 253-302 sequence outliers removed.
  • the last line of FIG. 4B identifies 3 highly conserved residues.
  • FIG. 5 depicts an alignment of Cas9 sequences from S. pyogenes and Neisseria meningitidis (N. meningitidis).
  • the N-terminal RuvC-like domain is boxed and indicated with a "Y”.
  • the other two RuvC-like domains are boxed and indicated with a "B”.
  • the HNH-like domain is boxed and indicated with a "G”.
  • Sp S. pyogenes
  • Nm N. meningitidis.
  • Motif this is a motif based on the two sequences: residues conserved in both sequences are indicated by a single amino acid designation; "*" indicates any amino acid found in the corresponding position of any of the two sequences; "-" indicates any amino acid, e.g., any of the 20 naturally occurring amino acids, and "-” indicates any amino acid, e.g., any of the 20 naturally occurring amino acids, or absent.
  • FIG. 6 shows a nucleic acid sequence encoding Cas9 of N. meningitidis (SEQ ID NO: 303). Sequence indicated by an "R” is an SV40 NLS; sequence indicated as “G” is an HA tag; sequence indicated by an “O” is a synthetic NLS sequence. The remaining (unmarked) sequence is the open reading frame (ORF).
  • Domain is used to describe segments of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional property.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”).
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences.
  • Modulator refers to an entity, e.g., a drug, that can alter the activity (e.g., enzymatic activity, transcriptional activity, or translational activity), amount, distribution, or structure of a subject molecule or genetic sequence.
  • modulation comprises cleavage, e.g., breaking of a covalent or non-covalent bond, or the forming of a covalent or non- covalent bond, e.g., the attachment of a moiety, to the subject molecule.
  • a modulator alters the, three dimensional, secondary, tertiary, or quaternary structure, of a subject molecule.
  • a modulator can increase, decrease, initiate, or eliminate a subject activity.
  • "Large molecule” refers to a molecule having a molecular weight of at least 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kD. Large molecules include proteins, polypeptides, nucleic acids, biologies, and carbohydrates.
  • Polypeptide refers to a polymer of amino acids having less than 100 amino acid residues. In an embodiment, it has less than 50, 20, or 10 amino acid residues.
  • Reference molecule e.g., a reference Cas9 molecule or reference gRNA, as used herein, refers to a molecule to which a subject molecule, e.g., a subject Cas9 molecule of subject gRNA molecule, e.g., a modified or candidate Cas9 molecule is compared.
  • a Cas9 molecule can be characterized as having no more than 10% of the nuclease activity of a reference Cas9 molecule.
  • reference Cas9 molecules include naturally occurring unmodified Cas9 molecules, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S.
  • the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology with the Cas9 molecule to which it is being compared.
  • the reference Cas9 molecule is a sequence, e.g., a naturally occurring or known sequence, which is the parental form on which a change, e.g., a mutation has been made.
  • Small molecule refers to a compound having a molecular weight less than about 2 kD, e.g., less than about 2 kD, less than about 1 .5 kD, less than about 1 kD, or less than about 0.75 kD.
  • Subject may mean either a human or non-human animal.
  • the term includes, but is not limited to, mammals (e.g., humans, other primates, pigs, rodents (e.g., mice and rats or hamsters), rabbits, guinea pigs, cows, horses, cats, dogs, sheep, and goats).
  • the subject is a human.
  • the subject is poultry.
  • Treatment mean the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting or preventing its development; (b) relieving the disease, i.e., causing regression of the disease state; or (c) curing the disease.
  • "X" as used herein in the context of an amino acid sequence refers to any amino acid (e.g., any of the twenty natural amino acids) unless otherwise specified.
  • a gRNA molecule refers to a nucleic acid that promotes the specific targeting or homing of a gRNA molecule/Cas9 molecule complex to a target nucleic acid.
  • gRNA molecules can be unimolecular (having a single RNA molecule), sometimes referred to herein as "chimeric" gRNAs, or modular (comprising more than one, and typically two, separate RNA molecules).
  • a gRNA molecule comprises a number of domains. The gRNA molecule domains are described in more detail below.
  • FIG. 1 Several exemplary gRNA structures, with domains indicated thereon, are provided in FIG. 1 . While not wishing to be bound by theory with regard to the three dimensional form, or intra- or inter-strand interactions of an active form of a gRNA, regions of high complementarity are sometimes shown as duplexes in FIG. 1 and other depictions provided herein.
  • a unimolecular, or chimeric, gRNA comprises, preferably from 5' to 3' :
  • a targeting domain (which is complementary to a target nucleic acid);
  • a tail domain optionally, a tail domain.
  • a modular gRNA comprises:
  • a first strand comprising, preferably from 5' to 3' ;
  • a targeting domain (which is complementary with a target sequence from a target nucleic acid disclosed herein, e.g., a sequence from: a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII- 20, VII-21 , VII-22, VI1-23, VII-24, VII-25, IX- 1 , IX- 1 A, LX-2, 1X-3, XlV- 1 , or Section VIII); and
  • a second strand comprising, preferably from 5' to 3': optionally, a 5' extension domain;
  • a tail domain optionally, a tail domain.
  • FIG. 1A-G provides examples of the placement of targeting domains.
  • the targeting domain comprises a nucleotide sequence that is complementary, e.g., at least 80, 85, 90, or 95% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid.
  • the targeting domain is part of an RNA molecule and will therefore comprise the base uracil (U), while any DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, it is believed that the
  • the targeting domain contributes to specificity of the interaction of the gRNA molecule/Cas9 molecule complex with a target nucleic acid. It is understood that in a targeting domain and target sequence pair, the uracil bases in the targeting domain will pair with the adenine bases in the target sequence.
  • the target domain itself comprises, in the 5' to 3' direction, an optional secondary domain, and a core domain.
  • the core domain is fully complementary with the target sequence.
  • the targeting domain is 5 to 50, e.g., 10 to 40, e.g., 10 to 30, e.g., 15 to 30, e.g., 15 to 25 nucleotides in length.
  • the targeting domain is 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides in length.
  • the strand of the target nucleic acid with which the targeting domain is complementary is referred to herein as the complementary strand.
  • Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.
  • the targeting domain is 16 nucleotides in length. [0195] In an embodiment, the targeting domain is 17 nucleotides in length.
  • the targeting domain is 1 8 nucleotides in length.
  • the targeting domain is 19 nucleotides in length.
  • the targeting domain is 20 nucleotides in length.
  • the targeting domain is 21 nucleotides in length.
  • the targeting domain is 22 nucleotides in length.
  • the targeting domain is 23 nucleotides in length.
  • the targeting domain is 24 nucleotides in length.
  • the targeting domain is 25 nucleotides in length.
  • FIG. lA-G provides examples of first complementarity domains.
  • the first complementarity domain is complementary with the second complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions.
  • the first complementarity domain is 5 to 30 nucleotides in length.
  • the first complementarity domain is 5 to 25 nucleotides in length.
  • the first complementary domain is 7 to 25 nucleotides in length.
  • the first complementary domain is 7 to 22 nucleotides in length. In an
  • the first complementary domain is 7 to 18 nucleotides in length.
  • the first complementary domain is 7 to 15 nucleotides in length.
  • the first complementary domain is 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 nucleotides in length.
  • the first complementarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain.
  • the 5' subdomain is 4-9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length.
  • the central subdomain is I , 2, or 3, e.g., 1 , nucleotide in length.
  • the 3' subdomain is 3 to 25, e.g., 4-22, 4- 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 1 5, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25, nucleotides ' ⁇ length.
  • the first complementarity domain can share homology with, or be derived from, a naturally occurring first complementarity domain. In an embodiment, it has at least 50% homology with a first complementarity domain disclosed herein, e.g., an S. pyogenes, or S. thennophilus, first complementarity domain.
  • nucleotides of the domain can have a modification, e.g., modification found in Section X herein.
  • FIG. 1B-E provides examples of linking domains.
  • a linking domain serves to link the first complementarity domain with the second complementarity domain of a unimolecular gRNA.
  • the linking domain can link the first and second complementarity domains covalently or non-covalently.
  • the linkage is covalent.
  • the linking domain covalently couples the first and second complementarity domains, see, e.g., FIG. 1 B-E.
  • the linking domain is, or comprises, a covalent bond inteiposed between the first complementarity domain and the second complementarity domain.
  • the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the two molecules can be associated by virtue of the hybridization of the complementarity domains, see e.g., FIG. 1A.
  • linking domains are suitable for use in unimolecular gRNA molecules.
  • Linking domains can consist of a covalent bond, or be as short as one or a few nucleotides, e.g., 1 , 2, 3, 4, or 5 nucleotides in length.
  • a linking domain is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in length. In an embodiment, a linking domain is 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, or 2 to 5 nucleotides in length. In an embodiment, a linking domain shares homology with, or is derived from, a naturally occurring sequence, e.g., the sequence of a tracrRNA that is 5' to the second complementarity domain. In an embodiment, the linking domain has at least 50% homology with a linking domain disclosed herein.
  • nucleotides of the domain can have a modification, e.g., modification found in Section X herein.
  • a modular gRNA can comprise additional sequence, 5' to the second complementarity domain, referred to herein as the 5' extension domain, see, e.g., FIG. 1A.
  • the 5' extension domain is, 2- 10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4 nucleotides in length.
  • the 5' extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.
  • FIG. 1 A-F provides examples of second complementarity domains.
  • the second complementarity domain is complementary with the first complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions.
  • the second complementarity domain can include sequence that lacks complementarity with the first complementarity domain, e.g., sequence that loops out from the duplexed region.
  • the second complementarity domain is 5 to 27 nucleotides in length. In an embodiment, it is longer than the first complementarity region.
  • the second complementary domain is 7 to 27 nucleotides in length. In an embodiment, the second complementary domain is 7 to 25 nucleotides in length. In an embodiment, the second complementary domain is 7 to 20 nucleotides in length. In an embodiment, the second complementary domain is 7 to 17 nucleotides in length. In an embodiment, the complementary domain is 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides in length. [0223] In an embodiment, the second complementarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain.
  • the 5' subdomain is 3 to 25, e.g., 4 to 22, 4 to l 8, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 nucleotides in length.
  • the central subdomain is 1 , 2, 3, 4 or 5, e.g., 3, nucleotides in length.
  • the 3' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length.
  • the 5' subdomain and the 3' subdomain of the first complementarity domain are respectively, complementaiy, e.g., fully complementary, with the 3' subdomain and the 5' subdomain of the second complementarity domain.
  • the second complementarity domain can share homology with or be derived from a naturally occurring second complementarity domain. In an embodiment, it has at least 50% homology with a second complementarity domain disclosed herein, e.g., an S. pyogenes, or S. thermophilus, first complementarity domain.
  • nucleotides of the domain can have a modification, e.g., modification found in Section X herein.
  • FIG. 1 A-F provides examples of proximal domains.
  • the proximal domain is 5 to 20 nucleotides in length.
  • the proximal domain can share homology with or be derived from a naturally occurring proximal domain. In an embodiment, it has at least 50% homology with a proximal domain disclosed herein, e.g., an S. pyogenes, or S. thermophilus, proximal domain.
  • nucleotides of the domain can have a modification, e.g., modification found in Section X herein.
  • FIG. 1 A and FIG. 1 C-F provide examples of tail domains.
  • tail domains in FIG. 1 A and FIG. 1C-F a broad spectrum of tail domains are suitable for use in gRNA molecules.
  • the tail domain is 0 (absent), 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.
  • the tail domain nucleotides are from or share homology with sequence from the 5' end of a naturally occurring tail domain, see e.g., FIG. ID or FIG. I E.
  • the tail domain includes sequences that are complementary to each other and which, under at least some physiological conditions, form a duplexed region.
  • the tail domain is absent or is 1 to 50 nucleotides in length.
  • the tail domain can share homology with or be derived from a naturally occurring proximal tail domain. In an embodiment, it has at least 50% homology with a tail domain disclosed herein, e.g., an S. pyogenes, or S. thermophilus, tail domain.
  • nucleotides of the domain can have a modification, e.g., modification found in Section X herein.
  • the tail domain includes nucleotides at the 3' end that are related to the method of in vitro or in vivo transcription.
  • these nucleotides may be any nucleotides present before the 3' end of the DNA template.
  • these nucleotides may be the sequence UUUUUU.
  • these nucleotides may be various numbers or uracil bases or may include alternate bases.
  • the "targeting domain" of the gRNA is complementary to the "target domain" on the target nucleic acid.
  • the strand of the target nucleic acid comprising the nucleotide sequence complementary to the core domain of the, gRNA is referred to herein as the "complementary strand" of the target nucleic acid.
  • Guidance on the selection of targeting domains can be found, e.g., in Fu Y el a!.. NAT BIOTECHNOL 2014 (doi: 10.1038/nbt.2808) and Sternberg SH el a/.. NATURE 2014 (doi: 10. 1038/naturel301 1).
  • the targeting domain is 16, 17, 18, 1 , 20, 21 , 22, 23, 24 or 25 nucleotides in length.
  • the targeting domain comprises 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
  • the targeting domain is 16 nucleotides in length. [0240] In an embodiment, the targeting domain is 17 nucleotides in length.
  • the targeting domain is 1 8 nucleotides in length.
  • the targeting domain is 19 nucleotides in length.
  • the targeting domain is 20 nucleotides in length.
  • the targeting domain is 21 nucleotides in length.
  • the targeting domain is 22 nucleotides in length.
  • the targeting domain is 23 nucleotides in length.
  • the targeting domain is 24 nucleotides in length.
  • the targeting domain is 25 nucleotides in length.
  • the targeting domain is 10 +/-5, 20+/-5, 30+/-5, 40+/-
  • the targeting domain is 20+/-5 nucleotides in length.
  • the targeting domain is 20+/- 10, 30+/- 10, 40+/-10, 50+/- 10, 60+/- 10, 70+/- 10, 80+/- 10, 90+/- 10, or 100+/- 10 nucleotides, in length.
  • the targeting domain is 30+/- 10 nucleotides in length.
  • the targeting domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. In other words,
  • the targeting domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.
  • the targeting domain has full complementarity with the target sequence.
  • the targeting domain has or includes 1 , 2, 3, 4, .5, 6, 7 or 8 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain.
  • the target domain includes 1 , 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5' end. In an embodiment, the target domain includes 1 , 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3' end. [0256] In an embodiment, the target domain includes 1 , 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5' end. In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3' end.
  • the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.
  • the targeting domain comprises two consecutive nucleotides that are not complementary to the target domain ("non-complementary nucleotides”), e.g., two consecutive noncomplementary nucleotides that are within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.
  • non-complementary nucleotides two consecutive nucleotides that are within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.
  • no two consecutive nucleotides within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain, are not complementary to the targeting domain.
  • the targeting domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X.
  • the targeting domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic.
  • the backbone of the targeting domain can be modified with a phosphorothioate, or other modification from Section X.
  • a nucleotide of the targeting domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' acetylation, e.g., a 2' methylation, or other modification from Section X.
  • the targeting domain includes 1 , 2, 3, 4, 5, 6, 7 or 8 or more modifications.
  • the targeting domain includes 1 , 2, 3, or 4 modifications within 5 nucleotides of its 5' end.
  • the targeting domain comprises as many as 1 , 2, 3, or 4 modifications within 5 nucleotides of its 3' end.
  • the targeting domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.
  • no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.
  • no nucleotide is modified within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.
  • Modifications in the targeting domain can be selected so as to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section ⁇ .
  • gRNA's having a candidate targeting domain having a selected length, sequence, degree of complementarity, or degree of modification can be evaluated in a system in Section III.
  • the candidate targeting domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
  • all of the modified nucleotides are complementary to and capable of hybridizing to corresponding nucleotides present in the target domain.
  • 1 , 2, 3, 4, 5, 6, 7 or 8 or more modified nucleotides are not complementary to or capable of hybridizing to corresponding nucleotides present in the target domain.
  • the targeting domain comprises, preferably in the 5' ⁇ 3' direction: a secondary domain and a core domain. These domains are discussed in more detail below.
  • the Core Domain and Secondary Domain of the Targeting Domain are discussed in more detail below.
  • the “core domain” of the targeting domain is complementary to the "core domain target" on the target nucleic acid.
  • the core domain comprises about 8 to about 13 nucleotides from the 3' end of the targeting domain (e.g., the most 3' 8 to 13 nucleotides of the targeting domain).
  • the core domain is 6 +/-2, 1+1-2, 8+/-2, 9+/-2, 10+/-2, 1 1+/-2, 12+/-2, 13+/-2, 14+/-2, 15+/-2, or 16+-2 nucleotides in length.
  • the core domain is 10+/- 2 nucleotides in length.
  • the core domain is 10+/-4 nucleotides in length.
  • the core domain is 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, or 16 nucleotides in length.
  • the core domain is 8 to 13, e.g., 8 to 12, 8 to 1 1 , 8 to 10, 8 to 9, 9 to 13, 9 to 12, 9 to 1 1 , or 9 to 10 nucleotides in length.
  • the core domain is 6 to 16, e.g., 6 to 15, 6 to 14, 6 to 13, 7 to 14, 7 to 13, 7 to 12, 7 to 1 1 , 7 to 10, 8 to 14, 8 to 13, 8 to 12, 8 to 1 1 , 8 to 10, or 8 to 9 nucleotides in length.
  • the core domain is complementary with the core domain target.
  • the core domain has exact complementarity with the core domain target.
  • the core domain can have 1 , 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the core domain.
  • the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.
  • the "secondary domain" of the targeting domain of the gRNA is complementary to the "secondary domain target" of the target nucleic acid.
  • the secondary domain is positioned 5' to the core domain.
  • the secondary domain is absent or optional.
  • the targeting domain is 25 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 12 to 17 nucleotides in length.
  • the targeting domain is 24 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length
  • the secondary domain is 1 1 to 16 nucleotides in length.
  • the targeting domain is 23 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length
  • the secondary domain is 10 to 15 nucleotides in length.
  • the targeting domain is 22 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length
  • the secondary domain is 9 to 14 nucleotides in length.
  • the targeting domain is 21 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length
  • the secondary domain is 8 to 13 nucleotides in length.
  • the targeting domain is 20 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length
  • the secondary domain is 7 to 12 nucleotides in length.
  • the targeting domain is 19 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length
  • the secondary domain is 6 to 1 1 nucleotides in length.
  • the targeting domain is 18 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length
  • the secondary domain is 5 to 10 nucleotides in length.
  • the targeting domain is 17 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length
  • the secondary domain is 4 to 9 nucleotides in length.
  • the targeting domain is 16 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length
  • the secondary domain is 3 to 8 nucleotides in length.
  • the secondary domain is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 nucleotides in length.
  • the secondary domain is complementary with the secondary domain target.
  • the secondary domain has exact complementarity with the secondary domain target.
  • the secondary domain can have 1 , 2, 3, 4 or 5 nucleotides that are not
  • the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.
  • the core domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X.
  • the core domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic.
  • the backbone of the core domain can be modified with a phosphorothioate, or other modification from Section X.
  • a nucleotide of the core domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X.
  • a core domain will contain no more than 1 , 2, or 3 modifications.
  • Modifications in the core domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III.
  • gRNA' s having a candidate core domain having a selected length, sequence, degree of complementarity, or degree of modification can be evaluated in the system described at Section ⁇ 1.
  • the candidate core domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule /Cas9 molecule system known to be functional with a selected target and evaluated.
  • the secondary domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X.
  • the secondary domain comprises one or more modifications, e.g., modifications that render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic.
  • the backbone of the secondary domain can be modified with a phosphorothioate, or other modification from Section X.
  • a nucleotide of the secondary domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X.
  • a secondary domain will contain no more than 1 , 2, or 3 modifications.
  • Modifications in the secondary domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III.
  • gRNA's having a candidate secondary domain having a selected length, sequence, degree of complementarity, or degree of modification can be evaluated in the system described at Section III.
  • the candidate secondary domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule /Cas9 molecule system known to be functional with a selected target and evaluated.
  • (1) the degree of complementarity between the core domain and its target, and (2) the degree of complementarity between the secondary domain and its target may differ.
  • ( 1) may be greater than (2).
  • (1 ) may be less than (2).
  • ( 1) and (2) may be the same, e.g., each may be completely
  • ( 1) the number of modifications (e.g., modifications from Section X) of the nucleotides of the core domain and (2) the number of modification (e.g., modifications from Section X) of the nucleotides of the secondaiy domain may differ.
  • (1) may be less than (2).
  • (1) may be greater than (2).
  • ( 1) and (2) may be the same, e.g., each may be free of modifications.
  • the first complementarity domain is complementary with the second complementarity domain.
  • the first domain does not have exact complementarity with the second complementarity domain target.
  • the first complementarity domain can have 1 , 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the second complementarity domain .
  • 1 , 2, 3, 4, 5 or 6, e.g., 3 nucleotides will not pair in the duplex, and, e.g., form a non-duplexed or looped-out region.
  • an unpaired, or loop-out, region e.g., a loop-out of 3 nucleotides, is present on the second complementarity domain.
  • the unpaired region begins 1 , 2, 3, 4, 5, or 6, e.g., 4, nucleotides from the 5' end of the second complementarity domain.
  • the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.
  • the first and second complementarity domains are:
  • the second complementarity domain is longer than the first complementarity domain, e.g., 2, 3, 4, 5, or 6, e.g., 6, nucleotides longer.
  • the first and second complementary domains independently, do not comprise modifications, e.g., modifications of the type provided in Section X.
  • the first and second complementary domains independently, comprise one or more modifications, e.g., modifications that the render the domain less susceptible to degradation or more bio-compatible, e.g., less immunogenic.
  • the backbone of the domain can be modified with a phosphorothioate, or other modification from Section X.
  • a nucleotide of the domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g. , a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X.
  • the first and second complementary domains independently, include 1 , 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the first and second complementary domains, independently, include 1 , 2, 3, or 4 modifications within 5 nucleotides of its 5' end. In an embodiment, the first and second complementary domains, independently, include as many as 1 , 2, 3, or 4 modifications within 5 nucleotides of its 3' end.
  • the first and second complementary domains independently, include modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the domain, within 5 nucleotides of the 3' end of the domain, or more than 5 nucleotides away from one or both ends of the domain.
  • the first and second complementary domains independently, include no two consecutive nucleotides that are modified, within 5 nucleotides of the 5' end of the domain, within 5 nucleotides of the 3' end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain.
  • the first and second complementary domains independently, include no nucleotide that is modified within 5 nucleotides of the 5' end of the domain, within 5 nucleotides of the 3' end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain.
  • Modifications in a complementarity domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III.
  • gRNA's having a candidate complementarity domain having a selected length, sequence, degree of complementarity, or degree of modification can be evaluated in the system described in Section III.
  • the candidate complementarity domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule /Cas9 molecule system known to be functional with a selected target and evaluated.
  • the first complementarity domain has at least 60, 70, 80, 85%, 90%, or 95% homology with, or differs by no more than 1 , 2, 3, 4, 5, or 6 nucleotides from, a reference first complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S.
  • a reference first complementarity domain e.g., a naturally occurring, e.g., an S. pyogenes, or S.
  • first complementarity domain or a first complementarity domain described herein, e.g., from FIG. 1 A-F.
  • the second complementarity domain has at least 60, 70, 80, 85%, 90%, or 95 % homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference second complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermopliilus, second complementarity domain, or a second complementarity domain described herein, e.g., from FIG. 1 A-F.
  • a reference second complementarity domain e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermopliilus
  • second complementarity domain e.g., from FIG. 1 A-F.
  • duplexed region formed by first and second complementarity domains is typically 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 base pairs in length (excluding any looped out or unpaired nucleotides).
  • the first and second complementarity domains when duplexed, comprise 1 1 paired nucleotides, for example, in the gRNA sequence (one paired strand underlined, one bolded):
  • the first and second complementarity domains when duplexed, comprise 15 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):
  • the first and second complementarity domains when duplexed, comprise 16 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):
  • the first and second complementarity domains when duplexed, comprise 21 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):
  • nucleotides are exchanged to remove poly-U tracts, for example in the gRNA sequences (exchanged nucleotides underlined):
  • a modular gRNA can comprise additional sequence, 5' to the second complementarity domain.
  • the 5' extension domain is 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4 nucleotides in length.
  • the 5' extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.
  • the 5' extension domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X.
  • the 5' extension domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic.
  • the backbone of the 5' extension domain can be modified with a phosphorothioate, or other modification from Section X.
  • a nucleotide of the 5' extension domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' - acetylation, e.g., a 2' methylation, or other modification from Section X.
  • a 2' modification e.g., a modification at the 2' position on ribose
  • a 2' - acetylation e.g., a 2' methylation
  • the 5' extension domain can comprise as many as 1 , 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the 5' extension domain comprises as many as 1 , 2, 3, or 4 modifications within 5 nucleotides of its 5' end, e.g., in a modular gRNA molecule. In an embodiment, the 5' extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end, e.g., in a modular gRNA molecule.
  • the 5' extension domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the 5' extension domain, within 5 nucleotides of the 3' end of the 5' extension domain, or more than 5 nucleotides away from one or both ends of the 5' extension domain.
  • no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the 5' extension domain, within 5 nucleotides of the 3' end of the 5' extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5' extension domain.
  • no nucleotide is modified within 5 nucleotides of the 5' end of the 5' • extension domain, within 5 nucleotides of the 3' end of the 5' extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5' extension domain.
  • Modifications in the 5' extension domain can be selected to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section ITT.
  • gRNAs having a candidate 5' extension domain having a selected length, sequence, degree of complementarity, or degree of modification can be evaluated in the system described at Section III.
  • the candidate 5' extension domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
  • the 5' extension domain has at least 60, 70, 80, 85, 90 or 95% homology with, or differs by no more than 1 , 2, 3, 4, 5, or 6 nucleotides from, a reference 5' extension domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus, 5' extension domain, or a 5' extension domain described herein, e.g., from FIG. 1 A and FIG. I F.
  • a reference 5' extension domain e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus
  • the linking domain is disposed between the first and second complementarity domains.
  • the two molecules are associated with one another by the complementarity domains.
  • the linking domain is 10 +/-5, 20+/-5, 30+/-5, 40+/-5, 50+/-5, 60+/-5, 70+/-5, 80+/-5, 90+/-5, or 100+/-5 nucleotides, in length.
  • the linking domain is 20+/- 10, 30+/- 10, 40+/- 10, 50+/- 10, 60+/- 10, 70+/-10, 80+/- 10, 90+/- 10, or 100+/- 10 nucleotides, in length.
  • the linking domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length.
  • the targeting domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.
  • the linking domain is 1 , 2, 3, 4, 5, . 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16 17, 18, 19, or 20 nucleotides in length.
  • the linking domain is a covalent bond.
  • the linking domain comprises a duplexed region, typically adjacent to or within 1 , 2, or 3 nucleotides of the 3' end of the first complementarity domain and/or the fiend of the second complementarity domain.
  • the duplexed region can be 20+/- 10, 30+/- 10, 40, +/- 10 or 50+/- 10 base pairs in length.
  • the duplexed region can be 10+/-5, 15+/-5, 20+/-5, or 30+/-5 base pairs in length.
  • the duplexed region can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, or 15 base pairs in length.
  • sequences forming the duplexed region have exact complementarity with one another, though in some embodiments as many as 1 , 2, 3, 4, 5, 6, 7 or 8 nucleotides are not complementary with the corresponding nucleotides.
  • the linking domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X.
  • the linking . domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic.
  • the backbone of the linking domain can be modified with a phosphorothioate, or other modification from Section X.
  • a nucleotide of the linking domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X.
  • a 2' modification e.g., a modification at the 2' position on ribose
  • a 2' -acetylation e.g., a 2' methylation
  • the linking domain can comprise as many as 1 , 2, 3, 4, 5, 6, 7 or 8 modifications.
  • Modifications in a linking domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section ⁇ .
  • gRNA's having a candidate linking domain having a selected length, sequence, degree of complementarity, or degree of modification can be evaluated a system described in Section III.
  • a candidate linking domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
  • the linking domain has at least 60, 70, 80, 85, 90 or 95% homology with, or differs by no more than 1 , 2, 3, 4, 5 ,or 6 nucleotides from, a reference linking domain, e.g., a linking domain described herein, e.g., from FIG. 1 B-E.
  • the proximal domain is 6 +1-2, ⁇ +/-2, 8+/-2, 9+/-2, 10+/-2, 1 1+/-2, 12+/-2, 13+/-2, 14+/-2, 14+/-2, 16+/-2, 17+/-2, 18+/-2, 19+/-2, or 20+/-2 nucleotides in length.
  • the proximal domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 16, 17, 18, 19, or 20 nucleotides in length.
  • the proximal domain is 5 to 20, 7, to 18, 9 to 16, or 10 to 14 nucleotides in length.
  • the proximal domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X.
  • the proximal domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic.
  • the backbone of the proximal domain can be modified with a phosphorothioate, or other modification from Section X.
  • a nucleotide of the proximal domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' - acetylation, e.g., a 2' methylation, or other modification from Section X.
  • a 2' modification e.g., a modification at the 2' position on ribose
  • a 2' - acetylation e.g., a 2' methylation
  • the proximal domain can comprise as many as 1 , 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the proximal domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5' end, e.g., in a modular gRNA molecule. In an embodiment, the target domain comprises as many as 1 , 2, 3, or 4 modifications within 5 nucleotides of its 3' end, e.g., in a modular gRNA molecule.
  • the pro imal domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the proximal domain, within 5 nucleotides of the 3' end of the proximal domain, or more than 5 nucleotides away from one or both ends of the proximal domain.
  • no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the proximal domain, within 5 nucleotides of the 3' end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain.
  • no nucleotide is modified within 5 nucleotides of the 5' end of the proximal domain, within 5 nucleotides of the 3' end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain.
  • Modifications in the proximal domain can be selected to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section III.
  • gRNA's having a candidate proximal domain having a selected length, sequence, degree of complementarity, or degree of modification can be evaluated in the system described at Section III.
  • the candidate proximal domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule /Cas9 molecule system known to be functional with a selected target and evaluated:
  • the proximal domain has at least 60%, 70%, 80%, 85%, 90%, or 95% homology with, or differs by no more thari 1 , 2, 3, 4, 5 ,or 6 nucleotides from, a reference proximal domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilics, proximal domain, or a proximal domain described herein, e.g., from FIG. 1A-F.
  • a reference proximal domain e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilics
  • proximal domain or a proximal domain described herein, e.g., from FIG. 1A-F.
  • the tail domain is 10 +/-5, 20+/-5, 30+/-5, 40+/-5, 50+/-5, 60+/-5, 70+/-5, 80+/-5, 90+/-5, or 100+/- 5 nucleotides, in length.
  • the tail domain is 20+/-5 nucleotides in length.
  • the tail domain is 20+/- 10, 30+/- 10, 40+/- 10, 50+/- 10, 60+/- 10, 70+/- 10, 80+/- 10, 90+/- 10, or 100+/- 10 nucleotides, in length.
  • the tail domain is 25+/- 10 nucleotides in length.
  • the tail domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. [0346] In other embodiments, the tail domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.
  • the tail domain is 1 to 20, 1 to 1 , 1 to 10, or 1 to 5 nucleotides in length.
  • the tail domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X.
  • the tail domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic.
  • the backbone of the tail domain can be modified with a phosphorothioate, or other modification from Section X.
  • a nucleotide of the tail domain can comprise a 2' modification (e.g., a modification at the position on ribose), e.g., a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X.
  • a 2' modification e.g., a modification at the position on ribose
  • a 2' -acetylation e.g., a 2' methylation
  • the tail domain can have as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications.
  • the target domain comprises as many as 1 , 2, 3, or 4 modifications within 5 nucleotides of its 5' end. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end.
  • the tail domain comprises a tail duplex domain, which can form a tail duplexed region.
  • the tail duplexed region can be 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 1 2 base pairs in length.
  • a further single stranded domain exists 3' to the tail duplexed domain.
  • this domain is 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In an embodiment, it is 4 to 6 nucleotides in length.
  • the tail domain has at least 60, 70, 80, or 90% homology with, or differs by no more than 1 , 2, 3, 4, 5 ,or 6 nucleotides from, a reference tail domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus, tail domain, or a tail domain described herein, e.g., from FIG. 1 A and FIG. 1 C-F.
  • a reference tail domain e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus
  • tail domain or a tail domain described herein, e.g., from FIG. 1 A and FIG. 1 C-F.
  • proximal and tail domain taken together comprise the following sequences:
  • AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU SEQ ID NO: 33
  • AAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGU GGUGC SEQ ID NO: 34
  • AAGGCUAGUCCGUUAUCA (SEQ ID NO: 37); or
  • the tail domain comprises the 3' sequence UUUUUU, e.g., if a U6 promoter is used for transcription.
  • the tail domain comprises the 3' sequence UUUU, e.g., if an H I promoter is used for transcription.
  • tail domain comprises variable numbers of 3' U's depending, e.g., on the termination signal of the pol-III promoter used.
  • the tail domain comprises variable 3' sequence derived from the DNA template if a T7 promoter is used.
  • the tail domain comprises variable 3' sequence derived from the DNA template, e.g., if in vitro transcription is used to generate the RNA molecule.
  • the tail domain comprises variable 3' sequence derived from the DNA template, e.g, if a pol-II promoter is used to drive transcription.
  • Modifications in the tail domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section ⁇ .
  • gRNA's having a candidate tail domain having a selected length, sequence, degree of complementarity, or degree of modification can be evaluated in the system described in Section ⁇ .
  • the candidate tail domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
  • the tail domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the tail domain, within 5 nucleotides of the 3' end of the tail domain, or more than 5 nucleotides away from one or both ends of the tail domain.
  • no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the tail domain, within 5 nucleotides of the 3' end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain.
  • no nucleotide is modified within 5 nucleotides of the 5' end of the tail domain, within 5 nucleotides of the 3' end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain.
  • a gRNA has the following structure:
  • the targeting domain comprises a core domain and optionally a secondary domain, and is
  • the first complementarity domain is 5 to 25 nucleotides in length and, in an embodiment has
  • the linking domain is 1 to 5 nucleotides in length;
  • the proximal domain is 5 to 20 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference proximal domain disclosed herein;
  • the tail domain is absent or a nucleotide sequence is 1 to 50 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference tail domain disclosed herein.
  • a unimolecular, or chimeric, gRNA comprises, preferably from 5' to 3' :
  • a targeting domain (which is complementary to a target nucleic acid);
  • the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.
  • the proximal and tail domain when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides in length.
  • the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length.
  • the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.
  • the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.
  • the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 1 nucleotides in length.
  • the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.
  • the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.
  • the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length.
  • the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.
  • the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.
  • the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length.
  • the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting . domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 17 nucleotides (e.g.; 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together;
  • the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31 , 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • 19 nucleotides e.g., 19 consecutive nucleotides having complementarity with the target domain
  • the targeting domain is 19 nucleotides in length
  • the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21, 26, 31 , 32, 36, 41, 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • 21 nucleotides e.g., 21 consecutive nucleotides having complementarity with the target domain
  • the targeting domain is 21 nucleotides in length
  • the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 5 1 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is
  • the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, ( or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41, 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is
  • the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41, 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain:
  • the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • a modular gRNA comprises:
  • a first strand comprising, preferably from 5' to 3' ;
  • a second strand comprising, preferably from 5' to 3' :
  • proximal and tail domain when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides;
  • the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.
  • the proximal and tail domain when taken together, comprise at least 15, 1 8, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 2 1 , 22, 23, 24 or 25 nucleotides in length.
  • the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length.
  • the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.
  • the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.
  • the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length.
  • the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.
  • the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.
  • the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the ' targeting domain is 22 nucleotides in length.
  • the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.
  • the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.
  • the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 5 nucleotides in length.
  • the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 1 8, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41, 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 1 8 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • 19 nucleotides e.g., 19 consecutive nucleotides having complementarity with the target domain
  • the targeting domain is 19 nucleotides in length
  • the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35-, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41, 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
  • the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
  • the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
  • Methods for designing gRNAs are described herein, including methods for selecting, designing and validating target domains. Exemplary targeting domains are also provided herein. Targeting Domains discussed herein can be incorporated into the gRNAs described herein.
  • a software tool can be used to optimize the choice of gRNA within a user's target sequence, e.g., to minimize total off-target activity across the genome. Off target activity may be other than cleavage.
  • the tool can identify all off-target sequences (e.g., preceding either NAG or NGG PAMs) across the genome that contain up to certain number (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs.
  • the cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme.
  • Each possible gRNA is then ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the least off-target cleavage.
  • Other functions e.g., automated reagent design for CR1SPR construction, primer design for the on-target Surveyor assay, and primer design for high-throughput detection and quantification of off-target cleavage via next-gen sequencing, can also be included in the tool.
  • Candidate gRNA molecules can be evaluated by art-known methods or as described in Section IV herein. II.
  • Cas9 molecules of a variety of species can be used in the methods and compositions described herein. While the S. pyogenes and S. thermophil s Cas9 molecules are the subject of much of the disclosure herein, Cas9 molecules of, derived from, or based on the Cas9 proteins of other species listed herein can be used as well. In other words, while the much of the description herein uses S. pyogenes and S. thermophilus Cas9 molecules, Cas9 molecules from the other species can replace them, e.g., Staphylococcus aureus and Neisseria meningitidis Cas9 molecules. Additional Cas9 species include: Acidovorax avenae, Actinobacillus
  • Methylosinus trichosporium Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica.
  • Neisseria sp. Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium
  • Streptococcus sp. Subdoligranulum sp., Tislrella mobilis, Treponema sp., or Verminephrobacter eiseniae.
  • a Cas9 molecule refers to a molecule that can interact with a gRNA molecule and, in concert with the gRNA molecule, localize (e.g., target or home) to a site which comprises a target domain and PAM sequence. .
  • the Cas9 molecule is capable of cleaving a target nucleic acid molecule.
  • a Cas9 molecule that is capable of cleaving a target nucleic acid molecule is referred to herein as an eaCas9 (an enzymatically active Cas9) molecule.
  • an eaCas9 molecule comprises one or more of the following activities:
  • nickase activity i.e., .the ability to cleave a single strand, e.g., the non-complementary strand or the complementary strand, of a nucleic acid molecule
  • a double stranded nuclease activity i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities;
  • a helicase activity i.e., the ability to unwind the helical structure of a double stranded nucleic acid.
  • an enzymatically active Cas9 or an eaCas9 molecule cleaves both DNA strands and results in a double stranded break.
  • an eaCas9 molecule cleaves only one strand, e.g., the strand to which the gRNA hybridizes to, or the strand complementary to the strand the gRNA hybridizes with.
  • an eaCas9 molecule comprises cleavage activity associated with an HNH-like domain.
  • an eaCas9 molecule comprises cleavage activity associated with an N-terminal RuvC-like domain.
  • an eaCas9 molecule comprises cleavage activity associated with an HNH-like domain and cleavage activity associated with an N-terminal RuvC-like domain.
  • an eaCas9 molecule comprises an active, or cleavage competent, HNH-like domain and an inactive, or cleavage incompetent, N-terminal RuvC-like domain.
  • an eaCas9 molecule comprises an inactive, or cleavage incompetent, HNH-like domain and an active, or cleavage competent, N-terminal RuvC-like domain.
  • the ability of an eaCas9 molecule to interact with and cleave a target nucleic acid is PAM sequence dependent.
  • a PAM sequence is a sequence in the target nucleic acid.
  • cleavage of the target nucleic acid occurs upstream from the PAM sequence.
  • EaCas9 molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences).
  • an eaCas9 molecule of S. pyogenes recognizes the sequence motif NGG and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence.
  • an eaCas9 molecule of S. mulans recognizes the sequence motif NGG or NAAR (R - A or G) and directs cleavage of a core target nucleic acid sequence 1 to 10, e.g., 3 to 5 base pairs, upstream from this sequence. See, e.g., Deveau et al. , J BACTERIOL 2008;
  • an eaCas9 molecule of N. meningitidis recognizes the sequence motif NNNNGATT and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Hou et al., PNAS EARLY EDITION 2013, 1 -6.
  • the ability of a Cas9 molecule to recognize a PAM sequence can be determined, e.g., using a transformation assay described in Jinek et al , SCIENCE 2012, 337:816.
  • Cas9 molecules have the ability to interact with a gRNA molecule, and in conjunction with the gRNA molecule home (e.g., targeted or localized) to a core target domain, but are incapable of cleaving the target nucleic acid, or incapable of cleaving at efficient rates.
  • Cas9 molecules having no, or no substantial, cleavage activity are referred to herein as an eiCas9 (an enzymatically inactive Cas9) molecule.
  • an eiCas9 molecule can lack cleavage activity or have substantially less, e.g., less than 20, 10, 5, 1 or 0.1 % of the cleavage activity of a reference Cas9 molecule, as measured by an assay described herein.
  • Exemplary naturally occurring Cas9 molecules are described in Chylinski et al , RNA Biology 2013; 10:5, 727-737.
  • Such Cas9 molecules include Cas9 molecules of a cluster 1 bacterial family, cluster 2 bacterial family, cluster 3 bacterial family, cluster 4 bacterial family, cluster 5 bacterial family, cluster 6 bacterial family, a cluster 7 bacterial family, a cluster 8 bacterial family, a cluster 9 bacterial family, a cluster 10 bacterial family, a cluster 1 1 bacterial family, a cluster 12 bacterial family, a cluster 13 bacterial family, a cluster 14 bacterial family, a cluster 1 bacterial family, a cluster 16 bacterial family, a cluster 17 bacterial family, a cluster 1 8 bacterial family, a cluster 19 bacterial family, a cluster 20 bacterial family, a cluster 21 bacterial family, a cluster 22 bacterial family, a cluster 23 bacterial family, a cluster 24 bacterial family, a cluster 25 bacterial family, a cluster 26 bacterial family, a cluster 27
  • Exemplary naturally occurring Cas9 molecules include a Cas9 molecule of a cluster 1 bacterial family.
  • Examples include a Cas9 molecule of: S. pyogenes (e.g., strain SF370, MGAS 10270, MGAS 10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI- 1), S. tliermophilus (e.g., strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. /nutans (e.g., strain UA 159, NN2025), S. macacae (e.g., strain NCTC1 1558), S.
  • S. pyogenes e.g., strain SF370, MGAS 10270, MGAS 10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131
  • gallolylicus e.g., strain UCN34, ATCC BAA-2069
  • S. equines e.g., strain ATCC 9812, MGCS 124
  • S. dysdalactiae e.g., strain GGS 124
  • S. bovis e.g., strain ATCC 70033
  • S. cmginosus e.g.; strain F021 1
  • S. agalactia* e.g., strain NEM316, A909
  • Listeria monocytogenes e.g., strain F6854
  • Listeria innocua L.
  • Additional exemplary Cas9 molecules are a Cas9 molecule of Neisseria meningitidis (Hou et ' al. PNAS Early Edition 2013, 1 -6) and a S. aureus Cas9 molecule.
  • a Cas9 molecule e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence: having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with;
  • the Cas9 molecule comprises one or more of the following activities: a nickase activity; a double stranded cleavage activity (e.g., an endonuclease and/or exonuclease activity); a helicase activity; or the ' ability, together with a gRNA molecule, to localize to a target nucleic acid.
  • a Cas9 molecule comprises the amino acid sequence of the consensus sequence of FIG. 2, wherein "*" indicates any amino acid found in the corresponding position in the amino acid sequence of a Cas9 molecule of S. pyogenes, S. thermophilus, S. mutatis and L. innocua, and "-" indicates any amino acid.
  • a Cas9 molecule differs from the sequence of the consensus sequence disclosed in Figure 2 by at least 1 , but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.
  • a Cas9 molecule comprises the amino acid sequence of SEQ ID NO:7 of FIG.
  • a Cas9 molecule differs from the sequence of SEQ ID NO:6 or 7 by at least 1 , but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.
  • region 1 (residues 1 to 1 80, or in the case of region ⁇ residues 120 to 180)
  • region 2 (residues 360 to 480);
  • region 3 (residues 660 to 720);
  • a Cas9 molecule comprises regions 1 -5, together with sufficient additional Cas9 molecule sequence to provide a biologically active molecule, e.g., a Cas9 molecule having at least one activity described herein.
  • each of regions 1 -6 independently, have, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with the corresponding residues of a Cas9 molecule described herein, e.g., a sequence from FIG. 2 or from FIG. 5.
  • a Cas9 molecule e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 1 :
  • a Cas9 molecule e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 1 ':
  • thermophilus S. mulans or, L. innocua, N. meningitidis, or S. aureus ; or
  • thermophilus S. mulans or, L. innocua, N. meningitidis, or S. aureus.
  • a Cas9 molecule e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 2: having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 997c homology with amino acids 360-480 (52% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus;
  • thermophilus S. mutans or, L. innocua, N. meningitidis, or S. aureus; or
  • thermophilus S. mutans or, L. innocua, N. meningitidis, or S. aureus.
  • a Cas9 molecule e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 3:
  • thermophilus S. mutans or, L: innocua, N. meningitidis, or S. aureus; or
  • thermophilus S. mutans or, L. innocua, N. meningitidis, or S. aureus.
  • a Cas9 molecule e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 4:
  • thermophilus S. mutans or, L. innocua, N. meningitidis, or S. aureus ; or
  • a Cas9 molecule e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 5:
  • thermophilus S. mutans or, L. innocua, N. meningitidis, or S. aureus; or
  • thermophilus S. mutans or, L. innocua, N. meningitidis, or S. aureus .
  • a Cas9 molecule comprises an HNH-like domain and an RuvC-like domain.
  • cleavage activity is dependent on a RuvC-like domain and an HNH- like domain.
  • a Cas9 molecule e.g., an eaCas9 or eiCas9 molecule, can comprise one or more of the following domains: a RuvC-like domain and an HNH-like domain.
  • a cas9 molecule is an eaCas9 molecule and the eaCas9 molecule comprises a RuvC-like domain, e.g., a RuvC-like domain described below, and/or an HNH-like domain, e.g., an HNH-like domain described below.
  • a Cas9 molecule is an eiCas9 molecule comprising one or more difference in an RuvC-like domain and/or in an HNH-like domain as compared to a reference Cas9 molecule, and the eiCas9 molecule does not cleave a nucleic acid, or cleaves with significantly less efficiency than does wildype, e.g., when compared with wild type in a cleavage assay, e.g., as described herein, cuts with less than 50, 25, 10, or 1 % of the a reference Cas9 molecule, as measured by an assay described herein.
  • a RuvC-like domain cleaves, a single strand, e.g., the non- complementary strand of the target nucleic acid molecule.
  • a Cas9 molecule can include more than one RuvC-like domain (e.g., one, two, three or more RuvC-like domains).
  • an RuvC-like domain is at least 5, 6, 7, 8 amino acids in length but not more than 20, 19, 1 8, 17, 16 or 15 amino acids in length.
  • the cas9 molecule comprises an N-terminal RuvC-like domain of about 10 to 20 amino acids, e.g., about 15 amino acids in length.
  • Cas9 molecules comprise more than one RuvC-like domain, with cleavage being dependent on the N-terminal RuvC-like domain. Accordingly, Cas9 molecules can comprise an N-terminal RuvC-like domain. Exemplary N-terminal RuvC-like domains are described below.
  • an eaCas9 molecule comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula I:
  • X 1 is selected from I, V, M, L and T (e.g., selected from I, V, and L);
  • X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);
  • X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);
  • X4 is selected from S, Y, N and F (e.g., S);
  • X5 is selected from V, I, L, C, T and F (e.g., selected from V, I and L);
  • X6 is selected from W, F, V, Y, S and ' L (e.g., W);
  • X7 is selected from A, S, C, V and G (e.g., selected from A and S);
  • X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L);
  • X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, ⁇ , F, S, A, Y, M and R, or, e.g., selected from T, V, I, L and ⁇ ).
  • the N-tenninal RuvC-like domain differs from a sequence of SEQ ID NO:8, by as many as 1 but no more than 2, 3, 4, or 5 residues.
  • N-terminal RuvC-like domain is cleavage competent.
  • N-terminal RuvC-like domain is cleavage incompetent.
  • an eaCas9 molecule comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula II:
  • X 1 is selected from I, V, M, L and T (e.g., selected from I, V, and L);
  • X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);
  • X3 is selected from N, S, G, A, D, T, R, and F (e.g., A or N);
  • X5 is selected from V, I, L, C, T and F (e.g., selected from V, I and L);
  • X6 is selected from W, F, V, Y, S and L (e.g., W);
  • X7 is selected from A, S, C, V and G (e.g., selected from A and S);
  • X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L);
  • X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, ⁇ , F, S, A, Y, M and R or selected from e.g., T, V, I, L and ⁇ ).
  • the N-temninal RuvC-like domain differs from a sequence of SEQ ID NO:9 by as many as 1 , but no more than 2, 3, 4, or 5 residues.
  • the N-terminal RuvC-like domain comprises an amino acid sequence of formula HI:
  • X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);
  • X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);
  • X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L);
  • X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, ⁇ , F, S, A, Y, M and R or selected from e.g., T, V, I, L and ⁇ ).
  • the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 10 by as many as 1 , but no more than, 2, 3, 4, or 5 residues.
  • the N-terminal RuvC-like domain comprises an amino acid sequence of formula 111:
  • X is a non-polar alkyl amino acid or a hydroxyl amino acid, e.g., X is selected from V, I, L and T (e.g., the eaCas9 molecule can comprise an N-terminal RuvC-like domain shown in FIG. 2 (depicted as "Y")).
  • the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 1 1 by as many as 1 but no more than, 2, 3, 4, or 5 residues.
  • the N-terminal RuvC-like domain differs from a sequence of an N- terminal RuvC-like domain disclosed herein, e.g., in FIG. 3 A or FIG. 5, as many as 1 , but no more than 2, 3, 4, or 5 residues. In an embodiment, 1 , 2, or all 3 of the highly conserved residues identified in FIG. 3A or FIG. 5 are present.
  • the N-terminal RuvC-like domain differs from a sequence of an N- terminal RuvC-like domain disclosed herein, e.g., in FIG. 3B, as many as 1 , but no more than 2, 3, 4, or 5 residues. In an embodiment, 1 , 2, 3 or all 4 of the highly conserved residues identified in FIG. 3B are present.
  • a Cas9 molecule in addition to the N-terminal RuvC-like domain, a Cas9 molecule, e.g., an eaCas9 molecule, can comprise one or more additional RuvC-like domains.
  • a Cas9 molecule can comprise two additional RuvC-like domains.
  • the additional RuvC-like domain is at least 5 amino acids in length and, e.g., less than 15 amino acids in length, e.g., 5 to 10 amino acids in length, e.g., 8 amino acids in length.
  • An additional RuvC-like domain can comprise an amino acid sequence:
  • X I is V or H
  • X2 is I, L or V (e.g., I or V);
  • X3 is or T.
  • the additional RuvC-like domain comprises the amino acid sequence:
  • X2 is I, L or V (e.g., I or V) (e.g., the eaCas9 molecule can comprise an additional RuvC- like domain shown in FIG. 2 or FIG. 5 (depicted as "B")).
  • An additional RuvC-like domain can comprise an amino acid sequence: H-H-A-X 1 -D-A-X2-X3 (SEQ ID NO: 14), wherein
  • X I is H or L
  • X2 is R or V; and X3 is E or V.
  • the additional RuvC-like domain comprises the amino acid sequence:. H-H-A-H-D-A-Y-L (SEQ ID NO: 15).
  • the additional RuvC-like domain differs from a sequence of SEQ ID NO: 13, 15, 12 or 14 by as many as 1 , but no more than 2, 3, 4, or 5 residues.
  • sequence flanking the N-terminal RuvC-like domain is a sequences of formula V:
  • X V is selected from and P,
  • X2' is selected from V, L, I, and F (e.g., V, I and L);
  • X3' is selected from G, A and S (e.g., G),
  • X4' is selected from L, I, V and F (e.g., L);
  • X9' is selected from D, E, N and Q;
  • Z is an N-terminal RuvC-like domain, e.g. , as described above.
  • an HNH-like domain cleaves a single stranded complementary domain, e.g., a complementary strand of a double stranded nucleic acid molecule.
  • an HNH-like domain is at least 15, 20, 25 amino acids in length but not more than 40, 35 or 30 amino acids in length, e.g., 20 to 35 amino acids in length, e.g., 25 to 30 amino acids in length. Exemplary HNH-like domains are described below.
  • an eaCas9 molecule comprises an HNH-like domain having an amino acid sequence of formula VI: X 1 -X2-X3-H-X4-X5-P-X6-X7-X8-X9-X 10-X 1 1 -X 12-X 13-X 14-X 15-N-X 16-X 17-X 18- X 19-X20-X21 -X22-X23-N (SEQ ID NO: 17), wherein
  • X I is selected from D, E, Q and N (e.g., D and E);
  • X2 is selected from L, I, R, Q, V, M and ;
  • X3 is selected from D and E;
  • X4 is selected from I, V, T, A and L (e.g., A, 1 and V);
  • X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);
  • X6 is selected from Q, H, R, , Y, I, L, F and W;
  • X7 is selected from S, A, D, T and K (e.g., S and A);
  • X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);
  • X9 is selected from L, R, T, I, V, S, C, Y, , F and G;
  • X 10 is selected from , Q, Y, T, F, L, W, M, A, E, G, and S;
  • X I 1 is selected from D, S, N, R, L and T (e.g., D);
  • X 12 is selected from D, N and S;
  • X 13 is selected from S, A, T, G and R (e.g., S);
  • X 14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);
  • X 15 is selected from D, S, 1, N, E, A, H, F, L, Q, M, G, Y and V;
  • X 16 is selected from K, L, R, M, T and F (e.g., L, R and K);
  • X 17 is selected from V, L, I, A and T;
  • XI 8 is selected from L, I, V and A (e.g., L and I);
  • X 19 is selected from T, V, C, E, S and A (e.g., T and V);
  • X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;
  • X21 is selected from S, P, R, , N, A, H, Q, G and L;
  • X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y;
  • X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.
  • a HNH-like domain differs from a sequence of SEQ ID NO: 1 7 by at least 1 , but no more than, 2, 3, 4, or 5 residues.
  • the HNH-like domain is cleavage competent.
  • an eaCas9 molecule comprises an HNH-like domain comprising an amino acid sequence of formula VII:
  • X I is selected from D and E;
  • X2 is selected from L, I, R, Q, V, M and K;
  • X3 is selected from D and E;
  • X4 is selected from I, V, T, A and L (e.g., A, I and V);
  • X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);
  • X6 is selected from Q, H, R, K, Y, I, L, F and W;
  • X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F); .
  • X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;
  • X 10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;
  • X 14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);
  • X I 5 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;
  • X19 is selected from T, V, C, E, S and A (e.g., T and V);
  • X20 is selected from R, F, T, W, E, L, N, C, , V, S, Q, I, Y, H and A;
  • X21 is selected from S, P, R, K, N, A, H, Q, G and L;
  • X22 is selected from D, G, T, N, S, , A, I, E, L, Q, R and Y;
  • X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.
  • the HNH-like domain differs from a sequence of SEQ ID NO: 18 by 1 , 2, 3, 4, or 5 residues.
  • an eaCas9 molecule comprises an HNH-like domain comprising an amino acid sequence of formula VII:
  • X 1 is selected from D and E;
  • X3 is selected from D and E;
  • X6 is selected from Q, H, R, K, Y, I, L and W;
  • X8 is selected from F, L, V, , Y, M, I, R, A, E, D and Q (e.g., F);
  • X9 is selected from L, R, T, I, V, S, C, Y, , F and G;
  • X 10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;
  • X 14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);
  • X 15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;
  • X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;
  • X21 is selected from S, P, R, K, N, A, H, Q, G and L;
  • X22 is selected from D, G, T, N, S, , A, I, E, L, Q, R and Y;
  • X23 is selected from , V, A, E, Y, I, C, L, S, T, G, , M, D and F.
  • the HNH-like domain differs from a sequence of SEQ ID NO: 19 by 1 , 2, 3, 4, or 5 residues.
  • an eaCas9 molecule comprises an HNH-like domain having an amino acid sequence of formula VIII:
  • X2 is selected from I and V;
  • X5 is selected from I and V;
  • X7 is selected from A and S;
  • X9 is selected from I and L;
  • X 10 is selected from K and T;
  • X 12 is selected from D and N;
  • X 16 is selected from R, K and L; X 19 is selected from T and V;
  • X20 is selected from S and R;
  • X22 is selected from K, D and A;
  • X23 is selected from E, K, G and N (e.g., the eaCas9 molecule can comprise an HNH- like domain as described herein).
  • the HNH-like domain differs from a sequence of SEQ ID NO:20 by as many as 1 , but no more than 2, 3, 4, or 5 residues.
  • an eaCas9 molecule comprises the amino acid sequence of formula IX:
  • X I ' is selected from K and R;
  • X2' is selected from V and T;
  • X3' is selected from G and D;
  • X4' is selected from E, Q and D;
  • X5' is selected from E and D;
  • X6' is selected from D, N and H;
  • X7' is selected from Y, R and N;
  • X8' is selected from Q, D and N; X9' is selected from G and E;
  • X 10' is selected from S and G;
  • X I 1 ' is selected from D and N;
  • Z is an HNH-like domain, e.g., as described above.
  • the eaCas9 molecule comprises an amino acid sequence that differs from a sequence of SEQ ID NO:21 by as many as 1 , but no more than 2, 3, 4, or 5 residues.
  • the HNH-like domain differs from a sequence of an HNH-like domain disclosed herein, e.g., in FIG. 4A or FIG. 5, as many as 1 , but no more than 2, 3, 4, or 5 residues.
  • the HNH -like domain differs from a sequence of an HNH-like domain disclosed herein, e.g., in FIG. 4B, by as many as 1 , but no more than 2, 3, 4, or 5 residues. In an embodiment, 1 , 2, all 3 of the highly conserved residues identified in FIG. 4B are present.
  • Naturally occurring Cas9 molecules possess a number of properties, including: nickase activity, nuclease activity (e.g., endonuclease and/or exonuclease activity); helicase activity; the ability to associate functionally with a gRNA molecule; and the ability to target (or localize to) a site on a nucleic acid (e.g., PAM recognition and specificity).
  • a Cas9 molecules can include all or a subset of these properties.
  • Cas9 molecules have the ability to interact with a gRNA molecule and, in concert with the gRNA molecule, localize to a site in a nucleic acid.
  • Other activities e.g., PAM specificity, cleavage activity, or helicase activity can vary more widely in Cas9 molecules.
  • Cas9 molecules with desired properties can be made in a number of ways, e.g., by alteration of a parental, e.g., naturally occurring Cas9 molecules to provide an altered Cas9 molecule having a desired property.
  • a parental e.g., naturally occurring Cas9 molecules
  • one or more mutations or differences relative to a parental Cas9 molecule can be introduced. Such mutations and differences comprise:
  • a Cas9 molecule can comprises one or more mutations or differences, e.g., at least 1 , 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mutations but less than 200, 100, or 80 mutations relative to a reference Cas9 molecule.
  • a mutation or mutations do not have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein.
  • a mutation or mutations have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein.
  • exemplary activities comprise one or more of PAM specificity, cleavage activity, and helicase activity.
  • a mutation(s) can be present, e.g., in: one or more RuvC-like domain, e.g., an N- terminal RuvC-like domain; an HNH-like domain; a region outside the RuvC-like domains and the HNH-like domain.
  • a mutation(s) is present in an N-terminal RuvC- like domain. In some embodiments, a mutation(s) is present in an HNH-like domain. In some embodiments, mutations are present in both an N-terminal RuvC-like domain and an HNH-like domain.
  • Whether or not a particular sequence, e.g., a substitution, may affect one or more activity, such as targeting activity, cleavage activity, etc, can be evaluated or predicted, e.g., by evaluating whether the mutation is conservative or by the method described in Section ⁇ .
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a Cas9 molecule, e.g., a naturally occurring Cas9 molecule, e.g., an eaCas9 molecule, without abolishing or more preferably, without substantially altering a Cas9 activity (e.g., cleavage activity), whereas changing an "essential" amino acid residue results in a substantial loss of activity (e.g., cleavage activity).
  • the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of S. pyogenes shown in the consensus sequence disclosed in Figure 2, and has one or more amino acids that differ from the amino acid sequence of S. pyogenes (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figure 2 or SEQ ID NO:7.
  • the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of S. pyogenes shown in the consensus sequence disclosed in Figure 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.
  • the altered Cas9 molecule comprises a sequence in which:
  • sequence corresponding to the fixed sequence of the consensus sequence disclosed in Figure 2 differs at no more than 1 , 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figure 2;
  • sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figure 2 differ at no more than 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the "*" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. pyogenes Cas9 molecule; and,
  • sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figure 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "- " residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. pyogenes Cas9 molecule.
  • the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of S. thermophilus shown in the consensus sequence disclosed in Figure 2, and has one or more amino acids that differ from the amino acid sequence of S. thermophilus (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figure 2.
  • the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of S. thermophilus shown in the consensus sequence disclosed in Figure 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.
  • the altered Cas9 molecule comprises a sequence in which:
  • the sequence corresponding to the fixed sequence of the consensus sequence disclosed in Figure 2 differs at no more than 1 , 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the
  • sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figure 2 differ at no more than 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the "*" " residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S.
  • thermophilus Cas9 molecule thermophilus Cas9 molecule
  • sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figure 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "- " residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. thermophilus Cas9 molecule.
  • the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of S. mutans shown in the consensus sequence disclosed in Figure 2, and has one or more amino acids that differ from the amino acid sequence of S. mutans (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figure 2.
  • the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of S. mutans shown in the consensus sequence disclosed in Figure 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.
  • the altered Cas9 molecule comprises a sequence in which:
  • the sequence corresponding to the fixed sequence of the consensus sequence disclosed in Figure 2 differs at no more than 1 , 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the
  • sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figure 2 differ at no more than 1 , 2, 3, 4, 5, 10, 1 , 20, 25, 30, 35, or 40% of the "*" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. mutatis Cas9 molecule; and,
  • sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figure 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "- " residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. mutatis Cas9 molecule.
  • the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of L. intiocula shown in the consensus sequence disclosed in Figure 2, and has one or more amino acids that differ from the amino acid sequence of L. innocula (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figure 2.
  • the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of L. innocul shown in the consensus sequence disclosed in Figure 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.
  • the altered Cas9 molecule comprises a sequence in which:
  • sequence corresponding to the fixed sequence of the consensus sequence disclosed in Figure 2 differs at no more than 1 , 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figure 2;
  • sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figure 2 differ at no more than 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the "*" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an L. innocula Cas9 molecule; and,
  • sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figure 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "- " residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an L. innocula Cas9 molecule.
  • the altered Cas9 molecule e.g., an eaCas9 molecule or an eiCas9 molecule
  • a fragment of a naturally occurring Cas9 molecule of one species can be fused to a fragment of a Cas9 molecule of a second species.
  • a fragment of Cas9 of S. pyogenes comprising an N-terminal RuvC-like domain can be fused to a fragment of Cas9 of a species other than S. pyogenes (e.g., S. thermophiliis) comprising an HNH-like domain.
  • Naturally occurring Cas9 molecules can recognize specific PAM sequences, for example the PAM recognition sequences described above for S. pyogenes, S. thermophilus,S. mutans, S. aureus and N. meningitidis.
  • a Cas9 molecule has the same PAM specificities as a naturally occurring Cas9 molecule.
  • a Cas9 molecule has a PAM specificity not associated with a naturally occurring Cas9 molecule, or a PAM specificity not associated with the naturally occurring Cas9 molecule to which it has the closest sequence homology.
  • a naturally occurring Cas9 molecule can be altered, e.g., to alter PAM recognition, e.g., to alter the PAM sequence that the Cas9 molecule recognizes to decrease off target sites and/or improve specificity; or eliminate a PAM recognition requirement.
  • a Cas9 molecule can be altered, e.g., to increase length of PAM recognition sequence and/or improve Cas9 specificity to high level of identity to decrease off target sites and increase specificity.
  • the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acids in length.
  • Cas9 molecules that recognize different PAM sequences and/or have reduced off- target activity can be generated using directed evolution. Exemplary methods and systems that can be used for directed evolution of Cas9 molecules are described, e.g., in Esvelt el ai , Nature 201 1 , 472(7344): 499-503.
  • Candidate Cas9 molecules can be evaluated, e.g., by methods described in Section III.
  • a Cas9 molecule comprises a cleavage property that differs from naturally occurring Cas9 molecules, e.g., that differs from the naturally occurring Cas9 molecule having the closest homology.
  • a Cas9 molecule can differ from naturally occurring Cas9 molecules, e.g., a Cas9 molecule of S. pyogenes, as follows: its ability to modulate, e.g., decreased or increased, cleavage of a double stranded break (endonuclease and/or exonuclease activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S.
  • pyogenes its ability to modulate, e.g., decreased or increased, cleavage of a single strand of a nucleic acid, e.g., a non-complimentary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); or the ability to cleave a nucleic acid molecule, e.g., a double stranded or single stranded nucleic acid molecule, can be eliminated.
  • an eaCas9 molecule comprises one or more of the following activities: cleavage activity associated with an N-terminal RuvC-like domain; cleavage activity associated with an HNH-like domain; cleavage activity associated with an HNH domain and cleavage activity associated with an N-terminal RuvC-like domain.
  • an eaCas9 molecule comprises an active, or cleavage competent, HNH-like domain (e.g., an HNH-like domain described herein, e.g., SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21 ) and an inactive, or cleavage incompetent, N-terminal RuvC-like domain.
  • HNH-like domain e.g., an HNH-like domain described herein, e.g., SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21
  • an inactive, or cleavage incompetent, N-terminal RuvC-like domain e.g., an HNH-like domain described herein, e.g., SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21
  • An exemplary inactive, or cleavage incompetent N- terminal RuvC-like domain can have a mutation of an aspartic acid in an N-terminal RuvC-like domain, e.g., an aspartic acid at position 9 of the consensus sequence disclosed in Figure 2 or an aspartic acid at position 10 of SEQ ID NO:7, e.g., can be substituted with an alanine.
  • the eaCas9 differs from wild type in the N-terminal RuvC-like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.
  • the reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S.
  • the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.
  • an eaCas9 molecule comprises an inactive, or cleavage incompetent, HNH domain and an active, or cleavage competent, N-terminal RuvC-like domain (e.g., an HNH-like domain described herein, e.g., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15).
  • an HNH-like domain described herein e.g., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • Exemplary inactive, or cleavage incompetent HNH-like domains can have a mutation at one or more of: a histidine in an HNH-like domain, e.g., a,histidine at position 856 of the consensus sequence disclosed in Figure 2, e.g., can be substituted with an alanine; and one or more asparagines in an HNH-like domain, e.g., an asparagine at position 870 of the consensus sequence disclosed in Figure 2 and/or at position 879 of the consensus sequence disclosed in Figure 2, e.g., can be substituted with an alanine.
  • the eaCas9 differs from wild type in the HNH- like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein.
  • the reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, or S. thermophilus.
  • the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.
  • the altered Cas9 molecule is an eiCas9 molecule which does not cleave a nucleic acid molecule (either double stranded or single stranded nucleic acid molecules) or cleaves a nucleic acid molecule with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1 % o the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein.
  • the reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of . . pyogenes, S.
  • thermophilus S. aureus or N. meningitidis.
  • the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.
  • the eiCas9 molecule lacks substantial cleavage activity associated with an N- terminal RuvC-like domain and cleavage activity associated with an HNH-like domain.
  • an eiCas9 molecule comprises an inactive, or cleavage incompetent, N-terminal RuvC-like domain.
  • An exemplary inactive, or cleavage incompetent N-terminal RuvC-like domain can have a mutation of an aspartic acid in an N-terminal RuvC-like domain, e.g., an aspartic acid at position 9 of the consensus sequence disclosed in Figure 2 or an aspartic acid at position 10 of SEQ ID NO:7, e.g., can be substituted with an alanine.
  • an eiCas9 molecule comprises an inactive, or cleavage incompetent, HNH domain (e.g., an HNH-like domain described herein, e.g., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15).
  • HNH domain e.g., an HNH-like domain described herein, e.g., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
  • Exemplary inactive, or cleavage incompetent HNH-like domains can have a mutation at one or more of: a histidine in an HNH-like domain, e.g., a histidine at position 856 of the consensus sequence disclosed in Figure 2, e.g., can be substituted with an alanine; and one or more asparagines in an HNH-like domain, e.g., an asparagine at position 870 of the consensus sequence disclosed in Figure 2 and/or at position 879 of the consensus sequence disclosed in Figure 2, e.g., can be substituted with an alanine.
  • a catalytically inactive Cas9 molecule may be fused with a transcription repressor.
  • An eiCas9 fusion protein complexes with a gRNA and localizes to a DNA sequence specified by gRNA's targeting domain, but, unlike an eaCas9, it will not cleave the target DNA. Fusion of an effector domain, such as a transcriptional repression domain, to an eiCas9 enables recmitment of the effector to any DNA site specified by the gRNA.
  • Site specific targeting of an eiCas9 or an eiCas9 fusion protein to a promoter region of a gene can block R A polymerase binding to the promoter region, a transcription factor (e.g., a transcription activator) and/or a transcriptional enhancer to inhibit transcription activation.
  • site specific targeting of an eiCas9- fusion to a transcription repressor to a promoter region of a gene can be used to decrease transcription activation.
  • Transcription repressors or transcription repressor domains that may be fused to an eiCas9 molecule can include ruppel associated box (KRAB or SKD), the Mad mSIN3 interaction domain (SID) or the ERF repressor domain (ERD).
  • KRAB or SKD ruppel associated box
  • SID Mad mSIN3 interaction domain
  • an eiCas9 molecule may be fused with a protein that modifies chromatin.
  • an eiCas9 molecule may be fused to heterochromatin protein 1 (HPl ), a histone lysine methyltransferase (e.g., SUV39H 1 , SUV39H2, G9A, ESET/SETDB l , Pr-SET7/8, SUV4-20H 1 , RIZ1), a histone lysine demethylates (e.g., LSD1/BHC1 10, SpLsdl/Sw, l/Safl 10, Su(var)3-3, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1 , JMJD2D, Rph l , JARID 1 A/RBP2, JARI DIB/PLU- I , JAR1D 1C/SMCX, JARID1 D/SMCY, Lid,
  • HPl heterochromatin protein
  • the heterologous sequence (e.g., the transcription repressor domain) may be fused to the N- or C-terminus of the eiCas9 protein.
  • the heterologous sequence (e.g., the transcription repressor domain) may be fused to an internal portion (i.e., a portion other than the N-terminus or C-terminus) of the eiCas9 protein.
  • the ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated, e.g., by the methods described herein in Section ⁇ .
  • the activity of a Cas9 molecule, either an eaCas9 or a eiCas9, alone or in a complex with a gRNA molecule may also be evaluated by methods well-known in the art, including, gene expression assays and chromatin-based assays, e.g., chromatin immunoprecipitation (ChiP) and chromatin in vivo assay (CiA).
  • Nucleic acids encoding the Cas9 molecules e.g., an eaCas9 molecule or an eiCas9 molecule are provided herein.
  • Exemplary nucleic acids encoding Cas9 molecules are described in Cong et al , SCIENCE 2013, 399(6121):819-823; Wang et al , CELL 2013, 153(4):910-918; Mali et al. , SCIENCE 2013, 399(6121):823-826; Jinek et al, SCIENCE 2012, 337(6096):816-821.
  • Another exemplary nucleic acid encoding a Cas9 molecule of N. meningitidis is shown in FIG. 6.
  • a nucleic acid encoding a Cas9 molecule can be a synthetic nucleic acid sequence.
  • the synthetic nucleic acid molecule can be chemically modified, e.g., as described in Section X.
  • the Cas9 mRNA has one or more of, e.g., all of the following properties: it is capped, polyadenylated, substituted with 5-methylcytidine and/or pseudouridine.
  • the synthetic nucleic acid sequence can be codon optimized, e.g., at least one non-common codon or less-common codon has been replaced by a common codon.
  • the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., described herein.
  • a nucleic acid encoding a Cas9 molecule may comprise a nuclear localization sequence (NLS).
  • NLS nuclear localization sequences are known in the art.

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Abstract

Methods and compositions useful in targeting a payload to or editing a target nucleic acid are disclosed herein.

Description

CRISPR-RELATED METHODS AND COMPOSITIONS
FIELD OF THE INVENTION
The invention relates to CRISPR-related methods and components for editing of, or delivery of a payload to, a target nucleic acid sequence.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S. Provisional Application No.
61/883,925, filed September 27, 2013; and U.S. Provisional Application No. 61/898,043, filed October 31 , 2013. The contents of each of which are hereby incorporated by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 25, 2014, is named "C2159-7000WO_SL.txt" and is 210 KB in size.
BACKGROUND OF THE INVENTION
[0003] CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) evolved in bacteria as an adaptive immune system to defend against viral attack. Upon exposure to a virus, short segments of viral DNA are integrated into the CRISPR locus. RNA is transcribed from a portion of the CRISPR locus that includes the viral sequence. That RNA, which contains sequence complimentary to the viral genome, mediates targeting of a Cas9 protein to the sequence in the viral genome. The Cas9 protein cleaves and thereby silences the viral target.
[0004] Recently, the CRISPR/Cas system has been adapted for genome editing in eukaryotic cells. The introduction of site-specific double strand breaks (DSBs) allows for target sequence alteration through one of two endogenous DNA repair mechanisms— either non-homologous end-joining (NHEJ) or homology-directed repair (HDR).. The CRISPR/Cas system has also been used for gene regulation including transcription repression and activation without altering the target sequence. Targeted gene regulation based on the CRISPR/Cas system uses an enzymatically inactive Cas9 (also known as a catalytically dead Cas9).
SUMMARY OF THE INVENTION
[0005] Methods and compositions disclosed herein, e.g., a Cas9 molecule complexed with a gRNA molecule, can be used to target a specific location in a target DNA. Depending on the Cas9 molecule/gRNA molecule complex used specific editing or the delivery of a payload can be effected.
[0006] In one aspect, the disclosure features a gRNA molecule comprising a targeting domain which is complementary with a target sequence from a target nucleic acid disclosed herein, e.g., a sequence from: a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, 1X-3, XIV- 1 , or Section VIII.
[0007] In another aspect, the disclosure features a composition, e.g., pharmaceutical
composition, comprising a gRNA molecule described herein.
[0008] In some embodiments, the composition further comprises a Cas9 molecule, e.g., an eaCas9 or an eiCas9 molecule. In some embodiments, said Cas9 molecule is an eaCas9 molecule. In other embodiments, said Cas9 molecule is an eiCas9 molecule.
[0009] In some embodiments, said composition comprises a payload, e.g., a payload described herein, e.g., in Section VI, e.g., in Table VI-1, VI-2, VI-3, VI-4, VI-5, VI-6, or VI-7.
[0010] In some embodiments, the payload comprises: an epigenetic modifier, e.g., a molecule that modifies DNA or chromatin; component, e.g., a molecule that modifies a histone, e.g., an epigenetic modifier described herein, e.g., in Section VI; a transcription factor, e.g., a
transcription factor described herein, e.g., in Section VI; a transcriptional activator domain; an inhibitor of a transcription factor, e.g., an anti-transcription factor antibody, or other inhibitors; a small molecule; an antibody; an enzyme; an enzyme that interacts with DNA, e.g., a helicase, restriction enzyme, ligase, or polymerase; and/or a nucleic acid, e.g., an enzymatically active nucleic acid, e.g., a ribozyme, or an mRNA, siRNA, of antisense oligonucleotide. In some embodiments, the composition further comprises a Cas9 molecule, e.g., an eiCas9, molecule. [0011 ] In some embodiments, said payload is coupled, e.g., covalently or noncovalently, to a Cas9 molecule, e.g., an eiCas9 molecule. In some embodiments, said payload is coupled to said Cas9 molecule by a linker. In some embodiments, said linker is or comprises a bond that is cleavable under physiological, e.g., nuclear, conditions. In some embodiments, said linker is, or comprises, a bond described herein, e.g., in Section XI. In some embodiments, said linker is, or comp ises, an ester bond. In some embodiments, said payload comprises a fusion partner fused to a Cas9 molecule, e.g., an eaCas9 molecule or an eiCas9 molecule.
[0012] In some embodiments, said payload is coupled, e.g., covalently or noncovalently, to the gRNA molecule. In some embodiments, said payload is coupled to said gRNA molecule by a linker. In some embodiments, said linker is or comprises a bond that is cleavable under physiological, e.g., nuclear, conditions. In some embodiments, said linker is, or comprises, a bond described herein, e.g., in Section XI. In some embodiments, said linker is, or comprises, an ester bond.
[0013] In some embodiments, the composition comprises an eaCas9 molecule. In some embodiments, the composition comprises an eaCas9 molecule which forms a double stranded break in the target nucleic acid.
[0014] In some embodiments, the composition comprises an eaCas9 molecule which forms a single stranded break in the target nucleic acid. In some embodiments, said single stranded break is formed in the complementary strand of the target nucleic acid. In some embodiments, said single stranded break is formed in the strand which is not the complementary strand of the target nucleic acid.
[0015] In some embodiments, the composition comprises HNH-like domain cleavage activity but having no, or no significant, N-terminal RuvC-like domain cleavage activity. In some embodiments, the composition comprises N-terminal RuvC-like domain cleavage activity but having no, or no significant, HNH-like domain cleavage activity.
[0016] In some embodiments, said double stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position. In some embodiments, said single stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position. [0017] In some embodiments, the composition further comprises a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
[0018] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, V1I- 16, VII- 17, VII-18, VII-19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- IA, IX-2, IX-3, XIV- 1 , or Section VIII.
[0019] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII-17, VII-18, VII-19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1 , IX-IA, IX-2, IX- 3, XIV- 1 , or Section VIII.
[0020] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, IX-2, IX-3, XIV- 1 , or Section VIII.
[0021] In some embodiments, the composition further comprises a second gRNA molecule, e.g., a second gRNA molecule described herein.
[0022] In some embodiments, said gRNA molecule and said second gRNA molecule mediate breaks at different sites in the target nucleic acid, e.g., flanking a target position. In some embodiments, said gRNA molecule and said second gRNA molecule are complementary to the same strand of the target. In some embodiments, said gRNA molecule and said second gRNA molecule are complementary to the different strands of the target.
[0023] In some embodiments, said Cas9 molecule mediates a double stranded break.
[0024] In some embodiments, said gRNA molecule and said second gRNA molecule are configured such that first and second break made by the Cas9 molecule flank a target position. In some embodiments, said double stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
[0025] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
[0026] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of a target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VI1B, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- IA, IX-2, IX-3, XIV- 1 , or Section VIII.
[0027] In some embodiments, the template nucleic acid is a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to'200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g.', in Section V11B, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- IA, IX-2, IX-3, XIV- 1 , or Section VIII.
[0028] In some embodiments, the template nucleic acid is a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX-I A, IX-2, IX- 3, XIV- 1 , or Section VIII.
[0029] In some embodiments, said Cas9 molecule mediates a single stranded break.
[0030] In some embodiments, said gRNA molecule and said second gRNA molecule are configured such that a first and second break are formed in the same strand of the nucleic acid target, e.g., in the case of transcribed sequence, the template strand or the non-template strand.
[0031 ] In some embodiments, said first and second break flank a target position.
[0032] In some embodiments, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position. [0033] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position. In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , ΓΧ- 1 Α, rX-2, IX-3, XIV- 1 , or Section VIII.
[0034] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX-1 , IX- I A, IX-2, IX- 3, XIV- 1 , or Section VIII.
[0035] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XTV- 1 , or Section VIII.
[0036] In some embodiments, said gRNA molecule and said second gRNA molecule are configured such that a first and a second breaks are formed in different strands of the target. In some embodiments, said first and second break flank a target position. In some embodiments, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
[0037] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
[0038] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII- 14, VII- 15, VII-16, VII-17, VII- 18, VII- 19, VII-20, VII-21 , VIl-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, IX-2, LX-3, XIV- 1 , or Section VIII.
[0039] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1 , IX-IA, IX-2, IX- 3, XIV- 1 , or Section VIII.
[0040] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII-19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1, LX- 1 A, IX-2, lX-3, XIV- 1 , or Section VIII.
[0041] In some embodiments, the composition comprises a second Cas9 molecule.
[0042] In some embodiments, one or both of said Cas9 molecule and said second Cas9 molecule are eiCas9 molecules. In some embodiments, said eiCas9 molecule is coupled to a payload by a linker and said second eiCas9 molecules is coupled to a second payload by a second linker.
[0043] In some embodiments, said payload and said second payload are the same. In some embodiments, said payload and said second payload are different. In some embodiments, said linker and said second linker are the same. In some embodiments, said linker and said second linker are different, e.g., have different release properties, e.g., different release rates.
[0044] In some embodiments, said payload and said second payload are each described herein, e.g., in Section VI, e.g., in Table VI- 1, VI-2, VI-3, VI-4, VI-5, VI-6, or VI-7. In some embodiments, said payload and said second payload can interact, e.g., they are subunits of a protein.
[0045] In some embodiments, one of both of said Cas9 molecule and said second Cas9 molecule are eaCas9 molecules.
[0046] In some embodiments, said eaCas9 molecule comprises a first cleavage activity and said second eaCas9 molecule comprises a second cleavage activity. In some embodiments, said cleavage activity and said second cleavage activity are the same, e.g., both are N-terminal RuvC- like domain activity or are both HNH-like domain activity. In some embodiments, said cleavage activity and said second cleavage activity are different, e.g., one is N-terminal RuvC-like domain activity and one is HNH-like domain activity.
[0047] In some embodiments, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for, e.g., NGGNG, NNAGAAW (W = A or T), or NAAR (R = A or G).
[0048] In some embodiments, said Cas9 molecule and said second Cas9 molecule both mediate double stranded breaks.
[0049] In some embodiments, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein. In some embodiments, said gRNA molecule and said second gRNA molecule are configured such that first and second break flank a target position. In some embodiments, one of said first and second double stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
[0050] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
[0051 ] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- IA, IX-2, IX-3, XIV- 1 , or Section VIII.
[0052] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, IX-2, IX- 3, XIV- 1 , or Section VIII. [0053] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, rX-2, IX-3, XIV- 1 , or Section VIII.
[0054] In some embodiments, one of said Cas9 molecule and said second Cas9 molecule mediates a double stranded break and the other mediates a single stranded break.
[0055] In some embodiments, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein. In some embodiments, said gRNA molecule and said second gRNA molecule are configured such that a first and second break flank a target position. In some embodiments, said first and second break flank a target position. In some embodiments, one of said first and second breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
[0056] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
[0057] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII-19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, IX-2, IX- 3, XIV- 1 , or Section VIII.
[0058] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VTT-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX- 3, XIV- 1 , or Section VIII. [0059] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XIV- 1 , or Section VIII.
[0060] In some embodiments, said Cas9 molecule and said second Cas9 molecule both mediate single stranded breaks.
[0061] In some embodiments, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein. In some embodiments, said first and second break flank a target position.
[0062] In some embodiments, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
[0063] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments,- the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
[0064] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19,' VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- IA, IX-2, IX-3, XIV- 1 , or Section VIII.
[0065] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, IX-2, IX- XIV- 1 , or Section VIII. [0066] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII-18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XIV- 1 , or Section VIII.
[0067] In some embodiments, said gRNA molecule, said second gRNA molecule are configured such that a first and second break are in the same strand.
[0068] In some embodiments, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein. In some embodiments, said gRNA molecule, said second gRNA molecule are configured such that a first and second break flank a target position. In some embodiments, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
[0069] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
[0070] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XIV- 1 , or Section VIII.
[0071] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX- 3, XIV- 1 , or Section VIII.
[0072] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% 6f its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , ΓΧ- 1Α, IX-2, IX-3, XIV- 1 , or Section VIII.
[0073] In some embodiments, said first and second break are on the different strands.
[0074] In some embodiments, said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another Pam described herein. In some embodiments, said gRNA molecule, said second gRNA molecule are configured such that a first and second break are on different strands.
[0075] In some embodiments, said gRNA molecule, said second gRNA molecule are configured such that a first and second break flank a target position. In some embodiments, said first and second break flank a target position.
[0076] In some embodiments, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
[0077] In some embodiments, the composition further comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
[0078] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XIV- 1 , or Section VIII. -
[0079] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII-18, VII- 19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX- 1 , IX-1A, IX-2, IX- 3, XIV- 1 , or Section VIII. [0080] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XIV- 1 , or Section VIII.
[0081] In yet another aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising a gRNA molecule and a second gRNA molecule described herein.
[0082] In some embodiments, the composition further comprises a nucleic acid, e.g., a DNA or mRNA, that encodes a Cas9 molecule described herein. In some embodiments, the composition further comprises a nucleic acid, e.g., a DNA or RNA, that encodes a second Cas9 molecule described herein. In some embodiments, the composition further comprises a template nucleic acid described herein.
[0083] In one aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising, nucleic acid sequence, e.g., a DNA, that encodes one or more gRNA molecules described herein.
[0084] In some embodiments, said nucleic acid comprises a promoter operably linked to the sequence that encodes a gRNA molecule, e.g., a promoter described herein.
[0085] In some embodiments, said nucleic acid comprises a second promoter operably linked to the sequence that encodes a second gRNA molecule, e.g., a promoter described herein. In some embodiments, the promoter and second promoter are different promoters. In some embodiments, the promoter and second promoter are the same.
[0086] In some embodiments, the nucleic acid further encodes a Cas9 molecule described herein. In some embodiments, the nucleic acid further encodes a second Cas9 molecule described herein.
[0087] In some embodiments, said nucleic acid comprises a promoter operably linked to the sequence that encodes a Cas9 molecule, e.g., a promoter described herein.
[0088] In some embodiments, said nucleic acid comprises a second promoter operably linked to the sequence that encodes a second Cas9 molecule, e.g., a promoter described herein. In some embodiments, the promoter and second promoter are different promoters. In some embodiments, the promoter and second promoter are the same.
[0089] In some embodiments, the composition further comprises a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.
[0090] In another aspect, the disclosure features a composition, e.g., a pharmaceutical composition, comprising nucleic acid sequence that encodes one or more of: a) a Cas9 molecule, b) a second Cas9 molecule, c) a gRNA molecule, and d) a second gRNA molecule.
[0091 ] In some embodiments, each of a), b)v c) and d) present are encoded on the same duplex molecule.
[0092] In some embodiments, a first sequence selected from of a), b), c) and d) is encoded on a first duplex molecule and a second sequence selected from a), b), c), and d) is encoded on a second duplex molecule.
[0093] In some embodiments, said nucleic acid encodes: a) and c); a), c), and d); or a), b), c), and d).
[0094] In some embodiments, the composition further comprises a Cas9 molecule, e.g., comprising one or more of the Cas9 molecules wherein said nucleic acid does not encode a Cas9 molecule.
[0095] In some embodiments, the composition further comprises an mRNA encoding Cas9 molecule, e.g., comprising one or more mRNAs encoding one or more of the Cas9 molecules wherein said nucleic acid does not encode a Cas9 molecule.
[0096] In some embodiments, the composition further comprises a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.
[0097] In yet another aspect, the disclosure features a nucleic acid described herein.
[0098] In one aspect, the disclosure features a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and c) optionally, a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV. [0099] In another aspect, the disclosure features a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.
[0100] In yet another aspect, the disclosure features a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.
[0101] In still another aspect, the disclosure features a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g. , in Section IV.
[0102] In one aspect, the disclosure features a method of altering a cell, e.g., altering the structure, e.g., sequence, of a target nucleic acid of a cell, comprising contacting said cell with:
1 ) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV;
2) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV;
3) a composition comprising:
a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; or
4) a composition comprising:
a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g.. in Section IV.
[0103] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by, one dosage form, mode of delivery, or formulation.
[0104] In some embodiments, a) a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by, a first dosage form, a first mode of delivery, or a first formulation; and b) an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.
[0105] In some embodiments, the cell is an animal or plant cell . In some embodiments, the cell is a mammalian, primate, or human cell. In some embodiments, the cell is a human cell, e.g., a cell from described herein, e.g., in Section VIIA. In some embodiments, the cell is: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blastocyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell. In some embodiments, the cell is a human cell, e.g., a cancer cell or other cell characterized by a disease or disorder.
[0106] In some embodiments, the target nucleic acid is a chromosomal nucleic acid. In some embodiments, the target nucleic acid is an organellar nucleic acid. In some embodiments, the target nucleic acid is a mitochondrial nucleic acid. In some embodiments, the target nucleic acid is a chloroplast nucleic acid.
[0107] In some embodiments, the cell is a cell of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
[0108] In some embodiments, the target nucleic acid is the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
[0109] In some embodiments, said method comprises: modulating the expression of a gene or inactivating a disease organism.
[0110] In some embodiments, said cell is a cell characterized by unwanted proliferation, e.g., a cancer cell. In some embodiments, said cell is a cell characterized by an unwanted genomic component, e.g., a viral genomic component. In some embodiments, the cell is a cell described herein, e.g., in Section ΠΑ. In some embodiments, a control or structural sequence of at least, 2 3, 4, or 5 genes is altered.
[0111] In some embodiments, the target nucleic acid is a rearrangement, a kinase, a
rearrangement that comprises a kinase, or a tumor suppressor.
[0112] In some embodiments, the method comprises cleaving a target nucleic acid within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position. In some embodiments, said composition comprises a template nucleic acid.
[0113] In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
[01 14] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, V1I-20, VII, 21 , VIl-22, Vl -23, VII-24, VII-25, IX- 1 , IX- IA, 1X-2, IX-3, XIV- 1 , or Section VIII.
[01 15] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VlI- 17, Vll-18, VII- 19, VII-20, VII-21, VII-22, VII-23, VII-24, VII-25, IX-1 , IX- I A, IX-2, IX- 3, XIV- 1 , or Section VIII.
[0116] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a con-esponding sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 1 8, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- I A, IX-2, IX-3, XIV- 1 , or Section VIII.
[01 17] In some embodiments,
a) a control region, e.g., a cis-acting or tans-acting control region, of a gene is cleaved; b) the sequence of a control region, e.g., a cis-acting or tans-acting control region, of a gene is altered, e.g., by an alteration that modulates, e.g., increases or decreases, expression a gene under control of the control region, e.g., a control sequence is disrupted or a new control sequence is inserted;
c) the coding sequence of a gene is cleaved;
d) the sequence of a transcribed region, e.g., a coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that increases expression of or activity of the gene product is effected, e.g., a mutation is corrected; and/or
e) the sequence of a transcribed region, e.g., the coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that decreases expression of or activity of the gene product is effected, e.g., a mutation is inserted, e.g., the sequence of one or more nucleotides is altered so as to insert a stop codon.
[0118] In some embodiments, a control region or transcribed region, e.g., a coding sequence, of at least 2, 3, 4, 5, or 6 genes are altered. [0119] In another aspect, the disclosure features a method of treating a subject, e.g., by altering the structure, e.g., altering the sequence, of a target nucleic acid, comprising administering to the subject, an effective amount of:
1 ) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule) ;
b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV;
2) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV;
3) a composition comprising:
a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; and/or
4) a composition comprising:
a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.
[0120] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation.
[0121] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage form, in a first mode of delivery, or first formulation; and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.
[0122] In some embodiments, the subject is an animal or plant. In some embodiments, the subject is a mammalian, primate, or human.
[0123] In some embodiments, the target nucleic acid is the nucleic acid of a human cell, e.g., a cell described herein, e.g., in Section VIIA. In some embodiments, the target nucleic acid is the nucleic acid of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.
[0124] In some embodiments, the target nucleic acid is a chromosomal nucleic acid. In some embodiments, the target nucleic acid is an organellar nucleic acid. In some embodiments, the nucleic acid is a mitochondrial nucleic acid. In some embodiments, the nucleic acid is a chloroplast nucleic acid.
[0125] In some embodiments, the target nucleic acid is the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite. In some embodiments, said method comprises modulating expression of a gene or inactivating a disease organism.
[0126] In some embodiments, the target nucleic acid is the nucleic acid of a cell characterized by unwanted proliferation, e.g., a cancer cell. In some embodiments, said target nucleic acid comprises an unwanted genomic component, e.g., a viral genomic component. In some embodiments, a control or structural sequence of at least, 2 3, 4, or 5 genes is altered. In some embodiments, the target nucleic acid is a rearrangement, a kinase, a rearrangement that comprises a kinase, or a rumor suppressor.
[0127] In some embodiments, the method comprises cleaving a target nucleic acid within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
[0128] In some embodiments, said composition comprises a template nucleic acid. In some embodiments, the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
[0129] In some embodiments, said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18. VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, IX-2, IX-3, XIV- 1 , or Section Vlll.
[0130] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table Vll- 13, VII- 14, VII- 15, VI1- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1, IX-I A, IX-2, IX- 3, XIV- 1 , or Section VIII.
[0131] In some embodiments, the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 1 0 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:
[0132] In some embodiments,
a) a control region, e.g., a cis-acting or trans-acting control region, of a gene is cleaved; b) the sequence of a control region, e.g., a cis-acting or trans-acting control region, of a gene is altered, e.g., by an alteration that modulates, e.g., increases or decreases, expression a gene under control of the control region, e.g., a control sequence is disrupted or a new control sequence is inserted;
c) the coding sequence of a gene is cleaved; d) the sequence of a transcribed region, e.g., a coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that increases expression of or activity of the gene product is effected, e.g., a mutation is corrected;
e) the non-coding sequence of a gene or an intergenic region between genes is cleaved; and/or
f) the sequence of a transcribed region, e.g., the coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that decreases expression of or activity of the gene product is effected, e.g., a mutation is inserted, e.g., the sequence of one or more nucleotides is altered so as to insert a stop codon.
[0133] In some embodiments, a control region or transcribed region, e.g., a coding sequence, of at least 2, 3, 4, 5, or 6 genes are altered.
[0134] In one aspect, the disclosure features a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload coupled, covalently or non- covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule or the gRNA molecule.
[0135] In another aspect, the disclosure features a composition comprising: a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is: coupled, covalently or non-covalently, the gRNA molecule; or a fusion partner with the Cas9 molecule.
[0136] In yet another aspect, the disclosure features a composition comprising: a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and c) a payload which is coupled, covalently or no -covalently, to the Cas9 molecule.
[0137] In still another aspect, the disclosure features a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA, encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) a payload which is a fusion partner with the Cas9 molecule.
[0138] In one aspect, the disclosure features a method of delivering a payload to a cell, e.g., by targeting a payload to target nucleic acid, comprising contacting said cell with:
1 ) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule or the gRNA molecule;
2) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload which is: coupled, covalently or non-covalently, the gRNA molecule; or a fusion partner with the Cas9 molecule;
3) a composition comprising:
a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload which is coupled, covalently or non-covalently, to the Cas9 molecule; and/or
4) a composition comprising: a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA .encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
c) a payload which is a fusion partner with the Cas9 molecule.
[0139] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation.
[0140] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage form, first mode of delivery, or first formulation; and a Cas9 molecule, or nucleic acid encoding a Cas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.
[0141] In some embodiments, the cell is an animal or plant cell. In some embodiments, the cell is a mammalian, primate, or human cell. In some embodiments, the cell is a human cell, e.g., a human cell described herein, e.g., in Section VIIA. In some embodiments, the cell is: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell. In some embodiments, the cell is a human cell, e.g., a cancer cell, a cell comprising an unwanted genetic element, e.g., all or part of a viral genome.
[0142] In some embodiments, the gRNA mediates targeting of a chromosomal nucleic acid. In some embodiments, the gRNA mediates targeting of a selected genomic signature. In some embodiments, the gRNA mediates targeting of an organellar nucleic acid. In some
embodiments, the gRNA mediates targeting of a mitochondrial nucleic acid. In some embodiments, the gRNA mediates targeting of a chloroplast nucleic acid.
[0143] In some embodiments, the cell is a cell of a disease causing organism, e.g., a vims, bacterium, fungus, protozoan, or parasite. [0144] In some embodiments, the gRNA mediates targeting of the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
[0145] In some embodiments, the payload comprises a payload described herein, e.g., in Section VI.
[0146] In some embodiments, said cell is a cell characterized by unwanted proliferation, e.g., a cancer cell. In some embodiments, said cell is characterized by an unwanted genomic component, e.g., a viral genomic component.
[0147] In some embodiments, a control or structural sequence of at least, 2 3, 4, or 5 genes is altered.
[0148] In some embodiments, the gRNA targets a selected genomic signature, e.g., a mutation, e.g., a germline or acquired somatic mutation. In some embodiments, the gRNA targets a rearrangement, a kinase, a rearrangement that comprises a kinase, or tumor suppressor. In some embodiments, the gRNA targets a cancer cell, e.g., a cancer cell disclosed herein, e.g., in Section VIIA. In some embodiments, the gRNA targets a cell which has been infected with a virus.
[0149] In another aspect, the disclosure features a method of treating a subject, e.g., by targeting a payload to target nucleic acid, comprising administering to the subject, an effective amount of:
1) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule;
2) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload which is:
coupled, covalently or non-covalently, the gRNA molecule; or is a fusion partner with the Cas9 molecule;
3) a composition comprising:
a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload which is coupled, covalently or non-covalently, to the Cas9 molecule; and/or
4) a composition comprising:
a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) a nucleic acid, e.g. a DNA or mRNA, encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and c) a payload which is a fusion partner with the Cas9 molecule.
[0150] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation.
[0151] In some embodiments, a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage, mode of delivery form or formulation; and a Cas9 molecule, or nucleic acid encoding a Cas9 molecule, is delivered in or by a second dosage form, mode of delivery, or formulation.
[0152] In some embodiments, the subject is an animal or plant cell. In some embodiments, the subject is a mammalian, primate, or human cell. [0153] In some embodiments, the gRNA mediates targeting of a human cell, e.g., a human cell described herein, e.g., in Section VIIA. In some embodiments, the gRNA mediates targeting of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell. In some embodiments, the gRNA mediates targeting of a cancer cell or a cell comprising an unwanted genomic element, e.g., all or part of a viral genome. In some embodiments, the gRNA mediates targeting of a chromosomal nucleic acid. In some embodiments, the gRNA mediates targeting of a selected genomic signature. In some embodiments, the gRNA mediates targeting of an organellar nucleic acid. In some embodiments, the gRNA mediates targeting of a mitochondrial nucleic acid. In some embodiments, the gRNA mediates targeting of a chloroplast nucleic acid. In some embodiments, the gRNA mediates targeting of the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite. In some embodiments, the gRNA targets a cell characterized by unwanted proliferation, e.g., a cancer cell, e.g., a cancer cell from Section VIIA, e.g., from Table VII- 1 1 . In some
embodiments, the gRNA targets a cell characterized by an unwanted genomic component, e.g., a viral genomic component.
[0154] In some embodiments, a control element, e.g., a promoter or enhancer, is targeted. In some embodiments, the gRNA targets a rearrangement, a kinase, a rearrangement that comprises a kinase, or a tumor suppressor. In some embodiments, the gRNA targets a selected genomic signature, e.g., a mutation, e.g., a germline or acquired somatic mutation.
[0155] In some embodiments, the gRNA targets a cancer cell. In some embodiments, the gRNA targets a cell which has been infected with a virus.
[0156] In some embodiments, at least one eaCas9 molecule and a payload are administered. In some embodiments, the payload comprises a payload described herein, e.g., in Section VI.
[0157] In one aspect, the disclosure features a reaction mixture comprising a composition described herein and a cell.
[0158] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
[0159] Headings, including numeric and alphabetical headings and subheadings, are for organization and presentation and are not intended to be limiting.
[0160] Other features and advantages of the invention will be apparent from the detailed description, drawings, and from the claims.
BRIEF DESCRIITION OF THE DRAWING
[0161] The Figures described below, that together make up the Drawing, are for illustration purposes only, not for limitation.
[0162] FIG. 1 A-G are representations of several exemplary gRNAs.
[0163] FIG. 1 A depicts a modular gRNA molecule derived in part (or modeled on a sequence in part) from Streptococcus pyogenes {S. pyogenes) as a duplexed structure (SEQ ID NOS 42 and 43, respectively, in order of appearance);
[0164] FIG. IB depicts a unimolecular (or chimeric) gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 44);
[0165] FIG. IC depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 45);
[0166] FIG. I D depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 46);
[0167] FIG. I E depicts a unimolecular gRNA molecule derived in part from S. pyogenes as a duplexed structure (SEQ ID NO: 47);
[0168] FIG. I F depicts a modular gRNA molecule derived in part from Streptococcus
Ihermophilus (S. Ihermophilus) as a duplexed structure (SEQ ID NOS 48 and 49, respectively, in order of appearance);
[0169] FIG. 1G depicts an alignment of modular gRNA molecules of S. pyogenes and S.
Ihermophilus (SEQ ID NOS 50-53, respectively, in order of appearance). [0170] FIG. 2 depicts an alignment of Cas9 sequences from Chylinski et al. , RNA BIOL. 2013; 10(5): 726-737. The N-terminal RuvC-like domain is boxed and indicated with a "Y". The other two RuvC-like domains are boxed and indicated with a "B". The HNH-like domain is boxed and indicated by a "G". Sm: S. mutatis (SEQ ID NO: 1 ); Sp: S. pyogenes (SEQ ID NO: 2); St: S. thermophil s (SEQ ID NO: 3); Li: L. innocua (SEQ ID NO: 4). Motif: this is a motif based on the four sequences: residues conserved in all four sequences are indicated by single letter amino acid abbreviation; "*" indicates any amino acid found in the corresponding position of any of the four sequences; and "-" indicates any amino acid, e.g., any of the 20 naturally occurring amino acids.
[0171] FIG. 3A shows an alignment of the N-terminal RuvC-like domain from the Cas9 molecules disclosed in Chylinski et al. (SEQ ID NOS 54-103, respectively, in order of appearance). The last line of FIG. 3A identifies 3 highly conserved residues.
[0172] FIG. 3B shows an alignment of the N-terminal RuvC-like domain from the Cas9 molecules disclosed in Chylinski et al. with sequence outliers removed (SEQ ID NOS 104-177, respectively, in order of appearance). The last line of FIG. 3B identifies 4 highly conserved residues.
[0173] FIG. 4A shows an alignment of the HNH-like domain from the Cas9 molecules disclosed in Chylinski et al. (SEQ ID NOS 178-252, respectively, in order of appearance). The last line of FIG. 4A identifies conserved residues.
[0174] FIG. 4B shows an alignment of the HNH-like domain from the Cas9 molecules disclosed in Chylinski et al. with sequence outliers removed (SEQ ID NOS 253-302, respectively, in order of appearance). The last line of FIG. 4B identifies 3 highly conserved residues.
[0175] FIG. 5 depicts an alignment of Cas9 sequences from S. pyogenes and Neisseria meningitidis (N. meningitidis). The N-terminal RuvC-like domain is boxed and indicated with a "Y". The other two RuvC-like domains are boxed and indicated with a "B". The HNH-like domain is boxed and indicated with a "G". Sp: S. pyogenes; Nm: N. meningitidis. Motif: this is a motif based on the two sequences: residues conserved in both sequences are indicated by a single amino acid designation; "*" indicates any amino acid found in the corresponding position of any of the two sequences; "-" indicates any amino acid, e.g., any of the 20 naturally occurring amino acids, and "-" indicates any amino acid, e.g., any of the 20 naturally occurring amino acids, or absent.
[0176] FIG. 6 shows a nucleic acid sequence encoding Cas9 of N. meningitidis (SEQ ID NO: 303). Sequence indicated by an "R" is an SV40 NLS; sequence indicated as "G" is an HA tag; sequence indicated by an "O" is a synthetic NLS sequence. The remaining (unmarked) sequence is the open reading frame (ORF).
DEFINITIONS
[01 77] "Domain", as used herein, is used to describe segments of a protein or nucleic acid. Unless otherwise indicated, a domain is not required to have any specific functional property.
[0178] Calculations of "homology" or "sequence identity" between two sequences (the terms are used interchangeably herein) are performed as follows. The sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, in some embodiments, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences.
[0179] "Modulator", as used herein, refers to an entity, e.g., a drug, that can alter the activity (e.g., enzymatic activity, transcriptional activity, or translational activity), amount, distribution, or structure of a subject molecule or genetic sequence. In an embodiment, modulation comprises cleavage, e.g., breaking of a covalent or non-covalent bond, or the forming of a covalent or non- covalent bond, e.g., the attachment of a moiety, to the subject molecule. In an embodiment, a modulator alters the, three dimensional, secondary, tertiary, or quaternary structure, of a subject molecule. A modulator can increase, decrease, initiate, or eliminate a subject activity. [0180] "Large molecule", as used herein, refers to a molecule having a molecular weight of at least 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kD. Large molecules include proteins, polypeptides, nucleic acids, biologies, and carbohydrates.
[0181] "Polypeptide", as used herein, refers to a polymer of amino acids having less than 100 amino acid residues. In an embodiment, it has less than 50, 20, or 10 amino acid residues.
[0182] "Reference molecule", e.g., a reference Cas9 molecule or reference gRNA, as used herein, refers to a molecule to which a subject molecule, e.g., a subject Cas9 molecule of subject gRNA molecule, e.g., a modified or candidate Cas9 molecule is compared. For example, a Cas9 molecule can be characterized as having no more than 10% of the nuclease activity of a reference Cas9 molecule. Examples of reference Cas9 molecules include naturally occurring unmodified Cas9 molecules, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S.
pyogenes, or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology with the Cas9 molecule to which it is being compared. In an embodiment, the reference Cas9 molecule is a sequence, e.g., a naturally occurring or known sequence, which is the parental form on which a change, e.g., a mutation has been made.
[0183] "Replacement", or "replaced", as used herein with reference to a modification of a molecule does not require a process limitation but merely indicates that the replacement entity is present.
[0184] "Small molecule", as used herein, refers to a compound having a molecular weight less than about 2 kD, e.g., less than about 2 kD, less than about 1 .5 kD, less than about 1 kD, or less than about 0.75 kD.
[0185] "Subject", as used herein, may mean either a human or non-human animal. The term includes, but is not limited to, mammals (e.g., humans, other primates, pigs, rodents (e.g., mice and rats or hamsters), rabbits, guinea pigs, cows, horses, cats, dogs, sheep, and goats). In an embodiment, the subject is a human. In other embodiments, the subject is poultry.
[0186] "Treat", "treating" and "treatment", as used herein, mean the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting or preventing its development; (b) relieving the disease, i.e., causing regression of the disease state; or (c) curing the disease. [0187] "X" as used herein in the context of an amino acid sequence, refers to any amino acid (e.g., any of the twenty natural amino acids) unless otherwise specified.
DETAILED DESCRIPTION
I . gRNA Molecules
[0188] A gRNA molecule, as that term is used herein, refers to a nucleic acid that promotes the specific targeting or homing of a gRNA molecule/Cas9 molecule complex to a target nucleic acid. gRNA molecules can be unimolecular (having a single RNA molecule), sometimes referred to herein as "chimeric" gRNAs, or modular (comprising more than one, and typically two, separate RNA molecules). A gRNA molecule comprises a number of domains. The gRNA molecule domains are described in more detail below.
[0189] Several exemplary gRNA structures, with domains indicated thereon, are provided in FIG. 1 . While not wishing to be bound by theory with regard to the three dimensional form, or intra- or inter-strand interactions of an active form of a gRNA, regions of high complementarity are sometimes shown as duplexes in FIG. 1 and other depictions provided herein.
[0190] In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5' to 3' :
a targeting domain (which is complementary to a target nucleic acid);
a first complementarity domain;
a linking domain;
a second complementarity domain (which is complementary to the first
complementarity domain);
a proximal domain; and
optionally, a tail domain.
In an embodiment, a modular gRNA comprises:
a first strand comprising, preferably from 5' to 3' ;
a targeting domain (which is complementary with a target sequence from a target nucleic acid disclosed herein, e.g., a sequence from: a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII- 20, VII-21 , VII-22, VI1-23, VII-24, VII-25, IX- 1 , IX- 1 A, LX-2, 1X-3, XlV- 1 , or Section VIII); and
a first complementarity domain; and
a second strand, comprising, preferably from 5' to 3': optionally, a 5' extension domain;
a second complementarity domain; and
a proximal domain; and
optionally, a tail domain.
[0191] The domains are discussed briefly below:
1) The Targeting Domain:
[0192] FIG. 1A-G provides examples of the placement of targeting domains.
[0193] The targeting domain comprises a nucleotide sequence that is complementary, e.g., at least 80, 85, 90, or 95% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid. The targeting domain is part of an RNA molecule and will therefore comprise the base uracil (U), while any DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, it is believed that the
complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA molecule/Cas9 molecule complex with a target nucleic acid. It is understood that in a targeting domain and target sequence pair, the uracil bases in the targeting domain will pair with the adenine bases in the target sequence. In an embodiment, the target domain itself comprises, in the 5' to 3' direction, an optional secondary domain, and a core domain. In an embodiment, the core domain is fully complementary with the target sequence. In an embodiment, the targeting domain is 5 to 50, e.g., 10 to 40, e.g., 10 to 30, e.g., 15 to 30, e.g., 15 to 25 nucleotides in length. In an embodiment, the targeting domain is 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides in length. The strand of the target nucleic acid with which the targeting domain is complementary is referred to herein as the complementary strand. Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.
[0194] In an embodiment, the targeting domain is 16 nucleotides in length. [0195] In an embodiment, the targeting domain is 17 nucleotides in length.
[0196] In an embodiment, the targeting domain is 1 8 nucleotides in length.
[0197] In an embodiment, the targeting domain is 19 nucleotides in length.
[0198] In an embodiment, the targeting domain is 20 nucleotides in length.
[0199] In an embodiment, the targeting domain is 21 nucleotides in length.
[0200] In an embodiment, the targeting domain is 22 nucleotides in length.
[0201] In an embodiment, the targeting domain is 23 nucleotides in length.
[0202] In an embodiment, the targeting domain is 24 nucleotides in length.
[0203] In an embodiment, the targeting domain is 25 nucleotides in length.
[0204] Targeting domains are discussed in more detail below.
2) The First Complementarity Domain:
[0205] FIG. lA-G provides examples of first complementarity domains.
[0206] The first complementarity domain is complementary with the second complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an · embodiment, the first complementarity domain is 5 to 30 nucleotides in length. In an embodiment, the first complementarity domain is 5 to 25 nucleotides in length. In an embodiment, the first complementary domain is 7 to 25 nucleotides in length. In an
embodiment, the first complementary domain is 7 to 22 nucleotides in length. In an
embodiment, the first complementary domain is 7 to 18 nucleotides in length. In an
embodiment, the first complementary domain is 7 to 15 nucleotides in length. In an
embodiment, the first complementary domain is 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 nucleotides in length.
[0207] In an embodiment, the first complementarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain. In an embodiment, the 5' subdomain is 4-9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length. In an embodiment, the central subdomain is I , 2, or 3, e.g., 1 , nucleotide in length. In an embodiment, the 3' subdomain is 3 to 25, e.g., 4-22, 4- 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 1 5, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25, nucleotides'^ length.
[0208] The first complementarity domain can share homology with, or be derived from, a naturally occurring first complementarity domain. In an embodiment, it has at least 50% homology with a first complementarity domain disclosed herein, e.g., an S. pyogenes, or S. thennophilus, first complementarity domain.
[0209] Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.
[0210] First complementarity domains are discussed in more detail below.
3) The Linking Domain
[0211] FIG. 1B-E provides examples of linking domains.
[0212] A linking domain serves to link the first complementarity domain with the second complementarity domain of a unimolecular gRNA. The linking domain can link the first and second complementarity domains covalently or non-covalently. In an embodiment, the linkage is covalent. In an embodiment, the linking domain covalently couples the first and second complementarity domains, see, e.g., FIG. 1 B-E. In an embodiment, the linking domain is, or comprises, a covalent bond inteiposed between the first complementarity domain and the second complementarity domain. Typically, the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
[0213] In modular gRNA molecules the two molecules can be associated by virtue of the hybridization of the complementarity domains, see e.g., FIG. 1A.
[0214] A wide variety of linking domains are suitable for use in unimolecular gRNA molecules. Linking domains can consist of a covalent bond, or be as short as one or a few nucleotides, e.g., 1 , 2, 3, 4, or 5 nucleotides in length.
[0215] In an embodiment, a linking domain is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in length. In an embodiment, a linking domain is 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, or 2 to 5 nucleotides in length. In an embodiment, a linking domain shares homology with, or is derived from, a naturally occurring sequence, e.g., the sequence of a tracrRNA that is 5' to the second complementarity domain. In an embodiment, the linking domain has at least 50% homology with a linking domain disclosed herein.
[0216] Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.
[0217] Linking domains are discussed in more detail below.
4) The 5' Extension Domain
[0218] In an embodiment, a modular gRNA can comprise additional sequence, 5' to the second complementarity domain, referred to herein as the 5' extension domain, see, e.g., FIG. 1A. In an embodiment, the 5' extension domain is, 2- 10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4 nucleotides in length. In an embodiment, the 5' extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.
5) The Second Complementarity Domain:
[021 ] FIG. 1 A-F provides examples of second complementarity domains.
[0220] The second complementarity domain is complementary with the first complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an embodiment, e.g., as shown in FIG. 1A or FIG. IB, the second complementarity domain can include sequence that lacks complementarity with the first complementarity domain, e.g., sequence that loops out from the duplexed region.
[0221] In an embodiment, the second complementarity domain is 5 to 27 nucleotides in length. In an embodiment, it is longer than the first complementarity region.
[0222] In an embodiment, the second complementary domain is 7 to 27 nucleotides in length. In an embodiment, the second complementary domain is 7 to 25 nucleotides in length. In an embodiment, the second complementary domain is 7 to 20 nucleotides in length. In an embodiment, the second complementary domain is 7 to 17 nucleotides in length. In an embodiment, the complementary domain is 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides in length. [0223] In an embodiment, the second complementarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain. In an embodiment, the 5' subdomain is 3 to 25, e.g., 4 to 22, 4 to l 8, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, or 25 nucleotides in length. In an embodiment, the central subdomain is 1 , 2, 3, 4 or 5, e.g., 3, nucleotides in length. In an embodiment, the 3' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length.
[0224] In an embodiment, the 5' subdomain and the 3' subdomain of the first complementarity domain, are respectively, complementaiy, e.g., fully complementary, with the 3' subdomain and the 5' subdomain of the second complementarity domain.
[0225] The second complementarity domain can share homology with or be derived from a naturally occurring second complementarity domain. In an embodiment, it has at least 50% homology with a second complementarity domain disclosed herein, e.g., an S. pyogenes, or S. thermophilus, first complementarity domain.
[0226] Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.
6) A Proximal Domain:
[0227] FIG. 1 A-F provides examples of proximal domains.
[0228] In an embodiment, the proximal domain is 5 to 20 nucleotides in length. In an embodiment, the proximal domain can share homology with or be derived from a naturally occurring proximal domain. In an embodiment, it has at least 50% homology with a proximal domain disclosed herein, e.g., an S. pyogenes, or S. thermophilus, proximal domain.
[0229] Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.
7) A Tail Domain:
[0230] FIG. 1 A and FIG. 1 C-F provide examples of tail domains.
[0231] As can be seen by inspection of the tail domains in FIG. 1 A and FIG. 1C-F, a broad spectrum of tail domains are suitable for use in gRNA molecules. In an embodiment, the tail domain is 0 (absent), 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In an embodiment, the tail domain nucleotides are from or share homology with sequence from the 5' end of a naturally occurring tail domain, see e.g., FIG. ID or FIG. I E. In an embodiment, the tail domain includes sequences that are complementary to each other and which, under at least some physiological conditions, form a duplexed region.
[0232] In an embodiment, the tail domain is absent or is 1 to 50 nucleotides in length. In an embodiment, the tail domain can share homology with or be derived from a naturally occurring proximal tail domain. In an embodiment, it has at least 50% homology with a tail domain disclosed herein, e.g., an S. pyogenes, or S. thermophilus, tail domain.
[0233] Some or all of the nucleotides of the domain can have a modification, e.g., modification found in Section X herein.
[0234] In an embodiment, the tail domain includes nucleotides at the 3' end that are related to the method of in vitro or in vivo transcription. When a T7 promoter is used for in vitro transcription of the gRNA, these nucleotides may be any nucleotides present before the 3' end of the DNA template. When a U6 promoter is used for in vivo transcription, these nucleotides may be the sequence UUUUUU. When altemate pol-III promoters are used, these nucleotides may be various numbers or uracil bases or may include alternate bases.
[0235] The domains of gRNA molecules are described in more detail below.
The Targeting Domain
[0236] The "targeting domain" of the gRNA is complementary to the "target domain" on the target nucleic acid. The strand of the target nucleic acid comprising the nucleotide sequence complementary to the core domain of the, gRNA is referred to herein as the "complementary strand" of the target nucleic acid. Guidance on the selection of targeting domains can be found, e.g., in Fu Y el a!.. NAT BIOTECHNOL 2014 (doi: 10.1038/nbt.2808) and Sternberg SH el a/.. NATURE 2014 (doi: 10. 1038/naturel301 1).
[0237] In an embodiment, the targeting domain is 16, 17, 18, 1 , 20, 21 , 22, 23, 24 or 25 nucleotides in length.
[0238] In an embodiment, the targeting domain comprises 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length.
[0239] In an embodiment, the targeting domain is 16 nucleotides in length. [0240] In an embodiment, the targeting domain is 17 nucleotides in length.
[0241 ] In an embodiment, the targeting domain is 1 8 nucleotides in length.
[0242] In an embodiment, the targeting domain is 19 nucleotides in length.
[0243] In an embodiment, the targeting domain is 20 nucleotides in length.
[0244] In an embodiment, the targeting domain is 21 nucleotides in length.
[0245] In an embodiment, the targeting domain is 22 nucleotides in length.
[0246] In an embodiment, the targeting domain is 23 nucleotides in length.
[0247] In an embodiment, the targeting domain is 24 nucleotides in length.
[0248] In an embodiment, the targeting domain is 25 nucleotides in length.
[0249] In an embodiment, the targeting domain is 10 +/-5, 20+/-5, 30+/-5, 40+/-
5, 70+/-5, 80+/-5, 90+/-5, or 100+/-5 nucleotides, in length.
[0250] In an embodiment, the targeting domain is 20+/-5 nucleotides in length.
[0251 ] In an embodiment, the targeting domain is 20+/- 10, 30+/- 10, 40+/-10, 50+/- 10, 60+/- 10, 70+/- 10, 80+/- 10, 90+/- 10, or 100+/- 10 nucleotides, in length.
[0252] In an embodiment, the targeting domain is 30+/- 10 nucleotides in length.
[0253] In an embodiment, the targeting domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. In other
embodiments, the targeting domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.
[0254] Typically the targeting domain has full complementarity with the target sequence. In some embodiments the targeting domain has or includes 1 , 2, 3, 4, .5, 6, 7 or 8 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain.
[0255] In an embodiment, the target domain includes 1 , 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5' end. In an embodiment, the target domain includes 1 , 2, 3, 4 or 5 nucleotides that are complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3' end. [0256] In an embodiment, the target domain includes 1 , 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 5' end. In an embodiment, the target domain includes 1, 2, 3, or 4 nucleotides that are not complementary with the corresponding nucleotide of the targeting domain within 5 nucleotides of its 3' end.
[0257] In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.
[0258] In some embodiments, the targeting domain comprises two consecutive nucleotides that are not complementary to the target domain ("non-complementary nucleotides"), e.g., two consecutive noncomplementary nucleotides that are within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.
[0259] In an embodiment, no two consecutive nucleotides within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain, are not complementary to the targeting domain.
[0260] In an embodiment, there are no noncomplementary nucleotides within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5'nucleotides away from one or both ends of the targeting domain.
[0261] In an embodiment, the targeting domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the targeting domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the targeting domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the targeting domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' acetylation, e.g., a 2' methylation, or other modification from Section X. [0262] In some embodiments, the targeting domain includes 1 , 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the targeting domain includes 1 , 2, 3, or 4 modifications within 5 nucleotides of its 5' end. In an embodiment, the targeting domain comprises as many as 1 , 2, 3, or 4 modifications within 5 nucleotides of its 3' end.
[0263] In some embodiments, the targeting domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or more than 5 nucleotides away from one or both ends of the targeting domain.
[0264] In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5' end of the targeting domain, within 5 nucleotides of the 3' end of the targeting domain, or within a region that is more than 5 nucleotides away from one or both ends of the targeting domain.
[0265] Modifications in the targeting domain can be selected so as to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section ΠΙ. gRNA's having a candidate targeting domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in a system in Section III. The candidate targeting domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
[0266] In some embodiments, all of the modified nucleotides are complementary to and capable of hybridizing to corresponding nucleotides present in the target domain. In other embodiments, 1 , 2, 3, 4, 5, 6, 7 or 8 or more modified nucleotides are not complementary to or capable of hybridizing to corresponding nucleotides present in the target domain.
[0267] In an embodiment, the targeting domain comprises, preferably in the 5'→3' direction: a secondary domain and a core domain. These domains are discussed in more detail below. The Core Domain and Secondary Domain of the Targeting Domain
[0268] The "core domain" of the targeting domain is complementary to the "core domain target" on the target nucleic acid. In an embodiment, the core domain comprises about 8 to about 13 nucleotides from the 3' end of the targeting domain (e.g., the most 3' 8 to 13 nucleotides of the targeting domain).
[0269] In an embodiment, the core domain is 6 +/-2, 1+1-2, 8+/-2, 9+/-2, 10+/-2, 1 1+/-2, 12+/-2, 13+/-2, 14+/-2, 15+/-2, or 16+-2 nucleotides in length.
[0270] In an embodiment, the core domain is 10+/- 2 nucleotides in length.
[0271] In an embodiment, the core domain is 10+/-4 nucleotides in length.
[0272] In an embodiment, the core domain is 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, or 16 nucleotides in length.
[0273] In an embodiment, the core domain is 8 to 13, e.g., 8 to 12, 8 to 1 1 , 8 to 10, 8 to 9, 9 to 13, 9 to 12, 9 to 1 1 , or 9 to 10 nucleotides in length.
[0274] In an embodiment, the core domain is 6 to 16, e.g., 6 to 15, 6 to 14, 6 to 13, 7 to 14, 7 to 13, 7 to 12, 7 to 1 1 , 7 to 10, 8 to 14, 8 to 13, 8 to 12, 8 to 1 1 , 8 to 10, or 8 to 9 nucleotides in length.
[0275] The core domain is complementary with the core domain target. Typically the core domain has exact complementarity with the core domain target. In some embodiments, the core domain can have 1 , 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the core domain. In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.
[0276] The "secondary domain" of the targeting domain of the gRNA is complementary to the "secondary domain target" of the target nucleic acid.
[0277] In an embodiment, the secondary domain is positioned 5' to the core domain.
[0278] In an embodiment, the secondary domain is absent or optional. [0279] In an embodiment, if the targeting domain is 25 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 12 to 17 nucleotides in length.
[0280] In an embodiment, if the targeting domain is 24 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 1 1 to 16 nucleotides in length.
[0281] In an embodiment, if the targeting domain is 23 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 10 to 15 nucleotides in length.
[0282] In an embodiment, if the targeting domain is 22 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 9 to 14 nucleotides in length.
[0283] In an embodiment, if the targeting domain is 21 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 8 to 13 nucleotides in length.
[0284] In an embodiment, if the targeting domain is 20 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 7 to 12 nucleotides in length.
[0285] In an embodiment, if the targeting domain is 19 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 6 to 1 1 nucleotides in length.
[0286] In an embodiment, if the targeting domain is 18 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 5 to 10 nucleotides in length.
[0287] In an embodiment, if the targeting domain is 17 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 4 to 9 nucleotides in length. [0288] In an embodiment, if the targeting domain is 16 nucleotides in length and the core domain (counted from the 3' end of the targeting domain) is 8 to 13 nucleotides in length, the secondary domain is 3 to 8 nucleotides in length.
[0289] In an embodiment, the secondary domain is 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14 or 15 nucleotides in length.
[0290] The secondary domain is complementary with the secondary domain target. Typically the secondary domain has exact complementarity with the secondary domain target. In some embodiments the secondary domain can have 1 , 2, 3, 4 or 5 nucleotides that are not
complementary with the corresponding nucleotide of the secondary domain. In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.
[0291] In an embodiment, the core domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the core domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the core domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the core domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X. Typically, a core domain will contain no more than 1 , 2, or 3 modifications.
[0292] Modifications in the core domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III. gRNA' s having a candidate core domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section Π1. The candidate core domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule /Cas9 molecule system known to be functional with a selected target and evaluated.
[0293] In an embodiment, the secondary domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the secondary domain comprises one or more modifications, e.g., modifications that render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the secondary domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the secondary domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X. Typically, a secondary domain will contain no more than 1 , 2, or 3 modifications.
[0294] Modifications in the secondary domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III. gRNA's having a candidate secondary domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section III. The candidate secondary domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule /Cas9 molecule system known to be functional with a selected target and evaluated.
[0295] In an embodiment, (1) the degree of complementarity between the core domain and its target, and (2) the degree of complementarity between the secondary domain and its target, may differ. In an embodiment, ( 1) may be greater than (2). In an embodiment, (1 ) may be less than (2). In an embodiment, ( 1) and (2) may be the same, e.g., each may be completely
complementary with its target.
[0296] In an embodiment, ( 1) the number of modifications (e.g., modifications from Section X) of the nucleotides of the core domain and (2) the number of modification (e.g., modifications from Section X) of the nucleotides of the secondaiy domain, may differ. In an embodiment, (1) may be less than (2). In an embodiment, (1) may be greater than (2). In an embodiment, ( 1) and (2) may be the same, e.g., each may be free of modifications.
The First and Second Complementarity Domains
[0297] The first complementarity domain is complementary with the second complementarity domain.
[0298] Typically the first domain does not have exact complementarity with the second complementarity domain target. In some embodiments, the first complementarity domain can have 1 , 2, 3, 4 or 5 nucleotides that are not complementary with the corresponding nucleotide of the second complementarity domain . In an embodiment, 1 , 2, 3, 4, 5 or 6, e.g., 3 nucleotides, will not pair in the duplex, and, e.g., form a non-duplexed or looped-out region. In an embodiment, an unpaired, or loop-out, region, e.g., a loop-out of 3 nucleotides, is present on the second complementarity domain. In an embodiment, the unpaired region begins 1 , 2, 3, 4, 5, or 6, e.g., 4, nucleotides from the 5' end of the second complementarity domain.
[0299] In an embodiment, the degree of complementarity, together with other properties of the gRNA, is sufficient to allow targeting of a Cas9 molecule to the target nucleic acid.
[0300] In an embodiment, the first and second complementarity domains are:
independently, 6 +1-2, 7+/-2, 8+/-2, 9+/-2, 10+/-2, 1 1+/-2, 12+/-2, 13+/-2, 14+/-2, 15+/-2, 16+/-2, 1 7+/-2, 1 8+/-2, 19+/-2, or 20+/-2, 21+/-2, 22+A2, 23+Λ2, or 24+/- 2 nucleotides in length; /
independently, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 14, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or 26 nucleotides in length; or
independently, 5 to 24, 5 to 23, 5 to 22, 5 to 21 , 5 to 20, 7 to 18, 9 to 16, or 10 to 14 nucleotides in length.
[0301 ] In an embodiment, the second complementarity domain is longer than the first complementarity domain, e.g., 2, 3, 4, 5, or 6, e.g., 6, nucleotides longer.
[0302] In an embodiment, the first and second complementary domains, independently, do not comprise modifications, e.g., modifications of the type provided in Section X.
[0303] In an embodiment, the first and second complementary domains, independently, comprise one or more modifications, e.g., modifications that the render the domain less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g. , a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X.
[0304] In an embodiment, the first and second complementary domains, independently, include 1 , 2, 3, 4, 5, 6, 7 or 8 or more modifications. In an embodiment, the first and second complementary domains, independently, include 1 , 2, 3, or 4 modifications within 5 nucleotides of its 5' end. In an embodiment, the first and second complementary domains, independently, include as many as 1 , 2, 3, or 4 modifications within 5 nucleotides of its 3' end.
[0305] In an embodiment, the first and second complementary domains, independently, include modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the domain, within 5 nucleotides of the 3' end of the domain, or more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no two consecutive nucleotides that are modified, within 5 nucleotides of the 5' end of the domain, within 5 nucleotides of the 3' end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain. In an embodiment, the first and second complementary domains, independently, include no nucleotide that is modified within 5 nucleotides of the 5' end of the domain, within 5 nucleotides of the 3' end of the domain, or within a region that is more than 5 nucleotides away from one or both ends of the domain.
[0306] Modifications in a complementarity domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section III. gRNA's having a candidate complementarity domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section III. The candidate complementarity domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule /Cas9 molecule system known to be functional with a selected target and evaluated.
[0307] In an embodiment, the first complementarity domain has at least 60, 70, 80, 85%, 90%, or 95% homology with, or differs by no more than 1 , 2, 3, 4, 5, or 6 nucleotides from, a reference first complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S.
lliermophilus, first complementarity domain, or a first complementarity domain described herein, e.g., from FIG. 1 A-F.
[0308] In an embodiment, the second complementarity domain has at least 60, 70, 80, 85%, 90%, or 95 % homology with, or differs by no more than 1, 2, 3, 4, 5, or 6 nucleotides from, a reference second complementarity domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermopliilus, second complementarity domain, or a second complementarity domain described herein, e.g., from FIG. 1 A-F.
[0309] The duplexed region formed by first and second complementarity domains is typically 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 base pairs in length (excluding any looped out or unpaired nucleotides).
[0310] In some embodiments, the first and second complementarity domains, when duplexed, comprise 1 1 paired nucleotides, for example, in the gRNA sequence (one paired strand underlined, one bolded):
NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAG UUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGC (SEQ ID NO: 5).
[0311] In some embodiments, the first and second complementarity domains, when duplexed, comprise 15 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):
NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGAAAAGC AUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA GUGGCACCGAGUCGGUGC (SEQ ID NO: 27).
[0312] In some embodiments the first and second complementarity domains, when duplexed, comprise 16 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):
NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGGAAACA GCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA AAGUGGCACCGAGUCGGUGC (SEQ ID NO: 28).
[0313] In some embodiments the first and second complementarity domains, when duplexed, comprise 21 paired nucleotides, for example in the gRNA sequence (one paired strand underlined, one bolded):
NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUGG AAACAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAU CAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 29). [0314] In some embodiments, nucleotides are exchanged to remove poly-U tracts, for example in the gRNA sequences (exchanged nucleotides underlined):
NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAGAAAUAGCAAG UUAAUAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGC (SEQ ID NO: 30);
NNNNNNNNNNNNNNNNNNNNGUUUAAGAGCUAGAAAUAGCAAG UUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGC (SEQ ID NO: 31); and
NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAUGCUGUAUUGG AAACAAUACAGCAUAGCAAGUUAAUAUAAGGCUAGUCCGUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 32).
The 5' Extension Domain
[0315] In an embodiment, a modular gRNA can comprise additional sequence, 5' to the second complementarity domain. In an embodiment, the 5' extension domain is 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4 nucleotides in length. In an embodiment, the 5' extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length.
[0316] In an embodiment, the 5' extension domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the 5' extension domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the 5' extension domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the 5' extension domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' - acetylation, e.g., a 2' methylation, or other modification from Section X.
[0317] In some embodiments, the 5' extension domain can comprise as many as 1 , 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the 5' extension domain comprises as many as 1 , 2, 3, or 4 modifications within 5 nucleotides of its 5' end, e.g., in a modular gRNA molecule. In an embodiment, the 5' extension domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end, e.g., in a modular gRNA molecule. [0318] In some embodiments, the 5' extension domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the 5' extension domain, within 5 nucleotides of the 3' end of the 5' extension domain, or more than 5 nucleotides away from one or both ends of the 5' extension domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the 5' extension domain, within 5 nucleotides of the 3' end of the 5' extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5' extension domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5' end of the 5' •extension domain, within 5 nucleotides of the 3' end of the 5' extension domain, or within a region that is more than 5 nucleotides away from one or both ends of the 5' extension domain.
[0319] Modifications in the 5' extension domain can be selected to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section ITT. gRNAs having a candidate 5' extension domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section III. The candidate 5' extension domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
[0320] In an embodiment, the 5' extension domain has at least 60, 70, 80, 85, 90 or 95% homology with, or differs by no more than 1 , 2, 3, 4, 5, or 6 nucleotides from, a reference 5' extension domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus, 5' extension domain, or a 5' extension domain described herein, e.g., from FIG. 1 A and FIG. I F.
The Linking Domain
[0321] In a unimolecular gRNA molecule the linking domain is disposed between the first and second complementarity domains. In a modular gRNA molecule, the two molecules are associated with one another by the complementarity domains.
[0322] In an embodiment, the linking domain is 10 +/-5, 20+/-5, 30+/-5, 40+/-5, 50+/-5, 60+/-5, 70+/-5, 80+/-5, 90+/-5, or 100+/-5 nucleotides, in length.
[0323] In an embodiment, the linking domain is 20+/- 10, 30+/- 10, 40+/- 10, 50+/- 10, 60+/- 10, 70+/-10, 80+/- 10, 90+/- 10, or 100+/- 10 nucleotides, in length. [0324] In an embodiment, the linking domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. In other embodiments, the targeting domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.
[0325] In an embodiment, the linking domain is 1 , 2, 3, 4, 5,.6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16 17, 18, 19, or 20 nucleotides in length.
[0326] In an embodiment, the linking domain is a covalent bond.
[0327] In an embodiment, the linking domain comprises a duplexed region, typically adjacent to or within 1 , 2, or 3 nucleotides of the 3' end of the first complementarity domain and/or the fiend of the second complementarity domain. In an embodiment, the duplexed region can be 20+/- 10, 30+/- 10, 40, +/- 10 or 50+/- 10 base pairs in length. In an embodiment, the duplexed region can be 10+/-5, 15+/-5, 20+/-5, or 30+/-5 base pairs in length. In an embodiment, the duplexed region can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, or 15 base pairs in length.
[0328] Typically the sequences forming the duplexed region have exact complementarity with one another, though in some embodiments as many as 1 , 2, 3, 4, 5, 6, 7 or 8 nucleotides are not complementary with the corresponding nucleotides.
[0329] In an embodiment, the linking domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment the linking . domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the linking domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the linking domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X.
[0330] In some embodiments, the linking domain can comprise as many as 1 , 2, 3, 4, 5, 6, 7 or 8 modifications.
[0331 ] Modifications in a linking domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section ΠΙ. gRNA's having a candidate linking domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated a system described in Section III. A candidate linking domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
[0332] In an embodiment, the linking domain has at least 60, 70, 80, 85, 90 or 95% homology with, or differs by no more than 1 , 2, 3, 4, 5 ,or 6 nucleotides from, a reference linking domain, e.g., a linking domain described herein, e.g., from FIG. 1 B-E.
The proximal domain
[0333] In an embodiment, the proximal domain is 6 +1-2, Ί+/-2, 8+/-2, 9+/-2, 10+/-2, 1 1+/-2, 12+/-2, 13+/-2, 14+/-2, 14+/-2, 16+/-2, 17+/-2, 18+/-2, 19+/-2, or 20+/-2 nucleotides in length.
[0334] In an embodiment, the proximal domain is 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 16, 17, 18, 19, or 20 nucleotides in length.
[0335] In an embodiment, the proximal domain is 5 to 20, 7, to 18, 9 to 16, or 10 to 14 nucleotides in length.
[0336] In an embodiment, the proximal domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment, the proximal domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the proximal domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the proximal domain can comprise a 2' modification (e.g., a modification at the 2' position on ribose), e.g., a 2' - acetylation, e.g., a 2' methylation, or other modification from Section X.
[0337] In some embodiments, the proximal domain can comprise as many as 1 , 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the proximal domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 5' end, e.g., in a modular gRNA molecule. In an embodiment, the target domain comprises as many as 1 , 2, 3, or 4 modifications within 5 nucleotides of its 3' end, e.g., in a modular gRNA molecule.
[0338] In some embodiments, the pro imal domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the proximal domain, within 5 nucleotides of the 3' end of the proximal domain, or more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the proximal domain, within 5 nucleotides of the 3' end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5' end of the proximal domain, within 5 nucleotides of the 3' end of the proximal domain, or within a region that is more than 5 nucleotides away from one or both ends of the proximal domain.
[0339] Modifications in the proximal domain can be selected to not interfere with gRNA molecule efficacy, which can be evaluated by testing a candidate modification in the system described in Section III. gRNA's having a candidate proximal domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described at Section III. The candidate proximal domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule /Cas9 molecule system known to be functional with a selected target and evaluated:
[0340] In an embodiment, the proximal domain has at least 60%, 70%, 80%, 85%, 90%, or 95% homology with, or differs by no more thari 1 , 2, 3, 4, 5 ,or 6 nucleotides from, a reference proximal domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilics, proximal domain, or a proximal domain described herein, e.g., from FIG. 1A-F.
The Tail Domain
[0341] In an embodiment, the tail domain is 10 +/-5, 20+/-5, 30+/-5, 40+/-5, 50+/-5, 60+/-5, 70+/-5, 80+/-5, 90+/-5, or 100+/- 5 nucleotides, in length.
[0342] In an embodiment, the tail domain is 20+/-5 nucleotides in length.
[0343] In an embodiment, the tail domain is 20+/- 10, 30+/- 10, 40+/- 10, 50+/- 10, 60+/- 10, 70+/- 10, 80+/- 10, 90+/- 10, or 100+/- 10 nucleotides, in length.
[0344] In an embodiment, the tail domain is 25+/- 10 nucleotides in length.
[0345] In an embodiment, the tail domain is 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 20 or 10 to 15 nucleotides in length. [0346] In other embodiments, the tail domain is 20 to 100, 20 to 90, 20 to 80, 20 to 70, 20 to 60, 20 to 50, 20 to 40, 20 to 30, or 20 to 25 nucleotides in length.
[0347] In an embodiment, the tail domain is 1 to 20, 1 to 1 , 1 to 10, or 1 to 5 nucleotides in length.
[0348] In an embodiment, the tail domain nucleotides do not comprise modifications, e.g., modifications of the type provided in Section X. However, in an embodiment the tail domain comprises one or more modifications, e.g., modifications that it render it less susceptible to degradation or more bio-compatible, e.g., less immunogenic. By way of example, the backbone of the tail domain can be modified with a phosphorothioate, or other modification from Section X. In an embodiment, a nucleotide of the tail domain can comprise a 2' modification (e.g., a modification at the position on ribose), e.g., a 2' -acetylation, e.g., a 2' methylation, or other modification from Section X.
[0349] In some embodiments, the tail domain can have as many as 1, 2, 3, 4, 5, 6, 7 or 8 modifications. In an embodiment, the target domain comprises as many as 1 , 2, 3, or 4 modifications within 5 nucleotides of its 5' end. In an embodiment, the target domain comprises as many as 1, 2, 3, or 4 modifications within 5 nucleotides of its 3' end.
[0350] In an embodiment, the tail domain comprises a tail duplex domain, which can form a tail duplexed region. In an embodiment, the tail duplexed region can be 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , or 1 2 base pairs in length. In an embodiment, a further single stranded domain, exists 3' to the tail duplexed domain. In an embodiment, this domain is 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In an embodiment, it is 4 to 6 nucleotides in length.
[0351] In an embodiment, the tail domain has at least 60, 70, 80, or 90% homology with, or differs by no more than 1 , 2, 3, 4, 5 ,or 6 nucleotides from, a reference tail domain, e.g., a naturally occurring, e.g., an S. pyogenes, or S. thermophilus, tail domain, or a tail domain described herein, e.g., from FIG. 1 A and FIG. 1 C-F.
[0352] In an embodiment, the proximal and tail domain, taken together comprise the following sequences:
AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCU (SEQ ID NO: 33); AAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGU GGUGC (SEQ ID NO: 34);
AAGGCUAGUCCGU UAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCGGAUC (SEQ ID NO: 35);
AAGGCUAGUCCGUUAUCAACUUGAAAAAGUG (SEQ ID NO: 36);
AAGGCUAGUCCGUUAUCA (SEQ ID NO: 37); or
AAGGCUAGUCCG (SEQ ID NO: 38).
[0353] In an embodiment, the tail domain comprises the 3' sequence UUUUUU, e.g., if a U6 promoter is used for transcription.
[0354] In an embodiment, the tail domain comprises the 3' sequence UUUU, e.g., if an H I promoter is used for transcription.
[0355] In an embodiment, tail domain comprises variable numbers of 3' U's depending, e.g., on the termination signal of the pol-III promoter used.
[0356] In an embodiment, the tail domain comprises variable 3' sequence derived from the DNA template if a T7 promoter is used.
[0357] In an embodiment, the tail domain comprises variable 3' sequence derived from the DNA template, e.g., if in vitro transcription is used to generate the RNA molecule.
[0358] In an embodiment, the tail domain comprises variable 3' sequence derived from the DNA template, e.g, if a pol-II promoter is used to drive transcription.
[0359] Modifications in the tail domain can be selected to not interfere with targeting efficacy, which can be evaluated by testing a candidate modification in the system described in Section ΠΙ. gRNA's having a candidate tail domain having a selected length, sequence, degree of complementarity, or degree of modification, can be evaluated in the system described in Section ΠΙ. The candidate tail domain can be placed, either alone, or with one or more other candidate changes in a gRNA molecule/Cas9 molecule system known to be functional with a selected target and evaluated.
[0360] In some embodiments, the tail domain comprises modifications at two consecutive nucleotides, e.g., two consecutive nucleotides that are within 5 nucleotides of the 5' end of the tail domain, within 5 nucleotides of the 3' end of the tail domain, or more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no two consecutive nucleotides are modified within 5 nucleotides of the 5' end of the tail domain, within 5 nucleotides of the 3' end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain. In an embodiment, no nucleotide is modified within 5 nucleotides of the 5' end of the tail domain, within 5 nucleotides of the 3' end of the tail domain, or within a region that is more than 5 nucleotides away from one or both ends of the tail domain.
[0361] In an embodiment a gRNA has the following structure:
[0362] 5' [targeting domain]-[first complementarity domain]-[linking domain]-[second complementarity domain]-[proximal domain]-[tail domain]-3'
wherein,
the targeting domain comprises a core domain and optionally a secondary domain, and is
10 to 50 nucleotides in length;
the first complementarity domain is 5 to 25 nucleotides in length and, in an embodiment has
at least 50, 60, 70, 80, 85, 90, or 95% homology with a reference first complementarity domain disclosed herein;
the linking domain is 1 to 5 nucleotides in length;.
the proximal domain is 5 to 20 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference proximal domain disclosed herein; and
the tail domain is absent or a nucleotide sequence is 1 to 50 nucleotides in length and, in an embodiment has at least 50, 60, 70, 80, 85, 90 or 95% homology with a reference tail domain disclosed herein.
Exemplary Chimeric gRNAs
[0363] In an embodiment, a unimolecular, or chimeric, gRNA comprises, preferably from 5' to 3' :
a targeting domain (which is complementary to a target nucleic acid);
a first complementarity domain; a linking domain;
a second complementarity domain (which is complementary to the first complementarity domain);
a proximal domain; and
a tail domain,
wherein,
(a) the proximal and tail domain, when taken together, comprise
at least 15, 1 8, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides;
(b) there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain; or
(c) there are at least 16, 19, 21 , 26, 31 , 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0364] In an embodiment, the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.
[0365] In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0366] In an embodiment, there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0367] In an embodiment, there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 5 1 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is
complementary to its corresponding nucleotide of the first complementarity domain.
[0368] In an embodiment, the targeting domain has, or consists of 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides in length.
[0369] In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length. [0370] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.
[0371 ] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.
[0372] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 1 nucleotides in length.
[0373] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.
[0374] In an embodiment, the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.
[0375] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length.
[0376] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.
[0377] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.
[0378] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length.
[0379] In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0380] In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting . domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0381] In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0382] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0383] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0384] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g.; 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0385] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together;
comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides. [0386] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0387] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0388] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0389] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0390] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21, 26, 31 , 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0391] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0392] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0393] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41, 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0394] In an embodiment, the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0395] In an embodiment, the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0396] In an embodiment, the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21, 26, 31 , 32, 36, 41, 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0397] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0398] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain. [0399] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 5 1 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is
complementary to its corresponding nucleotide of the first complementarity domain.
[0400] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0401 ] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, ( or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0402] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41, 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is
complementary to its corresponding nucleotide of the first complementarity domain.
[0403] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0404] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0405] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41, 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain:
[0406] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0407] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0408] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
Exemplary Modular gRNAs
[0409] In an embodiment, a modular gRNA comprises:
a first strand comprising, preferably from 5' to 3' ;
a targeting domain;
a first complementarity domain; and
a second strand, comprising, preferably from 5' to 3' :
optionally a 5' extension domain;
a second complementarity domain;
a proximal domain; and
a tail domain,
wherein:
(a) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides;
(b) there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain; or (c) there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0410] In an embodiment, the sequence from (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a naturally occurring gRNA, or with a gRNA described herein.
[041 1 ] In an embodiment, the proximal and tail domain, when taken together, comprise at least 15, 1 8, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0412] In an embodiment, there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0413] In an embodiment, there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is
complementary to its corresponding nucleotide of the first complementarity domain.
[0414] In an embodiment, the targeting domain has, or consists of, 16, 17, 18, 19, 20, 21 , 22, 23, 24 or 25 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16, 17, 18, 19, 20, 2 1 , 22, 23, 24 or 25 nucleotides in length.
[0415] >In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length.
[0416] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length.
[0417] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length.
[0418] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length. [0419] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length.
[0420] In an embodiment, the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length.
[0421] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the' targeting domain is 22 nucleotides in length.
[0422] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length.
[0423] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length.
[0424] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 5 nucleotides in length.
[0425] In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0426] In an embodiment, the targeting domain has, or consists of, 16 nucleotides (e.g., 16 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 16 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0427] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0428] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 15, 1 8, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0429] In an embodiment, the targeting domain has, or consists of, 17 nucleotides (e.g., 17 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 17 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41, 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0430] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0431 ] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 18 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0432] In an embodiment, the targeting domain has, or consists of, 18 nucleotides (e.g., 18 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 1 8 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain. [0433] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0434] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0435] In an embodiment, the targeting domain has, or consists of, 19 nucleotides (e.g., 19 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 19 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0436] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0437] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35-, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0438] In an embodiment, the targeting domain has, or consists of, 20 nucleotides (e.g., 20 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 20 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0439] In an embodiment, the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0440] In an embodiment, the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0441] In an embodiment, the targeting domain has, or consists of, 21 nucleotides (e.g., 21 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 21 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0442] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0443] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0444] In an embodiment, the targeting domain has, or consists of, 22 nucleotides (e.g., 22 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 22 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0445] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides. [0446] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0447] In an embodiment, the targeting domain has, or consists of, 23 nucleotides (e.g., 23 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 23 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41, 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0448] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0449] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0450] In an embodiment, the targeting domain has, or consists of, 24 nucleotides (e.g., 24 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 24 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
[0451] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides.
[0452] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain.
[0453] In an embodiment, the targeting domain has, or consists of, 25 nucleotides (e.g., 25 consecutive nucleotides) having complementarity with the target domain, e.g., the targeting domain is 25 nucleotides in length; and there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51, or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain.
Methods for Designing gRNAs
[0454] Methods for designing gRNAs are described herein, including methods for selecting, designing and validating target domains. Exemplary targeting domains are also provided herein. Targeting Domains discussed herein can be incorporated into the gRNAs described herein.
[0455] Methods for selection and validation of target sequences as well as off-target analyses are described, e.g., in. Mali el al. , 2013 SCIENCE 339(6121): 823-826; Hsu et al , 2013 NAT
BIOTECHNOL, 31 (9): 827-32; Fu et al , 2014 NAT BIOTECHNOL, doi: 10.1038/nbt.2808. PubMed PM ID: 24463574; Heigwer et al, 2014 NAT METHODS l l (2): 122-3. doi: 10.1038/nmeth.2812. PubMed PMID: 24481216; Bae el al , 2014 BIOINFORMATICS PubMed PMID: 24463181 ; Xiao A el al , 2014 BIOINFORMATICS PubMed PMID: 24389662.
[0456] For example, a software tool can be used to optimize the choice of gRNA within a user's target sequence, e.g., to minimize total off-target activity across the genome. Off target activity may be other than cleavage. For each possible gRNA choice e.g., using S. pyogenes Cas9, the tool can identify all off-target sequences (e.g., preceding either NAG or NGG PAMs) across the genome that contain up to certain number (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10) of mismatched base-pairs. The cleavage efficiency at each off-target sequence can be predicted, e.g., using an experimentally-derived weighting scheme. Each possible gRNA is then ranked according to its total predicted off-target cleavage; the top-ranked gRNAs represent those that are likely to have the greatest on-target and the least off-target cleavage. Other functions, e.g., automated reagent design for CR1SPR construction, primer design for the on-target Surveyor assay, and primer design for high-throughput detection and quantification of off-target cleavage via next-gen sequencing, can also be included in the tool. Candidate gRNA molecules can be evaluated by art-known methods or as described in Section IV herein. II. Cas9 Molecules
[0457] Cas9 molecules of a variety of species can be used in the methods and compositions described herein. While the S. pyogenes and S. thermophil s Cas9 molecules are the subject of much of the disclosure herein, Cas9 molecules of, derived from, or based on the Cas9 proteins of other species listed herein can be used as well. In other words, while the much of the description herein uses S. pyogenes and S. thermophilus Cas9 molecules, Cas9 molecules from the other species can replace them, e.g., Staphylococcus aureus and Neisseria meningitidis Cas9 molecules. Additional Cas9 species include: Acidovorax avenae, Actinobacillus
pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhiz' obium sp., Brevibacillus latemsporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lad, Candidatus Puniceispirillum, Clostridiu cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter sliibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacler diazotrophicus,
Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, llyobacler polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp.,
Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica. Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium
succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis,
Streptococcus sp., Subdoligranulum sp., Tislrella mobilis, Treponema sp., or Verminephrobacter eiseniae.
[0458] A Cas9 molecule, as that term is used herein, refers to a molecule that can interact with a gRNA molecule and, in concert with the gRNA molecule, localize (e.g., target or home) to a site which comprises a target domain and PAM sequence. .
[0459] In an embodiment, the Cas9 molecule is capable of cleaving a target nucleic acid molecule. A Cas9 molecule that is capable of cleaving a target nucleic acid molecule is referred to herein as an eaCas9 (an enzymatically active Cas9) molecule. In an embodiment, an eaCas9 molecule, comprises one or more of the following activities:
[0460] a nickase activity, i.e., .the ability to cleave a single strand, e.g., the non-complementary strand or the complementary strand, of a nucleic acid molecule;
a double stranded nuclease activity, i.e., the ability to cleave both strands of a double stranded nucleic acid and create a double stranded break, which in an embodiment is the presence of two nickase activities;
an endonuclease activity;
an exonuclease activity; and
a helicase activity, i.e., the ability to unwind the helical structure of a double stranded nucleic acid.
[0461] In an embodiment, an enzymatically active Cas9 or an eaCas9 molecule cleaves both DNA strands and results in a double stranded break. In an embodiment, an eaCas9 molecule cleaves only one strand, e.g., the strand to which the gRNA hybridizes to, or the strand complementary to the strand the gRNA hybridizes with. In an embodiment, an eaCas9 molecule comprises cleavage activity associated with an HNH-like domain. In an embodiment, an eaCas9 molecule comprises cleavage activity associated with an N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule comprises cleavage activity associated with an HNH-like domain and cleavage activity associated with an N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule comprises an active, or cleavage competent, HNH-like domain and an inactive, or cleavage incompetent, N-terminal RuvC-like domain. In an embodiment, an eaCas9 molecule comprises an inactive, or cleavage incompetent, HNH-like domain and an active, or cleavage competent, N-terminal RuvC-like domain.
[0462] In an embodiment, the ability of an eaCas9 molecule to interact with and cleave a target nucleic acid is PAM sequence dependent. A PAM sequence is a sequence in the target nucleic acid. In an embodiment, cleavage of the target nucleic acid occurs upstream from the PAM sequence. EaCas9 molecules from different bacterial species can recognize different sequence motifs (e.g., PAM sequences). In an embodiment, an eaCas9 molecule of S. pyogenes recognizes the sequence motif NGG and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Mali el ai, SCIENCE 2013; 339(6121): 823- 826. In an embodiment, an eaCas9 molecule of S. tliermophilus recognizes the sequence motif NGGNG and NNAG AAW (W = A or T) and directs cleavage of a core target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from these sequences. See, e.g., Horvath et al. , SCIENCE 2010; 327(5962): 167- 170, and Deveau et al , J BACTERIOL 2008; 190(4): 1390- 1400. In an embodiment, an eaCas9 molecule of S. mulans recognizes the sequence motif NGG or NAAR (R - A or G) and directs cleavage of a core target nucleic acid sequence 1 to 10, e.g., 3 to 5 base pairs, upstream from this sequence. See, e.g., Deveau et al. , J BACTERIOL 2008;
190(4): 1 390- 1400. In an embodiment, an eaCas9 molecule of S. aureus recognizes the sequence motif NNGRR (R = A or G) and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. In an embodiment, an eaCas9 molecule of N. meningitidis recognizes the sequence motif NNNNGATT and directs cleavage of a target nucleic acid sequence 1 to 10, e.g., 3 to 5, base pairs upstream from that sequence. See, e.g., Hou et al., PNAS EARLY EDITION 2013, 1 -6. The ability of a Cas9 molecule to recognize a PAM sequence can be determined, e.g., using a transformation assay described in Jinek et al , SCIENCE 2012, 337:816.
[0463] Some Cas9 molecules have the ability to interact with a gRNA molecule, and in conjunction with the gRNA molecule home (e.g., targeted or localized) to a core target domain, but are incapable of cleaving the target nucleic acid, or incapable of cleaving at efficient rates. Cas9 molecules having no, or no substantial, cleavage activity are referred to herein as an eiCas9 (an enzymatically inactive Cas9) molecule. For example, an eiCas9 molecule can lack cleavage activity or have substantially less, e.g., less than 20, 10, 5, 1 or 0.1 % of the cleavage activity of a reference Cas9 molecule, as measured by an assay described herein.
[0464] Exemplary naturally occurring Cas9 molecules are described in Chylinski et al , RNA Biology 2013; 10:5, 727-737. Such Cas9 molecules include Cas9 molecules of a cluster 1 bacterial family, cluster 2 bacterial family, cluster 3 bacterial family, cluster 4 bacterial family, cluster 5 bacterial family, cluster 6 bacterial family, a cluster 7 bacterial family, a cluster 8 bacterial family, a cluster 9 bacterial family, a cluster 10 bacterial family, a cluster 1 1 bacterial family, a cluster 12 bacterial family, a cluster 13 bacterial family, a cluster 14 bacterial family, a cluster 1 bacterial family, a cluster 16 bacterial family, a cluster 17 bacterial family, a cluster 1 8 bacterial family, a cluster 19 bacterial family, a cluster 20 bacterial family, a cluster 21 bacterial family, a cluster 22 bacterial family, a cluster 23 bacterial family, a cluster 24 bacterial family, a cluster 25 bacterial family, a cluster 26 bacterial family, a cluster 27 bacterial family, a cluster 28 bacterial family, a cluster 29 bacterial family, a cluster 30 bacterial family, a cluster 31 bacterial family, a cluster 32 bacterial family, a cluster 33 bacterial family, a cluster 34 bacterial family, a cluster 35 bacterial family, a cluster 36 bacterial family, a cluster 37 bacterial family, a cluster 38 bacterial family, a cluster 39 bacterial family, a cluster 40 bacterial family, a cluster 41 bacterial family, a cluster 42 bacterial family, a cluster 43 bacterial family, a cluster 44 bacterial family, a cluster 45 bacterial family, a cluster 46 bacterial family, a cluster 47 bacterial family, a cluster 48 bacterial family,. a cluster 49 bacterial family, a cluster 50 bacterial family, a cluster 5 1 bacterial family, a cluster 52 bacterial family, a cluster 53 bacterial family, a cluster 54 bacterial family, a cluster 55 bacterial family, a cluster 56 bacterial family, a cluster 57 bacterial family, a cluster 58 bacterial family, a cluster 59 bacterial family, a cluster 60 bacterial family, a cluster 61 bacterial family, a cluster 62 bacterial family, a cluster 63 bacterial family, a cluster 64 bacterial family, a cluster 65 bacterial family, a cluster 66 bacterial family, a cluster 67 bacterial family, a cluster 68 bacterial family, a cluster 69 bacterial family, a cluster 70 bacterial family, a cluster 71 bacterial family, a cluster 72 bacterial family, a cluster 73 bacterial family, a cluster 74 bacterial family, a cluster 75 bacterial family, a cluster 76 bacterial family, a cluster 77 bacterial family, or a cluster 78 bacterial family.
[0465] Exemplary naturally occurring Cas9 molecules include a Cas9 molecule of a cluster 1 bacterial family. Examples include a Cas9 molecule of: S. pyogenes (e.g., strain SF370, MGAS 10270, MGAS 10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI- 1), S. tliermophilus (e.g., strain LMD-9), S. pseudoporcinus (e.g., strain SPIN 20026), S. /nutans (e.g., strain UA 159, NN2025), S. macacae (e.g., strain NCTC1 1558), S.
gallolylicus (e.g., strain UCN34, ATCC BAA-2069), S. equines (e.g., strain ATCC 9812, MGCS 124), S. dysdalactiae (e.g., strain GGS 124), S. bovis (e.g., strain ATCC 700338), S. cmginosus (e.g.; strain F021 1 ), S. agalactia* (e.g., strain NEM316, A909), Listeria monocytogenes (e.g., strain F6854), Listeria innocua (L. innocua, e.g., strain Clip l 1262), EtUerococcus italicus (e.g., strain DSM 15952), or Enterococcus faecium (e.g., strain 1,231 ,408). Additional exemplary Cas9 molecules are a Cas9 molecule of Neisseria meningitidis (Hou et'al. PNAS Early Edition 2013, 1 -6) and a S. aureus Cas9 molecule.
[0466] In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence: having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology with;
differs at no more than, 2, 5, 10, 15, 20, 30, or 40% of the amino acid residues when compared with;
differs by at least 1, 2, 5, 10 or 20 amino acids but by no more than 100, 80, 70, 60, 50, 40 or 30 amino acids from; or
is identical to;
any Cas9 molecule sequence described herein or a naturally occurring Cas9 molecule sequence, e.g., a Cas9 molecule from a species listed herein or described in Chylinski et al. , RNA Biology 2013, 10:5, Ί2Ί-Τ,1 Hou et al. PNAS Early Edition 2013, 1-6. In an embodiment, the Cas9 molecule comprises one or more of the following activities: a nickase activity; a double stranded cleavage activity (e.g., an endonuclease and/or exonuclease activity); a helicase activity; or the' ability, together with a gRNA molecule, to localize to a target nucleic acid.
[0467] In an embodiment, a Cas9 molecule comprises the amino acid sequence of the consensus sequence of FIG. 2, wherein "*" indicates any amino acid found in the corresponding position in the amino acid sequence of a Cas9 molecule of S. pyogenes, S. thermophilus, S. mutatis and L. innocua, and "-" indicates any amino acid. In an embodiment, a Cas9 molecule differs from the sequence of the consensus sequence disclosed in Figure 2 by at least 1 , but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In an embodiment, a Cas9 molecule comprises the amino acid sequence of SEQ ID NO:7 of FIG. 5, wherein "*" indicates any amino acid found in the corresponding position in the amino acid sequence of a Cas9 molecule of S. pyogenes, or N. meningitidis, "-" indicates any amino acid, and "-" indicates any amino acid or absent. In an embodiment, a Cas9 molecule differs from the sequence of SEQ ID NO:6 or 7 by at least 1 , but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.
[0468] A comparison of the sequence of a number of Cas9 molecules indicate that certain regions are conserved. These are identified below as:
region 1 (residues 1 to 1 80, or in the case of region Γ residues 120 to 180)
region 2 (residues 360 to 480);
region 3 (residues 660 to 720);
region 4 (residues 817 to 900); and ; region 5 (residues 900 to 960). [0469] In an embodiment, a Cas9 molecule comprises regions 1 -5, together with sufficient additional Cas9 molecule sequence to provide a biologically active molecule, e.g., a Cas9 molecule having at least one activity described herein. In an embodiment, each of regions 1 -6, independently, have, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with the corresponding residues of a Cas9 molecule described herein, e.g., a sequence from FIG. 2 or from FIG. 5.
[0470] In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 1 :
having 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 1 - 180 (the numbering is according to the motif sequence in FIG. 2; 52% of residues in the four Cas9 sequences in Fig. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes;
differs by at least 1 , 2, 5, 10 or 20 amino acids but by no more than 90, 80, 70, 60, 50, 40 or 30 amino acids from amino acids 1 -180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mulans, L. innocua, N. meningitidis, or S. aureus; or
is identical to 1 - 180 of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mulans or, L. innocua, N. meningitidis, or S. aureus.
[0471 ] In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 1 ':
having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 120- 180 (55% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mulans or, L. innocua, N. meningitidis, or S. aureus;
differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 120- 180 of the amino acid sequence of Cas9 of S. pyogenes, S.
thermophilus, S. mulans or, L. innocua, N. meningitidis, or S. aureus ; or
is identical to 120- 180 of the amino acid sequence of Cas9 of S. pyogenes, S.
thermophilus, S. mulans or, L. innocua, N. meningitidis, or S. aureus.
[0472] In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 2: having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 997c homology with amino acids 360-480 (52% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus;
differs by at least 1 , 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 360-480 of the amino acid sequence of Cas9 of S. pyogenes, S.
thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus; or
is identical to 360-480 of the amino acid sequence of Cas9 of S. pyogenes, S.
thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus.
[0473] In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 3:
having 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 660-720 (56% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus;
differs by at least 1, 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 660-720 of the amino acid sequence of Cas9 of S. pyogenes, S.
thermophilus, S. mutans or, L: innocua, N. meningitidis, or S. aureus; or
is identical to 660-720 of the amino acid sequence of Cas9 of S. pyogenes, S.
thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus.
[0474] In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 4:
having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 817-900 (55% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus;
differs by at least 1 , 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 817-900 of the amino acid sequence of Cas9 of S. pyogenes, S.
thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus ; or
is identical to 817-900 of the amino acid sequence of Cas9 of S. pyogenes, S.
thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus. [0475] In an embodiment, a Cas9 molecule, e.g., an eaCas9 molecule or eiCas9 molecule, comprises an amino acid sequence referred to as region 5:
having 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with amino acids 900-960 (60% of residues in the four Cas9 sequences in FIG. 2 are conserved) of the amino acid sequence of Cas9 of S. pyogenes, S. thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus;
differs by at least 1 , 2, or 5 amino acids but by no more than 35, 30, 25, 20 or 10 amino acids from amino acids 900-960 of the amino acid sequence of Cas9 of S. pyogenes, S.
thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus; or
is identical to 900-960 of the amino acid sequence of Cas9 of S. pyogenes, S.
thermophilus, S. mutans or, L. innocua, N. meningitidis, or S. aureus .
A RuvC-like domain and an HNH-like domain
[0476] In an embodiment, a Cas9 molecule comprises an HNH-like domain and an RuvC-like domain. In an embodiment, cleavage activity is dependent on a RuvC-like domain and an HNH- like domain. A Cas9 molecule, e.g., an eaCas9 or eiCas9 molecule, can comprise one or more of the following domains: a RuvC-like domain and an HNH-like domain. In an embodiment, a cas9 molecule is an eaCas9 molecule and the eaCas9 molecule comprises a RuvC-like domain, e.g., a RuvC-like domain described below, and/or an HNH-like domain, e.g., an HNH-like domain described below. In an embodiment, a Cas9 molecule is an eiCas9 molecule comprising one or more difference in an RuvC-like domain and/or in an HNH-like domain as compared to a reference Cas9 molecule, and the eiCas9 molecule does not cleave a nucleic acid, or cleaves with significantly less efficiency than does wildype, e.g., when compared with wild type in a cleavage assay, e.g., as described herein, cuts with less than 50, 25, 10, or 1 % of the a reference Cas9 molecule, as measured by an assay described herein.
RuvC-like domains
[0477] In an embodiment, a RuvC-like domain cleaves, a single strand, e.g., the non- complementary strand of the target nucleic acid molecule. A Cas9 molecule can include more than one RuvC-like domain (e.g., one, two, three or more RuvC-like domains). In an
embodiment, an RuvC-like domain is at least 5, 6, 7, 8 amino acids in length but not more than 20, 19, 1 8, 17, 16 or 15 amino acids in length. In an embodiment, the cas9 molecule comprises an N-terminal RuvC-like domain of about 10 to 20 amino acids, e.g., about 15 amino acids in length.
N-terminal RuvC-like domains
[0478] Some naturally occurring Cas9 molecules comprise more than one RuvC-like domain, with cleavage being dependent on the N-terminal RuvC-like domain. Accordingly, Cas9 molecules can comprise an N-terminal RuvC-like domain. Exemplary N-terminal RuvC-like domains are described below.
[0479] In an embodiment, an eaCas9 molecule comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula I:
D-X 1-G-X2-X3-X4-X5-G-X6-X7-X8-X9 (SEQ ID NO: 8),
wherein,
X 1 is selected from I, V, M, L and T (e.g., selected from I, V, and L);
X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);
X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);
X4 is selected from S, Y, N and F (e.g., S);
X5 is selected from V, I, L, C, T and F (e.g., selected from V, I and L);
X6 is selected from W, F, V, Y, S and' L (e.g., W);
X7 is selected from A, S, C, V and G (e.g., selected from A and S);
X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and
X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R, or, e.g., selected from T, V, I, L and Δ).
[0480] In an embodiment, the N-tenninal RuvC-like domain differs from a sequence of SEQ ID NO:8, by as many as 1 but no more than 2, 3, 4, or 5 residues.
[0481] In embodiment the N-terminal RuvC-like domain is cleavage competent.
[0482] In embodiment the N-terminal RuvC-like domain is cleavage incompetent.
[0483] In an embodiment, an eaCas9 molecule comprises an N-terminal RuvC-like domain comprising an amino acid sequence of formula II:
D-X 1 -G-X2-X3-S-X5-G-X6-X7-X8-X9 (SEQ ID NO: 9), wherein
X 1 is selected from I, V, M, L and T (e.g., selected from I, V, and L);
X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);
X3 is selected from N, S, G, A, D, T, R, and F (e.g., A or N);
X5 is selected from V, I, L, C, T and F (e.g., selected from V, I and L);
X6 is selected from W, F, V, Y, S and L (e.g., W);
X7 is selected from A, S, C, V and G (e.g., selected from A and S);
X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and
X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R or selected from e.g., T, V, I, L and Δ).
[0484] In an embodiment, the N-temninal RuvC-like domain differs from a sequence of SEQ ID NO:9 by as many as 1 , but no more than 2, 3, 4, or 5 residues.
[0485] In an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence of formula HI:
D-I-G-X2-X3-S-V-G-W-A-X8-X9 (SEQ ID NO: 10),
wherein
X2 is selected from T, I, V, S, N, Y, E and L (e.g., selected from T, V, and I);
X3 is selected from N, S, G, A, D, T, R, M and F (e.g., A or N);
X8 is selected from V, I, L, A, M and H (e.g., selected from V, I, M and L); and
X9 is selected from any amino acid or is absent (e.g., selected from T, V, I, L, Δ, F, S, A, Y, M and R or selected from e.g., T, V, I, L and Δ).
[0486] In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 10 by as many as 1 , but no more than, 2, 3, 4, or 5 residues.
[0487] In an embodiment, the N-terminal RuvC-like domain comprises an amino acid sequence of formula 111:
D-I-G-T-N-S-V-G-W-A-V-X (SEQ ID NO: 1 1 ),
wherein
X is a non-polar alkyl amino acid or a hydroxyl amino acid, e.g., X is selected from V, I, L and T (e.g., the eaCas9 molecule can comprise an N-terminal RuvC-like domain shown in FIG. 2 (depicted as "Y")).
[0488] In an embodiment, the N-terminal RuvC-like domain differs from a sequence of SEQ ID NO: 1 1 by as many as 1 but no more than, 2, 3, 4, or 5 residues.
[0489] In an embodiment, the N-terminal RuvC-like domain differs from a sequence of an N- terminal RuvC-like domain disclosed herein, e.g., in FIG. 3 A or FIG. 5, as many as 1 , but no more than 2, 3, 4, or 5 residues. In an embodiment, 1 , 2, or all 3 of the highly conserved residues identified in FIG. 3A or FIG. 5 are present.
[0490] In an embodiment, the N-terminal RuvC-like domain differs from a sequence of an N- terminal RuvC-like domain disclosed herein, e.g., in FIG. 3B, as many as 1 , but no more than 2, 3, 4, or 5 residues. In an embodiment, 1 , 2, 3 or all 4 of the highly conserved residues identified in FIG. 3B are present.
Additional RuvC-like domains
[0491] In addition to the N-terminal RuvC-like domain, a Cas9 molecule, e.g., an eaCas9 molecule, can comprise one or more additional RuvC-like domains. In an embodiment, a Cas9 molecule can comprise two additional RuvC-like domains. Preferably, the additional RuvC-like domain is at least 5 amino acids in length and, e.g., less than 15 amino acids in length, e.g., 5 to 10 amino acids in length, e.g., 8 amino acids in length.
[0492] An additional RuvC-like domain can comprise an amino acid sequence:
I-X 1 -X2-E-X3-A-R-E (SEQ ID NO: 12), wherein
X I is V or H,
X2 is I, L or V (e.g., I or V); and
X3 is or T.
[0493] In an embodiment, the additional RuvC-like domain comprises the amino acid sequence:
I-V-X2-E-M-A-R-E (SEQ ID NO: 13), wherein
X2 is I, L or V (e.g., I or V) (e.g., the eaCas9 molecule can comprise an additional RuvC- like domain shown in FIG. 2 or FIG. 5 (depicted as "B")).
[0494] An additional RuvC-like domain can comprise an amino acid sequence: H-H-A-X 1 -D-A-X2-X3 (SEQ ID NO: 14), wherein
X I is H or L;
X2 is R or V; and X3 is E or V.
[0495] In an embodiment, the additional RuvC-like domain comprises the amino acid sequence:. H-H-A-H-D-A-Y-L (SEQ ID NO: 15).
[0496] In an embodiment, the additional RuvC-like domain differs from a sequence of SEQ ID NO: 13, 15, 12 or 14 by as many as 1 , but no more than 2, 3, 4, or 5 residues.
[0497] In some embodiments, the sequence flanking the N-terminal RuvC-like domain is a sequences of formula V:
-X r-Y-X2' -X3'-X4' -Z-T-D-X9'-Y (SEQ ID NO: 16),
wherein
X V is selected from and P,
X2' is selected from V, L, I, and F (e.g., V, I and L);
X3' is selected from G, A and S (e.g., G),
X4' is selected from L, I, V and F (e.g., L);
X9' is selected from D, E, N and Q; and
Z is an N-terminal RuvC-like domain, e.g. , as described above.
HN H-like domains
[0498] In an embodiment, an HNH-like domain cleaves a single stranded complementary domain, e.g., a complementary strand of a double stranded nucleic acid molecule. In an embodiment, an HNH-like domain is at least 15, 20, 25 amino acids in length but not more than 40, 35 or 30 amino acids in length, e.g., 20 to 35 amino acids in length, e.g., 25 to 30 amino acids in length. Exemplary HNH-like domains are described below.
[0499] In an embodiment, an eaCas9 molecule comprises an HNH-like domain having an amino acid sequence of formula VI: X 1 -X2-X3-H-X4-X5-P-X6-X7-X8-X9-X 10-X 1 1 -X 12-X 13-X 14-X 15-N-X 16-X 17-X 18- X 19-X20-X21 -X22-X23-N (SEQ ID NO: 17), wherein
X I is selected from D, E, Q and N (e.g., D and E);
X2 is selected from L, I, R, Q, V, M and ;
X3 is selected from D and E;
X4 is selected from I, V, T, A and L (e.g., A, 1 and V);
X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);
X6 is selected from Q, H, R, , Y, I, L, F and W;
X7 is selected from S, A, D, T and K (e.g., S and A);
X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F);
X9 is selected from L, R, T, I, V, S, C, Y, , F and G;
X 10 is selected from , Q, Y, T, F, L, W, M, A, E, G, and S;
X I 1 is selected from D, S, N, R, L and T (e.g., D);
X 12 is selected from D, N and S;
X 13 is selected from S, A, T, G and R (e.g., S);
X 14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);
X 15 is selected from D, S, 1, N, E, A, H, F, L, Q, M, G, Y and V;
X 16 is selected from K, L, R, M, T and F (e.g., L, R and K);
X 17 is selected from V, L, I, A and T;
XI 8 is selected from L, I, V and A (e.g., L and I);
X 19 is selected from T, V, C, E, S and A (e.g., T and V);
X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;
X21 is selected from S, P, R, , N, A, H, Q, G and L;
X22 is selected from D, G, T, N, S, K, A, I, E, L, Q, R and Y; and
X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.
[0500] In an embodiment, a HNH-like domain differs from a sequence of SEQ ID NO: 1 7 by at least 1 , but no more than, 2, 3, 4, or 5 residues.
[0501 ] In an embodiment, the HNH-like domain is cleavage competent.
[0502] In an embodiment, the HNH-like domain is cleavage incompetent. [0503] In an embodiment, an eaCas9 molecule comprises an HNH-like domain comprising an amino acid sequence of formula VII:
X 1-X2-X3-H-X4-X5-P-X6-S-X8-X9-X 10-D-D-S-X 14-X 15-N- -V-L-X 19-X20-X21 - X22-X23-N (SEQ ID NO: 18),
wherein
X I is selected from D and E;
X2 is selected from L, I, R, Q, V, M and K;
X3 is selected from D and E;
X4 is selected from I, V, T, A and L (e.g., A, I and V);
X5 is selected from V, Y, I, L, F and W (e.g., V, I and L);
X6 is selected from Q, H, R, K, Y, I, L, F and W;
X8 is selected from F, L, V, K, Y, M, I, R, A, E, D and Q (e.g., F); .
X9 is selected from L, R, T, I, V, S, C, Y, K, F and G;
X 10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;
X 14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);
X I 5 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;
X19 is selected from T, V, C, E, S and A (e.g., T and V);
X20 is selected from R, F, T, W, E, L, N, C, , V, S, Q, I, Y, H and A;
X21 is selected from S, P, R, K, N, A, H, Q, G and L;
X22 is selected from D, G, T, N, S, , A, I, E, L, Q, R and Y; and
X23 is selected from K, V, A, E, Y, I, C, L, S, T, G, K, M, D and F.
[0504] In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 18 by 1 , 2, 3, 4, or 5 residues.
[0505] In an embodiment, an eaCas9 molecule comprises an HNH-like domain comprising an amino acid sequence of formula VII:
X 1 -V-X3-H-I- V-P-X6-S-X8-X9-X 10-D-D-S-X 14-X 15-N- -V-L-T-X20-X21 -X22-X23- N (SEQ ID NO: 19),
wherein
X 1 is selected from D and E;
X3 is selected from D and E;
X6 is selected from Q, H, R, K, Y, I, L and W; X8 is selected from F, L, V, , Y, M, I, R, A, E, D and Q (e.g., F);
X9 is selected from L, R, T, I, V, S, C, Y, , F and G;
X 10 is selected from K, Q, Y, T, F, L, W, M, A, E, G, and S;
X 14 is selected from I, L, F, S, R, Y, Q, W, D, K and H (e.g., I, L and F);
X 15 is selected from D, S, I, N, E, A, H, F, L, Q, M, G, Y and V;
X20 is selected from R, F, T, W, E, L, N, C, K, V, S, Q, I, Y, H and A;
X21 is selected from S, P, R, K, N, A, H, Q, G and L;
X22 is selected from D, G, T, N, S, , A, I, E, L, Q, R and Y; and
X23 is selected from , V, A, E, Y, I, C, L, S, T, G, , M, D and F.
[0506] In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO: 19 by 1 , 2, 3, 4, or 5 residues.
[0507] In an embodiment, an eaCas9 molecule comprises an HNH-like domain having an amino acid sequence of formula VIII:
D-X2-D-H-I-X5-P-Q-X7-F-X9-X 10-D-X 12-S-I-D-N-X16-V-L-X 19-X20-S-X22-X23-N (SEQ ID NO:20),
wherein
X2 is selected from I and V;
X5 is selected from I and V;
X7 is selected from A and S;
X9 is selected from I and L;
X 10 is selected from K and T;
X 12 is selected from D and N;
X 16 is selected from R, K and L; X 19 is selected from T and V;
X20 is selected from S and R;
X22 is selected from K, D and A; and
X23 is selected from E, K, G and N (e.g., the eaCas9 molecule can comprise an HNH- like domain as described herein).
[0508] In an embodiment, the HNH-like domain differs from a sequence of SEQ ID NO:20 by as many as 1 , but no more than 2, 3, 4, or 5 residues. [0509] In an embodiment, an eaCas9 molecule comprises the amino acid sequence of formula IX:
L-Y-Y-L-Q-N-G-X 1 '-D-M-Y-X2'-X3'-X4'-X5'-L-D-I— X6'-X7'-L-S-X8'-Y-Z-N-R- X9'-K-X 10'-D-X 1 1 '-V-P (SEQ ID NO:21 ),
wherein
X I ' is selected from K and R;
X2' is selected from V and T;
X3' is selected from G and D;
X4' is selected from E, Q and D;
X5' is selected from E and D;
X6' is selected from D, N and H;
X7' is selected from Y, R and N;
X8' is selected from Q, D and N; X9' is selected from G and E;
X 10' is selected from S and G;
X I 1 ' is selected from D and N; and
Z is an HNH-like domain, e.g., as described above.
[0510] In an embodiment, the eaCas9 molecule comprises an amino acid sequence that differs from a sequence of SEQ ID NO:21 by as many as 1 , but no more than 2, 3, 4, or 5 residues.
[0511] In an embodiment, the HNH-like domain differs from a sequence of an HNH-like domain disclosed herein, e.g., in FIG. 4A or FIG. 5, as many as 1 , but no more than 2, 3, 4, or 5 residues.
[0512] In an embodiment, the HNH -like domain differs from a sequence of an HNH-like domain disclosed herein, e.g., in FIG. 4B, by as many as 1 , but no more than 2, 3, 4, or 5 residues. In an embodiment, 1 , 2, all 3 of the highly conserved residues identified in FIG. 4B are present.
Altered Cas9 Molecules
[0513] Naturally occurring Cas9 molecules possess a number of properties, including: nickase activity, nuclease activity (e.g., endonuclease and/or exonuclease activity); helicase activity; the ability to associate functionally with a gRNA molecule; and the ability to target (or localize to) a site on a nucleic acid (e.g., PAM recognition and specificity). In an embodiment, a Cas9 molecules can include all or a subset of these properties. In typical embodiments, Cas9 molecules have the ability to interact with a gRNA molecule and, in concert with the gRNA molecule, localize to a site in a nucleic acid. Other activities, e.g., PAM specificity, cleavage activity, or helicase activity can vary more widely in Cas9 molecules.
[0514] Cas9 molecules with desired properties can be made in a number of ways, e.g., by alteration of a parental, e.g., naturally occurring Cas9 molecules to provide an altered Cas9 molecule having a desired property. For example, one or more mutations or differences relative to a parental Cas9 molecule can be introduced. Such mutations and differences comprise:
substitutions (e.g., conservative substitutions or substitutions of non-essential amino acids); insertions; or deletions. In an embodiment, a Cas9 molecule can comprises one or more mutations or differences, e.g., at least 1 , 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mutations but less than 200, 100, or 80 mutations relative to a reference Cas9 molecule.
[0515] In an embodiment, a mutation or mutations do not have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein. In an embodiment, a mutation or mutations have a substantial effect on a Cas9 activity, e.g. a Cas9 activity described herein. In an embodiment, exemplary activities comprise one or more of PAM specificity, cleavage activity, and helicase activity. A mutation(s) can be present, e.g., in: one or more RuvC-like domain, e.g., an N- terminal RuvC-like domain; an HNH-like domain; a region outside the RuvC-like domains and the HNH-like domain. In some embodiments, a mutation(s) is present in an N-terminal RuvC- like domain. In some embodiments, a mutation(s) is present in an HNH-like domain. In some embodiments, mutations are present in both an N-terminal RuvC-like domain and an HNH-like domain.
[0516] Whether or not a particular sequence, e.g., a substitution, may affect one or more activity, such as targeting activity, cleavage activity, etc, can be evaluated or predicted, e.g., by evaluating whether the mutation is conservative or by the method described in Section ΠΙ. In an embodiment, a "non-essential" amino acid residue, as used in the context of a Cas9 molecule, is a residue that can be altered from the wild-type sequence of a Cas9 molecule, e.g., a naturally occurring Cas9 molecule, e.g., an eaCas9 molecule, without abolishing or more preferably, without substantially altering a Cas9 activity (e.g., cleavage activity), whereas changing an "essential" amino acid residue results in a substantial loss of activity (e.g., cleavage activity).
[0517] In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of S. pyogenes shown in the consensus sequence disclosed in Figure 2, and has one or more amino acids that differ from the amino acid sequence of S. pyogenes (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figure 2 or SEQ ID NO:7. In an embodiment, the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of S. pyogenes shown in the consensus sequence disclosed in Figure 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.
[0518] In an embodiment, the altered Cas9 molecule comprises a sequence in which:
the sequence corresponding to the fixed sequence of the consensus sequence disclosed in Figure 2 differs at no more than 1 , 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figure 2;
the sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figure 2 differ at no more than 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the "*" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. pyogenes Cas9 molecule; and,
the sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figure 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "- " residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. pyogenes Cas9 molecule.
[0519] In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of S. thermophilus shown in the consensus sequence disclosed in Figure 2, and has one or more amino acids that differ from the amino acid sequence of S. thermophilus (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figure 2. In an embodiment, the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of S. thermophilus shown in the consensus sequence disclosed in Figure 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.
[0520] In an embodiment the altered Cas9 molecule comprises a sequence in which:
the sequence corresponding to the fixed sequence of the consensus sequence disclosed in Figure 2 differs at no more than 1 , 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the
consensus sequence disclosed in Figure 2;
the sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figure 2 differ at no more than 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the "*" " residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S.
thermophilus Cas9 molecule; and,
the sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figure 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "- " residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. thermophilus Cas9 molecule.
[0521] In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of S. mutans shown in the consensus sequence disclosed in Figure 2, and has one or more amino acids that differ from the amino acid sequence of S. mutans (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figure 2. In an embodiment, the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of S. mutans shown in the consensus sequence disclosed in Figure 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.
[0522] In an embodiment the altered Cas9 molecule comprises a sequence in which:
the sequence corresponding to the fixed sequence of the consensus sequence disclosed in Figure 2 differs at no more than 1 , 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the
consensus sequence disclosed in Figure 2;
the sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figure 2 differ at no more than 1 , 2, 3, 4, 5, 10, 1 , 20, 25, 30, 35, or 40% of the "*" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. mutatis Cas9 molecule; and,
the sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figure 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "- " residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an S. mutatis Cas9 molecule.
[0523] In an embodiment, the altered Cas9 molecule is an eaCas9 molecule comprising the fixed amino acid residues of L. intiocula shown in the consensus sequence disclosed in Figure 2, and has one or more amino acids that differ from the amino acid sequence of L. innocula (e.g., has a substitution) at one or more residue (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) represented by an "-" in the consensus sequence disclosed in Figure 2. In an embodiment, the altered Cas9 molecule is an eiCas9 molecule wherein one or more of the fixed amino acid residues of L. innocul shown in the consensus sequence disclosed in Figure 2 (e.g., 2, 3, 5, 10, 15, 20, 30, 50, 70, 80, 90, 100, 200 amino acid residues) is mutated.
[0524] In an embodiment the altered Cas9 molecule comprises a sequence in which:
the sequence corresponding to the fixed sequence of the consensus sequence disclosed in Figure 2 differs at no more than 1 , 2, 3, 4, 5, 10, 15, or 20% of the fixed residues in the consensus sequence disclosed in Figure 2;
the sequence corresponding to the residues identified by "*" in the consensus sequence disclosed in Figure 2 differ at no more than 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, or 40% of the "*" residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an L. innocula Cas9 molecule; and,
the sequence corresponding to the residues identified by "-" in the consensus sequence disclosed in Figure 2 differ at no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 55, or 60% of the "- " residues from the corresponding sequence of naturally occurring Cas9 molecule, e.g., an L. innocula Cas9 molecule.
[0525] In an embodiment, the altered Cas9 molecule, e.g., an eaCas9 molecule or an eiCas9 molecule, can be a fusion, e.g., of two of more different Cas9 molecules, e.g., of two or more naturally occurring Cas9 molecules of different species. For example, a fragment of a naturally occurring Cas9 molecule of one species can be fused to a fragment of a Cas9 molecule of a second species. As an example, a fragment of Cas9 of S. pyogenes comprising an N-terminal RuvC-like domain can be fused to a fragment of Cas9 of a species other than S. pyogenes (e.g., S. thermophiliis) comprising an HNH-like domain.
Cas9 Molecules with altered PAM recognition or no PAM recognition
[0526] Naturally occurring Cas9 molecules can recognize specific PAM sequences, for example the PAM recognition sequences described above for S. pyogenes, S. thermophilus,S. mutans, S. aureus and N. meningitidis.
[0527] In an embodiment, a Cas9 molecule has the same PAM specificities as a naturally occurring Cas9 molecule. In other embodiments, a Cas9 molecule has a PAM specificity not associated with a naturally occurring Cas9 molecule, or a PAM specificity not associated with the naturally occurring Cas9 molecule to which it has the closest sequence homology. For example, a naturally occurring Cas9 molecule can be altered, e.g., to alter PAM recognition, e.g., to alter the PAM sequence that the Cas9 molecule recognizes to decrease off target sites and/or improve specificity; or eliminate a PAM recognition requirement. In an embodiment, a Cas9 molecule can be altered, e.g., to increase length of PAM recognition sequence and/or improve Cas9 specificity to high level of identity to decrease off target sites and increase specificity. In an embodiment, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10 or 15 amino acids in length. Cas9 molecules that recognize different PAM sequences and/or have reduced off- target activity can be generated using directed evolution. Exemplary methods and systems that can be used for directed evolution of Cas9 molecules are described, e.g., in Esvelt el ai , Nature 201 1 , 472(7344): 499-503. Candidate Cas9 molecules can be evaluated, e.g., by methods described in Section III.
Non-Cleaving and Modified-Cleavage Cas9 Molecules
[0528] In an embodiment, a Cas9 molecule comprises a cleavage property that differs from naturally occurring Cas9 molecules, e.g., that differs from the naturally occurring Cas9 molecule having the closest homology. For example, a Cas9 molecule can differ from naturally occurring Cas9 molecules, e.g., a Cas9 molecule of S. pyogenes, as follows: its ability to modulate, e.g., decreased or increased, cleavage of a double stranded break (endonuclease and/or exonuclease activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); its ability to modulate, e.g., decreased or increased, cleavage of a single strand of a nucleic acid, e.g., a non-complimentary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (nickase activity), e.g., as compared to a naturally occurring Cas9 molecule (e.g., a Cas9 molecule of S. pyogenes); or the ability to cleave a nucleic acid molecule, e.g., a double stranded or single stranded nucleic acid molecule, can be eliminated.
Modified Cleavage eaCas9 Molecules
[0529] In an embodiment, an eaCas9 molecule comprises one or more of the following activities: cleavage activity associated with an N-terminal RuvC-like domain; cleavage activity associated with an HNH-like domain; cleavage activity associated with an HNH domain and cleavage activity associated with an N-terminal RuvC-like domain.
[0530] In an embodiment an eaCas9 molecule comprises an active, or cleavage competent, HNH-like domain (e.g., an HNH-like domain described herein, e.g., SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 or SEQ ID NO:21 ) and an inactive, or cleavage incompetent, N-terminal RuvC-like domain. An exemplary inactive, or cleavage incompetent N- terminal RuvC-like domain can have a mutation of an aspartic acid in an N-terminal RuvC-like domain, e.g., an aspartic acid at position 9 of the consensus sequence disclosed in Figure 2 or an aspartic acid at position 10 of SEQ ID NO:7, e.g., can be substituted with an alanine. In an embodiment, the eaCas9 differs from wild type in the N-terminal RuvC-like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0. 1 % of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S.
pyogenes, or S. ihermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.
[0531] In an embodiment, an eaCas9 molecule comprises an inactive, or cleavage incompetent, HNH domain and an active, or cleavage competent, N-terminal RuvC-like domain (e.g., an HNH-like domain described herein, e.g., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15). Exemplary inactive, or cleavage incompetent HNH-like domains can have a mutation at one or more of: a histidine in an HNH-like domain, e.g., a,histidine at position 856 of the consensus sequence disclosed in Figure 2, e.g., can be substituted with an alanine; and one or more asparagines in an HNH-like domain, e.g., an asparagine at position 870 of the consensus sequence disclosed in Figure 2 and/or at position 879 of the consensus sequence disclosed in Figure 2, e.g., can be substituted with an alanine. In an embodiment, the eaCas9 differs from wild type in the HNH- like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of S. pyogenes, or S. thermophilus. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.
Non-Cleaving eiCas9 Molecules
[0532] In an embodiment, the altered Cas9 molecule is an eiCas9 molecule which does not cleave a nucleic acid molecule (either double stranded or single stranded nucleic acid molecules) or cleaves a nucleic acid molecule with significantly less efficiency, e.g., less than 20, 10, 5, 1 or 0.1 % o the cleavage activity of a reference Cas9 molecule, e.g., as measured by an assay described herein. The reference Cas9 molecule can by a naturally occurring unmodified Cas9 molecule, e.g., a naturally occurring Cas9 molecule such as a Cas9 molecule of . . pyogenes, S. thermophilus, S. aureus or N. meningitidis. In an embodiment, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology. In an embodiment, the eiCas9 molecule lacks substantial cleavage activity associated with an N- terminal RuvC-like domain and cleavage activity associated with an HNH-like domain.
[0533] In an embodiment, an eiCas9 molecule comprises an inactive, or cleavage incompetent, N-terminal RuvC-like domain. An exemplary inactive, or cleavage incompetent N-terminal RuvC-like domain can have a mutation of an aspartic acid in an N-terminal RuvC-like domain, e.g., an aspartic acid at position 9 of the consensus sequence disclosed in Figure 2 or an aspartic acid at position 10 of SEQ ID NO:7, e.g., can be substituted with an alanine.
[0534] In an embodiment an eiCas9 molecule comprises an inactive, or cleavage incompetent, HNH domain (e.g., an HNH-like domain described herein, e.g., SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15). Exemplary inactive, or cleavage incompetent HNH-like domains can have a mutation at one or more of: a histidine in an HNH-like domain, e.g., a histidine at position 856 of the consensus sequence disclosed in Figure 2, e.g., can be substituted with an alanine; and one or more asparagines in an HNH-like domain, e.g., an asparagine at position 870 of the consensus sequence disclosed in Figure 2 and/or at position 879 of the consensus sequence disclosed in Figure 2, e.g., can be substituted with an alanine.
[0535] A catalytically inactive Cas9 molecule may be fused with a transcription repressor. An eiCas9 fusion protein complexes with a gRNA and localizes to a DNA sequence specified by gRNA's targeting domain, but, unlike an eaCas9, it will not cleave the target DNA. Fusion of an effector domain, such as a transcriptional repression domain, to an eiCas9 enables recmitment of the effector to any DNA site specified by the gRNA. Site specific targeting of an eiCas9 or an eiCas9 fusion protein to a promoter region of a gene can block R A polymerase binding to the promoter region, a transcription factor (e.g., a transcription activator) and/or a transcriptional enhancer to inhibit transcription activation. Alternatively, site specific targeting of an eiCas9- fusion to a transcription repressor to a promoter region of a gene can be used to decrease transcription activation.
[0536] Transcription repressors or transcription repressor domains that may be fused to an eiCas9 molecule can include ruppel associated box (KRAB or SKD), the Mad mSIN3 interaction domain (SID) or the ERF repressor domain (ERD).
[0537] In another embodiment, an eiCas9 molecule may be fused with a protein that modifies chromatin. For example, an eiCas9 molecule may be fused to heterochromatin protein 1 (HPl ), a histone lysine methyltransferase (e.g., SUV39H 1 , SUV39H2, G9A, ESET/SETDB l , Pr-SET7/8, SUV4-20H 1 , RIZ1), a histone lysine demethylates (e.g., LSD1/BHC1 10, SpLsdl/Sw, l/Safl 10, Su(var)3-3, JMJD2A/JHDM3A, JMJD2B, JMJD2C/GASC1 , JMJD2D, Rph l , JARID 1 A/RBP2, JARI DIB/PLU- I , JAR1D 1C/SMCX, JARID1 D/SMCY, Lid, Jhn2, Jmj2), a histone lysine deacetylases (e.g., HDAC 1 , HDAC2, HDAC3, HDAC8, Rpd3, Hos l , Cir6, HDAC4, HDAC5, HDAC7, HDAC9, Hdal , Cir3, SIRT 1 , SIRT2, Sir2, Hst l , Hst2, Hst3, Hst4, HDAC 1 1 ) and a DNA methylases (DNMT1 , DNMT2a/DMNT3b, MET1). An eiCas9-chomatin modifying molecule fusion protein can be used to alter chromatin status to reduce expression a target gene.
[0538] The heterologous sequence (e.g., the transcription repressor domain) may be fused to the N- or C-terminus of the eiCas9 protein. In an alternative embodiment, the heterologous sequence (e.g., the transcription repressor domain) may be fused to an internal portion (i.e., a portion other than the N-terminus or C-terminus) of the eiCas9 protein.
[0539] The ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated, e.g., by the methods described herein in Section ΠΙ. The activity of a Cas9 molecule, either an eaCas9 or a eiCas9, alone or in a complex with a gRNA molecule may also be evaluated by methods well-known in the art, including, gene expression assays and chromatin-based assays, e.g., chromatin immunoprecipitation (ChiP) and chromatin in vivo assay (CiA).
Nucleic Acids Encoding Cas9 Molecules
[0540] Nucleic acids encoding the Cas9 molecules, e.g., an eaCas9 molecule or an eiCas9 molecule are provided herein.
[0541] Exemplary nucleic acids encoding Cas9 molecules are described in Cong et al , SCIENCE 2013, 399(6121):819-823; Wang et al , CELL 2013, 153(4):910-918; Mali et al. , SCIENCE 2013, 399(6121):823-826; Jinek et al, SCIENCE 2012, 337(6096):816-821. Another exemplary nucleic acid encoding a Cas9 molecule of N. meningitidis is shown in FIG. 6.
[0542] In an embodiment, a nucleic acid encoding a Cas9 molecule can be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be chemically modified, e.g., as described in Section X. In an embodiment, the Cas9 mRNA has one or more of, e.g., all of the following properties: it is capped, polyadenylated, substituted with 5-methylcytidine and/or pseudouridine.
[0543] In addition or alternatively, the synthetic nucleic acid sequence can be codon optimized, e.g., at least one non-common codon or less-common codon has been replaced by a common codon. For example, the synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, e.g., optimized for expression in a mammalian expression system, e.g., described herein.
[0544] In addition, or alternatively, a nucleic acid encoding a Cas9 molecule may comprise a nuclear localization sequence (NLS). Nuclear localization sequences are known in the art.
[0545] Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of ,S'. pyogenes.
ATGGATAAAA AGTACAGCAT CGGGCTGGAC ATCGGTACAA ACTCAGTGGG
GTGGGCCGTG ATTACGGACG AGTACAAGGT ACCCTCCAAA AAATTTAAAG
TGCTGGGTAA CACGGACAGA CACTCTATAA AG AAAAAT C T TATTGGAGCC
T TGC TGTTCG ACTCAGGCGA GACAGCCGAA GCCACAAGGT TGAAGCGGAC
CGCCAGGAGG CGGTATACCA GGAGAAAGAA CCGCATATGC TACCTGCAAG
AAATCTTCAG TAACGAGATG GCAAAGGTTG ACGATAGCTT TTTCCATCGC
CTGGAAGAAT . CCTTTCTTGT TGAGGAAGAC AAGAAGCACG AACGGCACCC
CATC TTTGGC AATATTGTCG ACGAAGTGGC ATATCACGAA AAGTACCCGA
C TATCTACCA CCTCAGGAAG AAGCTGGTGG ACTOTACCGA TAAGGCGGAC
CTCAGACTTA TTTATTTGGC ACTCGCCCAC ATGATTAAAT T TAGAGGACA
TTTCTTGATC GAGGGCGACC TGAACCCGGA CAACAGTGAC GTCGATAAGC
TGT TCATCCA ACTTGTGCAG ACCTACAATC AACTGTTCGA AGAAAACCCT
ATAAATGCTT CAGGAGTCGA CGCTAAAGCA ATCCTGTCCG CGCGCCTCTC
AAAATCTAGA AGACTTGAGA ATCTGATTGC TCAGTTGCCC GGGGAAAAGA
AAAATGGATT GT T TGGCAAC C TGATCGCCC TCAGTCTCGG ACTGACCCCA
AATTTCAAAA GTAACTTCGA CCTGGCCGAA GACGCTAAGC TCCAGCTGTC
CAAGGACACA TACGATGACG ACCTCGACAA TCTGCTGGCC CAGATTGGGG
ATCAGTACGC CGATCTCT TT T TGGCAGCAA AGAACCTGTC CGACGCCATC
CTGT TGAGCG ATATCTTGAG AGTGAACACC GAAATTACTA AAGCACCCCT
TAGCGCATCT ATGATCAAGC GGTACGACGA GCATCATCAG GATCTGACCC
TGC TGAAGGC TCTTGTGAGG CAACAGCTCC CCGAAAAATA CAAGGAAATC
T TCT TTGACC AG AG C AAA A A CGGCTACGCT GGCTATATAG ATGGTGGGGC
GAGTCAGGAG GAATTCTATA AAT TCATCAA GCCCATTCTC GAGAAAATGG
ACGGCACAGA GGAGTTGCTG GTCAAACTTA ACAGGGAGGA CCTGCTGCGG
AAGCAGCGGA CCTTTGACAA CGGGTCTATC CCCCACCAGA T TCATCTGGG
CGAACTGCAC GCAATCCTGA GGAGGCAGGA GGATTTTTAT CCTTTTCTTA
AAGATAACCG CGAGAAAATA GAAAAGATTC TTACATTCAG GATCCCGTAC
TACGTGGGAC CTCTCGCCCG GGGCAATTCA CGGTTTGCCT GGATGACAAG
GAAGTCAGAG GAGACTATTA CACCTTGGAA CTTCGAAGAA GTGGTGGACA
AGGGTGCATC TGCCCAGTC T TTCATCGAGC GGATGACAAA TTTTGACAAG
AACCTCCCTA ATGAGAAGGT GCTGCCCAAA CATTCTCTGC TCTACGAGTA
CTTTACCGTC TACAATGAAC TGACTAAAGT CAAGTACGTC ACCGAGGGAA
TGAGGAAGCC GGCATTCCTT AGTGGAGAAC AGAAGAAGGC GATTGTAGAC
CTGTTGTTCA AGACCAACAG GAAGGTGACT GTGAAGCAAC TTAAAGAAGA CTAC TTTAAG AAGATCGAAT GTTT TGACAG TGTGGAAAT T TCAGGGGTTG AAGACCGC TT CAATGCGTCA TTGGGGACTT ACCATGATC T TCTCAAGATC ATAAAGGACA AAGACTTCCT GGACAACGAA GAAAATGAGG ATATTCTCGA AGACATCGTC CTCACCCTGA CCCTGTTCGA AGACAGGGAA ATGATAGAAG AGCGCTTGAA AACCTATGCC CACCTCTTCG ACGATAAAGT TATGAAGCAG CTGAAGCGCA GGAGATACAC AGGATGGGGA AGATTGTCAA GGAAGCTGAT CAATGGAATT AGGGATAAAC AGAGTGGCAA GACCATACTG . GATTTCCTCA AATCTGATGG CTTCGCCAAT AGGAACTTCA TGCAACTGAT TCACGATGAC TCTCTTACCT TCAAGGAGGA CATTCAAAAG GCTCAGGTGA GCGGGCAGGG AGACTCCCTT CATGAACACA TCGCGAATTT GGCAGGTTCC CCCGCTATTA AAAAGGGCAT CCTTCAAACT GTCAAGGTGG TGGATGAATT GGTCAAGGTA ATGGGCAGAC ATAAGCCAGA AAATATTGTG ATCGAGATGG CCCGCGAAAA CCAGACCACA CAGAAGGGCC AGAAAAATAG TAGAGAGCGG ATGAAGAGGA TCGAGGAGGG CATCAAAGAG C TGGGATCTC AGATTCTCAA AGAACACCCC GTAGAAAACA CACAGCTGCA GAACGAAAAA TTGTACTTGT ACTATCTGCA GAACGGCAGA GACATGTACG TCGACCAAGA ACTTGATATT AATAGACTGT CCGAC TATGA CGTAGACCAT ATCGTGCCCC AGTCCTTCCT GAAGGACGAC TCCAT TGATA ACAAAGTCTT GACAAGAAGC GACAAGAACA GGGGTAAAAG TGATAATGTG CCTAGCGAGG AGGTGGTGAA AAAAATGAAG AACTACTGGC GACAGCTGCT TAATGCAAAG CTCATTACAC AACGGAAGT T ' CGATAATCTG ACGAAAGCAG AGAGAGGTGG CTTGTCTGAG TTGGACAAGG- CAGGGTTTAT TAAGCGGCAG CTGGTGGAAA CTAGGCAGAT CACAAAGCAC GTGGCGCAGA T TTTGGACAG CCGGATGAAC ACAAAATACG ACGAAAATGA TAAACTGATA CGAGAGGTCA AAGTTATCAC GCTGAAAAGC AAGCTGGTGT CCGATTTTCG GAAAGACTTC CAGTTCTACA AAGT TCGCGA GATTAATAAC TACCATCATG C TCACGATGC GTACCTGAAC GCTGTTGTCG GGACCGCCTT GATAAAGAAG TACCCAAAGC TGGAATCCGA GTTCGTATAC GGGGATTACA AAGTGTACGA TGTGAGGAAA ATGATAGCCA AG.TCCGAGCA GGAGATTGGA AAGGCCACAG CTAAGTACTT CTTTTATTCT AACATCATGA ATTTTTTTAA GACGGAAATT ACCC TGGCCA ACGGAGAGAT CAGAAAGCGG CCCCTTATAG AGACAAATGG TGAAACAGGT GAAATCGTCT GGGATAAGGG CAGGGATTTC GCTACTGTGA GGAAGGTGCT GAGTATGCCA CAGGTAAATA TCGTGAAAAA AACCGAAGTA CAGACCGGAG GATTTTCCAA GGAAAGCATT TTGCCTAAAA GAAACTCAGA CAAGCTCATC GCCCGCAAGA AAGATTGGGA CCCTAAGAAA TACGGGGGAT TTGACTCACC CACCGTAGCC TATTCTGTGC TGGTGGTAGC TAAGGTGGAA AAAGGAAAGT CTAAGAAGC T GAAGTCCGTG AAGGAACTC T TGGGAATCAC TATCATGGAA AGATCATCCT TTGAAAAGAA CCCTATCGAT TTCCTGGAGG C TAAGGGTTA CAAGGAGGTC AAGAAAGACC TCATCATTAA ACTGCCAAAA TACTCTCTCT TCGAGCTGGA AAATGGCAGG AAGAGAATGT TGGCCAGCGC CGGAGAGCTG CAAAAGGGAA ACGAGCTTGC TCTGCCCTCC AAATATGTTA ATTTTC TCTA TCTCGCTTCC CACTATGAAA AGCTGAAAGG GTC TCCCGAA GATAACGAGC AGAAGCAGCT GT TCGTCGAA CAGCACAAGC ACTATCTGGA TGAAATAATC GAACAAATAA GCGAGTTCAG CAAAAGGGTT ATCCTGGCGG ATGC TAATTT GGACAAAGTA C TGTCTGCTT ATAACAAGCA CCGGGATAAG CC TATTAGGG AACAAGCCGA GAATATAATT CACCTCTTTA CACTCACGAA TCTCGGAGCC CCCGCCGCCT TCAAATACTT TGATACGACT ATCGACCGGA AACGGTATAC CAGTACCAAA GAGGTCCTCG ATGCCACCCT CATCCACCAG TCAATTACTG GCCTGTACGA AACACGGATC GACCTCTCTC AACTGGGCGG CGACTAG
(SEQ ID NO: 22)
[0546] Provided below is the corresponding amino acid sequence of a S. pyogenes Cas9 molecule.
DKKYSIGLDIGTNSVG AVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRL
KRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAY
HEKYPTIYHLRKKLVDSTDKADLRLI YLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY
NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNF
DLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSAS
MIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD
GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRI
PYYVGPLARGNSRFA MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHS
LLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
SVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYA
HLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF
KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEi ARENQ
TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINR
LSDYDVDHIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRK
FDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV VITLKS
KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAK
SEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIV DKGRDFATVRKVLS
MPQV I VKKTEVQTGGFSKES ILPKRNSDKLIARKKD DPKKYGGFDSPTVAYSVLVVAKVEKG
KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKRMLAS
AGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQI SEFSKRV
ILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
ATLIHQS ITGLYETRIDLSQLGGD*
(SEQ ID NO: 23)
[0547] Provided below is an exemplary codon optimized nucleic acid sequence encoding a Cas9 molecule of N. meningitidis.
ATGGCCGCCTTCAAGCCCAACCCCATCAACTACATCCTGGGCCTGGACATCGGCATCGCCAGCG TGGGCTGGGCCATGGTGGAGATCGACGAGGACGAGAACCCCATCTGCCTGATCGACCTGGGTGT GCGCGTGTTCGAGCGCGCTGAGGTGCCCAAGACTGGTGACAGTCTGGCTATGGCTCGCCGGCTT GCTCGCTCTGTTCGGCGCCTTACTCGCCGGCGCGCTCACCGCCTTCTGCGCGCTCGCCGCCTGC TGAAGCGCGAGGGTGTGCTGCAGGCTGCCGACTTCGACGAGAACGGCCTGATCAAGAGCCTGCC CAACACTCCTTGGCAGCTGCGCGCTGCCGCTCTGGACCGCAAGCTGACTCCTCTGGAGTGGAGC GCCGTGCTGCTGCACCTGATCAAGCACCGCGGCTACCTGAGCCAGCGCAAGAACGAGGGCGAGA CCGCCGACAAGGAGCTGGGTGCTCTGCTGAAGGGCGTGGCCGACAACGCCCACGCCCTGCAGAC TGGTGACTTCCGCACTCCTGCTGAGCTGGCCCTGAACAAGTTCGAGAAGGAGAGCGGCCACATC CGCAACCAGCGCGGCGACTACAGCCACACCTTCAGCCGCAAGGACCTGCAGGCCGAGCTGATCC TGCTGTTCGAGAAGCAGAAGGAGTTCGGCAACCCCCACGTGAGCGGCGGCCTGAAGGAGGGCAT CGAGACCCTGCTGATGACCCAGCGCCCCGCCCTGAGCGGCGACGCCGTGCAGAAGATGCTGGGC
CACTGCACCTTCGAGCCAGCCGAGCCCAAGGCCGCCAAGAACACC TACACCGCCGAGCGCT TCA
TCTGGCTGACCAAGCTGAACAACCTGCGCATCCTGGAGCAGGGCAGCGAGCGCCCCCTGACCGA
CACCGAGCGCGCCACCCTGATGGACGAGCCCTACCGCAAGAGCAAGCTGACCTACGCCCAGGCC
CGCAAGCTGCTGGGTCTGGAGGACACCGCCTTCTTCAAGGGCCTGCGCTACGGCAAGGACAACG
CCGAGGCCAGCACCCTGATGGAGATGAAGGCCTACCACGCCATCAGCCGCGCCCTGGAGAAGGA
GGGCCTGAAGGACAAGAAGAGTCCTCTGAACCTGAGCCCCGAGCTGCAGGACGAGATCGGCACC
GCCTTCAGCCTGTTCAAGACCGACGAGGACATCACCGGCCGCCTGAAGGACCGCATCCAGCCCG
AGATCCTGGAGGCCCTGCTGAAGCACATCAGCTTCGACAAGTTCGTGCAGATCAGCCTGAAGGC
CCTGCGCCGCATCGTGCCCCTGATGGAGCAGGGCAAGCGCTACGACGAGGCCTGCGCCGAGATC
TACGGCGACCACTACGGCAAGAAGAACACCGAGGAGAAGATC TACCTGCCTCCTATCCCCGCCG
ACGAGATCCGCAACCCCGTGGTGCTGCGCGCCCTGAGCCAGGCCCGCAAGGTGATCAACGGCGT
GGTGCGCCGCTACGGCAGCCCCGCCCGCATCCACATCGAGACCGCCCGCGAGGTGGGCAAGAGC
TTCAAGGACCGCAAGGAGATCGAGAAGCGCCAGGAGGAGAACCGCAAGGACCGCGAGAAGGCCG
CCGCCAAGTTCCGCGAGTAC TTCCCCAACTTCGTGGGCGAGCCCAAGAGCAAGGACATCCTGAA
GC TGCGCCTGTACGAGCAGCAGCACGGCAAGTGCCTGTACAGCGGCAAGGAGATCAACCTGGGC
CGCC TGAACGAGAAGGGCTACGTGGAGATCGACCACGCCCTGCCCTTCAGCCGCACCTGGGACG
ACAGCTTCAACAACAAGGTGCTGGTGCTGGGCAGCGAGAACCAGAACAAGGGCAACCAGACCCC
CTACGAGTACTTCAACGGCAAGGACAACAGCCGCGAGTGGCAGGAGTTCAAGGCCCGCGTGGAG
ACCAGCCGCTTCCCCCGCAGCAAGAAGCAGCGCATCCTGCTGCAGAAGTTCGACGAGGACGGCT
TCAAGGAGCGCAACCTGAACGACACCCGCTACGTGAACCGCTTCCTGTGCCAGTTCGTGGCCGA
CCGCATGCGCCTGACCGGCAAGGGCAAGAAGCGCGTGTTCGCCAGCAACGGCCAGATCACCAAC
CTGC TGCGCGGCTTCTGGGGCCTGCGCAAGGTGCGCGCCGAGAACGACCGCCACCACGCCCTGG
ACGCCGTGGTGGTGGCCTGCAGCACCGTGGCCATGCAGCAGAAGATCACCCGCTTCGTGCGCTA
CAAGGAGATGAACGCCTTCGACGGTAAAACCATCGACAAGGAGACCGGCGAGGTGCTGCACCAG
AAGACCCACTTCCCCCAGCCCTGGGAGTTCTTCGCCCAGGAGGTGATGATCCGCGTGTTCGGCA
AGCCCGACGGCAAGCCCGAGTTCGAGGAGGCCGACACCCCCGAGAAGCTGCGCACCCTGCTGGC
CGAGAAGCTGAGCAGCCGCCCTGAGGCCGTGCACGAGTACGTGACTCCTCTGTTCGTGAGCCGC
GCCCCCAACCGCAAGATGAGCGGTCAGGGTCACATGGAGACCGTGAAGAGCGCCAAGCGCC TGG
ACGAGGGCGTGAGCGTGCTGCGCGTGCCCCTGACCCAGCTGAAGCTGAAGGACCTGGAGAAGAT
GGTGAACCGCGAGCGCGAGCCCAAGCTGTACGAGGCCCTGAAGGCCCGCCTGGAGGCCCACAAG
GACGACCCCGCCAAGGCCTTCGCCGAGCCCTTCTACAAGTACGACAAGGCCGGCAACCGCACCC
AGCAGGTGAAGGCCGTGCGCGTGGAGCAGGTGCAGAAGACCGGCGTGTGGGTGCGCAACCACAA
CGGCATCGCCGACAACGCCACCATGGTGCGCGTGGACGTGTTCGAGAAGGGCGACAAGTACTAC
C TGGTGCCCATCTACAGCTGGCAGGTGGCCAAGGGCATCCTGCCCGACCGCGCCGTGGTGCAGG
GCAAGGACGAGGAGGACTGGCAGCTGATCGACGACAGCTTCAACT TCAAGTTCAGCCTGCACCC
CAACGACCTGGTGGAGGTGATCACCAAGAAGGCCCGCATGTTCGGCTACTTCGCCAGCTGCCAC
CGCGGCACCGGCAACATCAACATCCGCATCCACGACCTGGACCACAAGATCGGCAAGAACGGCA
TCCTGGAGGGCATCGGCGTGAAGACCGCCCTGAGCTTCCAGAAGTACCAGATCGACGAGCTGGG
CAAGGAGATCCGCCCCTGCCGCCTGAAGAAGCGCCCTCCTGTGCGCTAA
(SEQ ID NO: 24)
[0548] Provided below is the corresponding amino acid sequence of a N. meningitidis Cas9 molecule.
MAAFKPNP INY I LGLD I G I ASVG AMVE I DEDENP I CL I DLGVRVFERAEVPKTGDS LAMARRL ARSVRRLTRRRAHRLLRARRLLKREGVLQAADFDENGL I KS LPNTPWQLRAAALDRKLTPLE S AVLLHLIKHRGYLSQRKNEGETADKELGALLKGVADNAHALQTGDFRTPAELALNKFEKESGHI
RNQRGDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGLKEGIETLL TQRPALSGDAVQK LG
HCTFEPAEPKAA NTYTAERFI LTKLNNLRILEQGSERPLTDTERATLMDEPYRKSKLTYAQA
RKLLGLEDTAFFKGLRYGKDNAEASTL EMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGT
AFSLFKTDEDITGRLKDRIQPEILEALLKHI SFDKFVQI SLKALRRIVPLMEQGKRYDEACAEI
YGDHYGKKNTEEKIYLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKS
FKDRKEIEKRQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLG
RLNEKGYVEIDHALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQEFKARVE
TSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNGQITN
LLRGF GLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETGEVLHQ
KTHFPQP EFFAQEVMIRVFGKPDGKPEFEEADTPEKLRTLLAEKLSSRPEAVHEYVTPLFVSR
APNRKMSGQGH ETV SAKRLDEGVSVLRVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHK
DDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRVDVFEKGDKYY
LVPIYS QVAKGILPDRAVVQGKDEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCH
RGTG INIRIHDLDHKIGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR*
(SEQ ID NO: 25)
[0549] Provided below is an amino acid sequence of a S. aureus Cas9 molecule.
MKRNYILGLDIGITSVGYGI IDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHRI QRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVEEDT GNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGS INRFKTSDYVKEAKQLLKVQKAYHQ LDQSFI DTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKYAYNADLY NALNDLNNLVITRDENEKLEYYEKFQI IENVFKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGK PEFTNLKVYHDIKDITARKEI IENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQI S NLKGYTGTHNLSLKAINLILDEL HTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSP VVKRSFIQSIKVINAI IKKYGLPNDI I IELAREKNSKDAQKMINEMQKRNRQTNERIEEI IRTT GKENAKYLIEKIKLHDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHI IPRSVSFDNSFNNKVLVK QEENSKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKD FINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRK KFKKERNKGYKHHAED ALIIANADFIFKE KKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKD YKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSPEKLLMYHH DPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVIKKIKYYGNKLNAHLDITDD YPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSKCYEEAKKLKKISNQA EFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMNDKRPPRI IKTIASKT QSIKKYSTDILGNLYEVKSKKHPQI IKKG*
(SEQ ID NO: 26)
[0550] If any of the above Cas9 sequences are fused with a peptide or polypeptide at the C- temninus (e.g., an eiCas9 fused with a transcripon repressor at the C-terminus), it is understood that the stop codon will be removed.
Other Cas Molecules [0551] Various types of Cas molecules can be used to practice the inventions disclosed herein. In some embodiments, Cas molecules of Type II Cas systems are used. In other embodiments, Cas molecules of other Cas systems are used. For example, Type I or Type III Cas molecules may be used. Exemplary Cas molecules (and Cas systems) are described, e.g., in Haft et ai, PLoS COMPUTATIONAL BIOLOGY 2005, 1(6): e60 and Makarova et al , NATURE REVIEW
MICROBIOLOGY 201 1 , 9:467-477, the contents of both references are incorporated herein by reference in their entirety. Exemplary Cas molecules (and Cas systems) are also shown in Table H-1.
Figure imgf000102_0001
Tabic ΙΙ·1 : Cas Systems
(iciic System type Nainc from Haft Structure of Families (and Representatives name* or subtype et al.% encoded protein siiperl'amily) of
(PDB encoded
accessions)1 protein
cas6 • Subtype 1- cas6 and cinx6 314H COG 1583 and PF1 131 and slr7014
A COG555 1
• Subtype I- (RAMP)
B
• Subtype I- D
• Subtype
ΙΙΙ-Α·
Subtype III- B
cas6e • Subtype I- cse3 1 WJ9 (RAMP) ygcH
E
cas6f • Subtype I- csy4 2XL.I (RAMP) y l 727
F
cas7 • Subtype I- csa2, csJ2, cst'4, NA COG 1857 and devR and ygc.l
A csh2, cspl and COG3649
• Subtype I- csl2 (RAMP)
B
. · Subtype I- C
• Subtype I- E
c.asHa I • Subtype I- cinxl, c.stl , csxtt, NA BH0338-like LA3191 § and
A** csx 13 and PG2018
cxxc-cxxc
cas8a2 • Subtype I- csa4 and csx9 NA PH0918 AF0070, AF1873,
A** MJ0385, PF0637,
PH0918 nd SSO 1401 casSb • Subtype I- cshl and NA BH0338-like MTH 1090 and
B** TM 1802 TM 1802 casSc • Subtype 1- csdl and csp2 NA BH0338-like B 1 10338
C**
cas9 • Type II** csnl and csx J 2 NA COG3513 FTN_0757 and
SPyl046 cas 10 • Type III** cmr2, csml and NA COG 1353 ΜΊΉ326, Rv2823c5§ csx 11 and TM1794§ r.aslOd • Subtype I- c.sc.3 NA COG 1353 slr701 1
D**
csyJ • Subtype 1- csyJ NA yl 724-like y l724
F**
csy2 • Subtype I- csy2 NA (RAMP) yl 725
F
csy3 • Subtype I- csy3 NA (RAMP) y ! 726 Table I I-l : Cas Systems
Gene .System type Name from Waft Structure of Families (and RepresentativGs name* or subtype el al * ; encoded protein ;ii supcrlamily) of
(P B encoded
accessions)1 protein
I-'
csel • Subtype I- csel NA YgcL-like ygcL
E»
cse2 • Subtype I- cse2 2ZCA YgcK-like ygcK
E
cscl • Subtype I- cscl NA alrl 563-like air 1563
n (RAMP)
c.sc.2 • Subtype I- cscl and csc2 NA COG 1337 slr7012
D (RAMP)
csa5 • Subtype I- csa5 NA AF1870 API 870, MJ0380.
Λ PF0643 and SSO1308 csn2 • Sublype II- csn2 NA SPyl 049-like SPyl049
A
csm2 • Subtype csm2 NA COG 1421 MTH I081 and
II1-A" SERP2460 csin3 • Subtype csc2 aud csm3 NA COG 1337 MTH 1080 and
ΠΙ-Α (RAMP) SERP2459 csin4 • Subtype csm4 NA COG 1567 MTH 1079 and
III-A (RAMP) SERP2458
• Sublype csniS NA COG 1332 MTH 1078 and III-A (RAMP) SERP2457 csni6 • Sublype ΛΡΕ2256 and 2WTE COG 1517 ΛΡΕ2256 and
III-A csm6 SS01445 cnirl • Sublype c.mrl NA COG 1367 PF1 130
III-B (RAMP)
cinr3 • Sublype cmr3 NA COG 1769 PF1 128
III-B (RAMP)
cmr4 • Subtype cmr4 NA COG 1336 PF1 126
III-B (RAMP)
cinrS • Sublype c.mr5 2ZOP and 20EB COG3337 MTH324 and PF1 125
III-B**
cmr6 • Subtype cinr6 NA COG 1604 PF1 124
III-B (RAMP) /
csbl • Subtype 1- GSU0053 NA (RAMP) Balac_ 1306 and
U GSU0053 csb2 • Sublype 1- NA NA (RAMP) Balac_1305 and
GSU0054 csb3 • Subtype I- NA NA (RAMP) Balac_1303 §
U
csx 17 • Subtype I- NA NA NA Btns_2683 Table I I- 1 : Cas Systems
Genc System type Name from II ift Structure of Families (and Representatives V naiiie* : or subtype et al.s encoded protein supcrfamily) of
(PDB encoded
accessions)1 protein
U
csx 14 • Subtype 1- NA NA NA GSU0052
U
cs.\ 10 • Subtype I- csx 10 NA (RAMP) Caur_2274
U
csx 16 • Subtype VVA 1548 NA NA VVA1548
III-U
csaX • Subtype. csaX NA NA SS01438
III-U
csx3 • Subtype csx3 NA NA AF1864
III-U
csxl • Subtype csa3, csxl. csx2, I XMX and 217 1 COG 15 17 and M.I 1666, NE01 13.
III-U DXTHG. COG4006 PF1 127 and TM 1812
NE01 13 and
TIGR02710
csx 15 • Unknown NA NA TTE2665 ΤΓΕ2665 csfl • Type U csfl NA NA AFE_1038 csfl • Type U sfl NA (RAMP) AFE_1039 cs/3 • Type U csf3 NA (RAMP) AFE_1040 cs/4 • Type U csf4 NA NA AFE_1037
III. Functional Analysis of Candidate Molecules
[0552] Candidate Cas9 molecules, candidate gRNA molecules, candidate Cas9 molecule/gRNA molecule complexes, can be evaluated by art-known methods or as described herein. For example, exemplary methods for evaluating the endonuclease activity of Cas9 molecule are described, e.g., in Jinek el ciL , SCIENCE 2012; 337(6096):8 16-821 .
Binding and Cleavage Assay: Testing the endonuclease activity of Cas9 molecule
[0553] The ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in a plasmid cleavage assay. In this assay, synthetic or in vitro- transcribed gRNA molecule is pre-annealed prior to the reaction by heating to 95°C and slowly cooling down to room temperature. Native or restriction digest-linearized plasmid DNA (300 ng (~8 nM)) is incubated for 60 min at 37°C with purified Cas9 protein molecule (50-500 nM) and gRNA (50-500 nM, 1 : 1 ) in a Cas9 plasmid cleavage buffer (20 mM HEPES pH 7.5, 150 mM KC1, 0.5 mM DTT, 0. 1 mM EDTA) with or without 10 mM MgCl2. The reactions are stopped with 5X DNA loading buffer (30% glycerol, 1 .2% SDS, 250 mM EDTA), resolved by a 0.8 or 1 % agarose gel electrophoresis and visualized by ethidium bromide staining. The resulting cleavage products indicate whether the Cas9 molecule cleaves both DNA strands, or only one of the two strands. For example, linear DNA products indicate the cleavage of both DNA strands. Nicked open circular products indicate that only one of the two strands is cleaved.
[0554] Alternatively, the ability of a Cas9 molecule/gRNA molecule complex to bind to and cleave a target nucleic acid can be evaluated in an oligonucleotide DNA cleavage assay. In this assay, DNA oligonucleotides (10 pmol) are radiolabeled by incubating with .5 units T4 polynucleotide kinase and -3-6 pmol (-20-40 mCi) [γ-32Ρ]-ΑΤΡ in IX T4 polynucleotide kinase reaction buffer at 37°C for 30 min, in a 50 μΙ_, reaction. After heat inactivation (65°C for 20 min), reactions are purified through a column to remove unincorporated label. Duplex substrates ( 100 nM) are generated by annealing labeled oligonucleotides with equimolar amounts of unlabeled complementary oligonucleotide at 95°C for 3 min, followed by slow cooling to room temperature. For cleavage assays, gRNA molecules are annealed by heating to 95°C for 30 s, followed by slow cooling to room temperature. Cas9 (500 nM final concentration) is pre- incubated with the annealed gRNA molecules (500 nM) in cleavage assay buffer (20 mM
HEPES pH 7.5, 100 mM C1, 5 mM MgC12, 1 mM DTT, 5% glycerol) in a total volume of 9 μΐ. Reactions are initiated by the addition of 1 μΐ target DNA (10 nM) and incubated for 1 h at 37°C. Reactions are quenched by the addition of 20 μΐ of loading dye (5 mM EDTA, 0.025% SDS, 5% glycerol in formamide) and heated to 95°C for 5 min. Cleavage products are resolved on 12% denaturing polyacrylamide gels containing 7 M urea and visualized by phosphorimaging. The resulting cleavage products indicate that whether the complementary strand, the non- complementary strand, or both, are cleaved.
[0555] One or both of these assays can be used to evaluate the suitability of a candidate gRNA molecule or candidate Cas9 molecule. Binding Assay: Testing the binding of Cas9 molecule to target DNA
[0556] Exemplary methods for evaluating the binding of Cas9 molecule to target DNA are described, e.g., in Jinek et al , SCIENCE 2012; 337(6096):816-821.
[0557] For example, in an electrophoretic mobility shift assay, target DNA duplexes are formed by mixing of each strand ( 10 nmol) in deionized water, heating to 95°C for 3 min and slow cooling to room temperature. All DNAs are purified on 8% native gels containing IX TBE. DNA bands are visualized by UV shadowing, excised, and eluted by soaking gel pieces in DEPC-treated H20. Eluted DNA is ethanol precipitated and dissolved in DEPC-treated H20. DNA samples are 5' end labeled with [γ-32Ρ]-ΑΤΡ using T4 polynucleotide kinase for 30 min at 37°C. Polynucleotide kinase is heat denatured at 65°C for 20 min, and unincorporated radiolabel is removed using a column. Binding assays are performed in buffer containing 20 mM HEPES pH 7.5, 100 mM KC1, 5 mM MgCl2, 1 mM DTT and 10% glycerol in a total volume of 10 μΐ. Cas9 protein molecule is programmed with equimolar amounts of pre-annealed gRNA molecule and titrated from 100 pM to 1 μΜ . Radiolabeled DNA is added to a final concentration of 20 pM. Samples are incubated for 1 h at 37°C and resolved at 4°C on an 8% native polyacrylamide gel containing 1 X TBE and 5 mM MgCl2. Gels are dried and DNA visualized by
phosphorimaging.
IV. Template Nucleic Acids (Genome Editing Approaches)
The terms "template nucleic acid" and "swap nucleic acid" are used interchangeably and have identical meaning in this document and its priority documents.
[0558] Mutations in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VH- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII- 25, IX- 1 , IX- 1 A, D<-2, IX-3, XIV- 1 , or Section VIII, may be corrected using one of the approaches discussed herein. In an embodiment, a mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VH- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX-1 A, IX-2, IX-3, XIV- 1 , or Section VIII, is corrected by homology directed repair (HDR) using a template nucleic acid (see Section IV. 1 ). In an embodiment, a mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII-17, VII- 18, VII- 19, VII-20, VII-21 , VII- 22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XIV- 1 , or Section VIII, is corrected by Non-Homologous End Joining (NHEJ) repair using a template nucleic acid (see Section IV.2).
IV.l HDR Repair and Template Nucleic Acids
[0559] As described herein, nuclease-induced homology directed repair (HDR) can be used to alter a target sequence and correct (e.g., repair or edit) a mutation in the genome. W ile not wishing to be bound by theory, it is believed that alteration of the target sequence occurs by homology-directed repair (HDR) with a donor template or template nucleic acid. For example, the donor template or the template nucleic acid provides for alteration of the target sequence. It is contemplated that a plasmid donor can be used as a template for homologous recombination. It is further contemplated that a single stranded donor template can be used as a template for alteration of the target sequence by alternate methods of homology directed repair (e.g., single strand annealing) between the target sequence and the donor template. Donor template-effected alteration of a target sequence depends on cleavage by a Cas9 molecule. Cleavage by Cas9 can comprise a double strand break or two single strand breaks.
[0560] In an embodiment, a mutation can be corrected by either a single double-strand break or two single strand breaks. In an embodiment, a mutation can be corrected by (1 ) a single double- strand break, (2) two single strand breaks, (3) two double stranded breaks with a break occurring on each side of the target sequence, (4) one double stranded breaks and two single strand breaks with the double strand break and two single strand breaks occurring on each side of the target sequence or (5) four single stranded breaks with a pair of single stranded breaks occurring on each side of the target sequence.
Double strand break mediated correction
[0561 ] In an embodiment, double strand cleavage is effected by a Cas9 molecule having cleavage activity associated with an HNH-like domain and cleavage activity associated with a RuvC-like domain, e.g., an N-termina] RuvC-like domain, e.g., a wild type Cas9. Such embodiments require only a single gRNA. Single strand break mediated correction
[0562] In other embodiments, two single strand breaks, or nicks, are effected by a Cas9 molecule having nickase activity, e.g., cleavage activity associated with an HNH-like domain or cleavage activity associated with an N-terminal RuvC-like domain. Such embodiments require two gRNAs, one for placement of each single strand break. In an embodiment, the Cas9 molecule having nickase activity cleaves the strand to which the gRNA hybridizes, but not the strand that is complementary to the strand to which the gRNA hybridizes. In an embodiment, the Cas9 molecule having nickase activity does not cleave the strand to which the gRNA hybridizes, but rather cleaves the strand that is complementary to the strand to which the gRNA hybridizes.
[0563] In an embodiment, the nickase has HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D 10, e.g., the D I OA mutation. D I OA inactivates RuvC; therefore, the Cas9 nickase has (only) HN H activity and will cut on the strand to which the gRNA hybridizes (e.g., the complementary strand, which does not have the NGG PAM on it). In other embodiments, a Cas9 molecule having an H840, e.g., an H840A, mutation can be used as a nickase. H840A inactivates HNH; therefore, the Cas9 nickase has (only) RuvC activity and cuts on the non-complementary strand (e.g., the strand that has the NGG PAM and whose sequence is identical to the gRNA).
[0564] In an embodiment, in which a nickase and two gRNAs are used to position two single strand nicks, one nick is on the + strand and one nick is on the - strand of the target nucleic acid. The PAMs are outwardly facing. The gRNAs can be selected such that the gRNAs are separated by, from about 0-50, 0- 100, or 0-200 nucleotides. In an embodiment, there is no overlap between the target sequence that is complementary to the targeting domains of the two gRNAs. In an embodiment, the gRNAs do not overlap and are separated by as much as 50, 100, or 200 nucleotides. In an embodiment, the use of two gRNAs can increase specificity, e.g., by decreasing off-target binding (Ran el cil., CELL 20 13).
[0565] In an embodiment, a single nick can be used to induce HDR. It is contemplated herein that a single nick can be used to increase the ratio of HR to NHEJ at a given cleavage site. Placement of the double strand break or a single strand break relative to target position
[0566] The double strand break or single strand break in one of the strands should be sufficiently close to target position such that correction occurs. In an embodiment, the distance is not more than 50, 100, 200, 300, 350 or 400 nucleotides. While not wishing to be bound by theory, it is believed that the break should be sufficiently close to target position such that the break is within the region that is subject to exonuclease-mediated removal during end resection. If the distance between the target position and a break is too great, the mutation may not be included in the end resection and, therefore, may not be corrected, as donor sequence may only be used to correct sequence within the end resection region.
[0567] In an embodiment, in which a gRNA (unimolecular (or chimeric) or modular gRNA) and Cas9 nuclease induce a double strand break for the purpose of inducing HDR-mediated correction, the cleavage site is between 0-200 bp (e.g., 0 to 175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 1 25, 75 to 100 bp) away from the target position. In an embodiment, the cleavage site is between 0- 100 bp (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the target position.
[0568] In an embodiment, in which two gRNAs (independently, unimolecular (or chimeric) or modular gRNA) complexing with Cas9 nickases induce two single strand breaks for the purpose of inducing HDR-mediated correction, the closer nick is between 0-200 bp (e.g., 0 to 175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp) away from the target position and the two nicks will ideally be within 25-55 bp of each other (e.g., 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 55, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 55, 35 to 50, 35 to 45, 35 to 40, 40 to 55, 40 to 50, 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 bp away from each other). In an embodiment, the cleavage site is between 0- 100 bp (e.g., 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the target position. [0569] In one embodiment, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double-strand break on both sides of a target position. In an alternate embodiment, three gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (i.e., one gRNA complexes with a cas9 nuclease) and two single strand breaks or paired single stranded breaks (i.e., two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5') of the target positionand the second gRNA is used to target downstream (i.e., 3') of the target position). In another embodiment, four gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single stranded breaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5') of the target position and the second gRNA is used to target downstream (i.e., 3') of the target position). The double strand break(s) or the closer of the two single strand nicks in a pair will . ideally be within 0-500 bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, l OO, 50 or 25 bp from the target position). When nickases are used, the two nicks in a pair are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35. to 50, 40 to 50 , 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).
[0570] In one embodiment, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double-strand break on both sides of a target position. In an alternate embodiment, three gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (i.e., one gRNA complexes with a cas9 nuclease) and two single strand breaks or paired single stranded breaks (i.e., two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5') of the mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII- 20, VII-21 , VII-22, VII-23, VU-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XIV- 1 , or Section VIII and the second gRNA is used to target downstream (i.e., 3') of the mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, IX-2, IX-3, XIV- 1 , or Section VIII). In another embodiment, four gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single stranded breaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5') of the mutation in a gene or pathway described herein, and the second gRNA is used to target downstream (i.e., 3') of the mutation in a gene or pathway described herein). The double strand break(s) or the closer of the two single strand nicks in a pair will ideally be within 0-500 bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50 or 25 bp from the target position). When nickases are used, the two nicks in a pair are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50 , 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).
Length of the homology arms
[0571] The homology arm should extend at least as far as the region in which end resection may occur, e.g., in order to allow the resected single stranded overhang to find a complementary region within the donor template. The overall length could be limited by parameters such as plasmid size or viral packaging limits. In an embodiment, a homology arm does not extend into repeated elements, e.g., ALU repeats, LINE repeats.
[0572] Exemplary homology arm lengths include a least 50, 100, 250, 500, 750 or 1000 nucleotides.
[0573] Target position, as used herein, refers to a site on a target nucleic acid (e.g., the chromosome) that is modified by a Cas9 molecule-dependent process. For example, the target position can be a modified Cas9 molecule cleavage of the target nucleic acid and template nucleic acid directed modification, e.g., correction, of the target position. In an embodiment, a target position can be a site between two nucleotides, e.g., adjacent nucleotides, on the target nucleic acid into which one or more nucleotides is added. The target position may comprise one or more nucleotides that are altered, e.g., corrected, by a template nucleic acid. In an embodiment, the target position is within a target sequence (e.g., the sequence to which the gRN A binds). In an embodiment, a target position is upstream or downstream of a target sequence (e.g., the sequence to which the gRNA binds).
[0574] A template nucleic acid, as that term is used herein, refers to a nucleic acid sequence which can be used in conjunction with a Cas9 molecule and a gRNA molecule to alter the structure of a target position. In an embodiment, the target nucleic acid is modified to have some or all of the sequence of the template nucleic acid, typically at or near cleavage site(s). In an embodiment, the template nucleic acid is single stranded. In an alternate embodiment, the tempolate nuceic acid is double stranded. In an embodiment, the template nucleic acid is DNA, e.g., double stranded DNA. In an alternate embodiment, the template nucleic acid is single stranded DNA.
[0575] In an embodiment, the template nucleic acid alters the structure of the target position by participating in a homology directed repair event. In an embodiment, the template nucleic acid alters the sequence of the target position. In an embodiment, the template nucleic acid results in the incorporation of a modified, or non-naturally occurring base into the target nucleic acid.
[0576] Typically, the template sequence undergoes a breakage mediated or catalyzed recombination with the target sequence. In an embodiment, the template nucleic acid includes sequence that corresponds to a site on the target sequence that is cleaved by an eaCas9 mediated cleavage event. In an embodiment, the template nucleic acid includes sequence that corresponds to both, a first site on the target sequence that is cleaved in a first Cas9 mediated event, and a second site on the target sequence that is cleaved in a second Cas9 mediated event.
[0577] In an embodiment, the template nucleic acid can include sequence which results in an alteration in the coding sequence of a translated sequence, e.g., one which results in the substitution of one amino acid for another in a protein product, e.g., transforming a mutant allele into a wild type allele, transforming a wild type allele into a mutant allele, and/or introducing a stop codon, insertion of an amino acid residue, deletion of an amino acid residue, or a nonsense mutation.
[0578] In other embodiments, the template nucleic acid can include sequence which results in an alteration in a non-coding sequence, e.g., an alteration in an exon or in a 5' or 3' non-translated or non-transcribed region. Such alterations include
an alteration in a control element, e.g., a promoter, enhancer, and an alteration in a cis-acting or trans-acting control element.
[0579] A template nucleic acid having homology with a target position in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table Vll- 13, VII- 14, VII- 1 5, VII- 16, VII- 17, VlI- 18, VII- 19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1, IX- IA, IX-2, IX-3, XIV- 1 , or Section VIII, can be used to alter the structure of a target sequence. The template sequence can be used to alter an unwanted structure, e.g., an unwanted or mutant nucleotide.
[0580] The template nucleic acid can include sequence which, when integrated, results in:
decreasing the activity of a positive control element;
increasing the activity of a positive control element;
decreasing the activity of a negative control element;
increasing the activityof a negative control element;
decreasing the expression of a gene;
increasing the expression of a gene;
increasing resistance to a disorder or disease;
increasing resistance to viral entry;
correcting a mutation or altering an unwanted amino acid residue
conferring, increasing, abolishing or decreasing a biological property of a gene product, e.g., increasing the enzymatic activity of an enzyme, or increasing the ability of a gene product to interact with another molecule.
[0581] The template nucleic acid can include sequence which results in:
a change in sequence of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or more nucleotides of the target sequence.
[0582] In an embodiment, the template nucleic acid is 20+/- 10, 30+/- 10, 40+/- 10, 50+/- 10, 60+/- 10, 70+/- 10, 80+/- 10, 90+/- 10, 100+/- 10, 1 10+/- 10, 120+/- 10, 130+/- 10, 140+/- 10, 150+/- 10, 160+/- 10, 170+/- 10, 1 80+/- 10, 190+/- 10, 200+/- 10, 210+/-10, of 220+/- 10 nucleotides in length.
[0583] In an embodiment, the template nucleic acid is 30+/-20, 40+/-20, 50+/-20, 60+/-20, 70+/- 20, 80+/-20, 90+/-20, 100+/-20, 1 10+/-20, 120+/-20, 130+/-20, 140+/-20, I 50+/-20, 160+/-20, 170+/-20, 180+/-20, 190+/-20, 200+/-20, 210+/-20, of 220+/-20 nucleotides in length. [0584] In an embodiment, the template nucleic acid is 10 to 1 ,000, 20 to 900, 30 to 800, 40 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to300, 50 to 200, or 50 to 100 nucleotides in length.
[0585] A template nucleic acid comprises the following components:
[5' homology arm]-[replacement sequence]-[3' homology arm].
[0586] The homology arms provide for recombination into the chromosome, thus replacing the undesired element, e.g., a mutation or signature, with the replacement sequence. In an embodiment, the homology arms flank the most distal cleavage sites.
[0587] In an embodiment, the 3' end of the 5' homology arm is the position next to the 5' end of the replacement sequence. In an embodiment, the 5' homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5' from the 5' end of the replacement sequence.
[0588] In an embodiment, the 5' end of the 3' homology arm is the position next to the 3' end of the replacement sequence. In an embodiment, the 3' homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 3' from the 3' end of the replacement sequence.
[0589] It is contemplated herein that one or both homology arms may be shortened to avoid including certain sequence repeat elements, e.g., Alu repeats, LINE elements. For example, a 5' homology arm may be shortened to avoid a sequence repeat element. In other embodiments, a 3' homology arm may be shortened to avoid a sequence repeat element. In some embodiments, both the 5' and the 3' homology arms may be shortened to avoid including certain sequence repeat elements.
[0590] It is contemplated herein that template nucleic acids for correcting a mutation may designed for use as a single-stranded oligonucleotide (ssODN). When using a ssODN, 5' and 3' homology arms may range up to about 200 base pairs (bp) in length, e.g., at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in length. Longer homology arms are also contemplated for ssODNs as improvements in oligonucleotide synthesis continue to be made.
[0591] In an embodiment, an ssODN may be used to correct a mutation in a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 1 , VII- 16, VII- 1 7, Vll- 1 8, VI1- 19, VII-20, Vll-21 , Vll-22, VII-23, VII-24, VII-25, IX- 1 , 1X- 1A, IX-2, 1X-3, XlV- 1 , or Section VIII.
IV.2 NHEJ Approaches for Gene Targeting
[0592] As described herein, nuclease-induced non-homologous end-joining (NHEJ) can be used to target gene-specific knockouts. Nuclease-induced NHEJ can also be used to remove (e.g., delete) sequence in a gene of interest.
[0593] While not wishing to be bound by theory, it is believed that, in an embodiment, the genomic alterations associated with the methods described herein rely on nuclease-induced NHEJ and the error-prone nature of the NHEJ repair pathway. NHEJ repairs a double-strand break in the DNA by joining together the two ends; however, generally, the original sequence is restored only if two compatible ends, exactly as they were formed by the double-strand break, are perfectly ligated. The DNA ends of the double-strand break are frequently the subject of enzymatic processing, resulting in the addition or removal of nucleotides, at one or both strands, prior to rejoining of the ends. This results in the presence of insertion and/or deletion (indel) mutations in the DNA sequence at the site of the NHEJ repair. Two-thirds of these mutations typically alter the reading frame and, therefore, produce a non-functional protein. Additionally, mutations that maintain the reading frame, but which insert or delete a significant amount of sequence, can destroy functionality of the protein. This is locus dependent as mutations in critical functional domains are likely less tolerable than mutations in non-critical regions of the protein.
[0594] The indel mutations generated by NHEJ are unpredictable in nature; however, at a given break site certain indel sequences are favored and are over represented in the population, likely due to small regions of microhomology. The lengths of deletions can vary widely; most .
commonly in the 1 -50 bp range, but they can easily reach greater than 100-200 bp. Insertions tend to be shorter and often include short duplications of the sequence immediately surrounding the break site. However, it is possible to obtain large insertions, and in these cases, the inserted sequence has often been traced to other regions of the genome or to plasmid DNA present in the cells. [0595] Because N HEJ is a mutagenic process, it can also be used to delete small sequence motifs as long as the generation of a specific final sequence is not required. If a double-strand break is targeted near to a short target sequence, the deletion mutations caused by the NHEJ repair often span, and therefore remove, the unwanted nucleotides. For the deletion of larger DNA segments, introducing two double-strand breaks, one on each side of the sequence, can result in NHEJ between the ends with removal of the entire intervening sequence. Both of these approaches can be used to delete specific DNA sequences; however, the error-prone nature of NHEJ may still produce indel mutations at the site of repair.
[0596] Both double strand cleaving eaCas9 molecules and single strand, or nickase, eaCas9 molecules can be used in the methods and compositions described herein to generate NHEJ- mediated indels. NHEJ-mediated indels targeted to the gene, e.g., a coding region, e.g., an early coding region of a gene of interest can be used to knockout (i.e., eliminate expression of) a gene■ of interest. For example, early coding region of a gene of interest includes sequence
immediately following a transcription start site, within a first exon of the coding sequence, or within 500 bp of the transcription start site (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp).
Placement of double strand or single strand breaks relative to the target position
[0597] In an embodiment, in which a gRNA and Cas9 nuclease generate a double strand break for the purpose of inducing NHEJ-mediated indels, a gRNA, e.g., a unimolecular (or chimeric) or modular gRNA molecule, is configured to position one double-strand break in close proximity to a nucleotide of the target position. In an embodiment, the cleavage site is between 0-500 bp away from the target position (e.g., less than 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from the target position).
[0598] In an embodiment, in which two gRNAs complexing with Cas9 nickases induce two single strand breaks for the puipose of inducing NHEJ-mediated indels, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position two single-strand breaks to provide for NHEJ repair a nucleotide of the target position. In an embodiment, the gRNAs are configured to position cuts at the same position, or within a few nucleotides of one another, on different strands, essentially mimicking a double strand break. In an embodiment, the closer nick is between 0-30 bp away from the target position (e.g., less than 30, 25, 20, 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 bp from the target position), and the two nicks are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50 , 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp). In an embodiment, the gRNAs are configured to place a single strand break on either side of a nucleotide of the target position.
[0599] Both double strand cleaving eaCas9 molecules and single strand, or nickase, eaCas9 molecules can be used in the methods and compositions described herein to generate breaks both sides of a target position. Double strand or paired single strand breaks may be generated on both sides of a target position (e.g., of a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VII- 13, VII- 14, VII- 15, VII- 16, VII- 17, VII- 18, VII-19, VII-20, VII-21 , VII-22, VII-23, VII-24, VII-25, IX- 1 , IX- 1 A, ΓΧ-2, IX-3, XIV- 1 , or Section VIII) to remove the nucleic acid sequence between the two cuts (e.g., the region between the two breaks is deleted). In one embodiment, two gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double-strand break on both sides of a target position (e.g., the first gRNA is used to target upstream (i.e., 5') of the mutation in a gene or pathway described herein, and the second gRNA is used to target downstream (i.e., 3') of the mutation in a gene or pathway described herein). In an alternate embodiment, three gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break (i.e., one gRNA complexes with a cas9 nuclease) and two single strand breaks or paired single stranded breaks (i.e., two gRNAs complex with Cas9 nickases) on either side of a target position (e.g., the fu st gRNA is used to target upstream (i.e., 5') of the mutation in a gene or pathway described herein, and the second gRNA is used to target downstream (i.e., 3') of the mutation in a gene or pathway described herein). In another embodiment, four gRNAs, e.g., independently, unimolecular (or chimeric) or modular gRNA, are configured to generate two pairs of single stranded breaks (i.e., two pairs of two gRNAs complex with Cas9 nickases) on either side of the target position (e.g., the first gRNA is used to target upstream (i.e., 5') of the mutation in a gene or pathway described herein, and the second gRNA is used to target downstream (i.e., 3') of the mutation in a gene or pathway described herein). The double strand break(s) or the closer of the two single strand nicks in a pair will ideally be within 0-500 bp of the target position (e.g., no more than 450, 400, 350, 300, 250, 200, 150, 100, 50 or 25 bp from the target position). When nickases are used, the two nicks in a pair are within 25-55 bp of each other (e.g., between 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 50 to 55, 45 to 55, 40 to 55, 35 to 55, 30 to 55, 30 to 50, 35 to 50, 40 to 50 , 45 to 50, 35 to 45, or 40 to 45 bp) and no more than 100 bp away from each other (e.g., no more than 90, 80, 70, 60, 50, 40, 30, 20 or 10 bp).
IV.3 Targeted Knockdown
[0600] Unlike CRISPR/Cas-mediated gene knockout, which permanently eliminates expression by mutating the gene at the DNA level, CRISPR/Cas knockdown allows for temporary reduction of gene expression through the use of artificial transcription factors. Mutating key residues in both DNA cleavage domains of the Cas9 protein (e.g. the D IOA and H840A mutations) results in the generation of a catalytically inactive Cas9 (eiCas9 which is also known as dead Cas9 or dCas9). A catalytically inactive Cas9 complexes with a gRNA and localizes to the DNA sequence specified by that gRNA's targeting domain, however, it does not cleave the target DNA. Fusion of the dCas9 to an effector domain, e.g., a transcription repression domain, enables recruitment of the effector to any DNA site specified by the gRNA. While it has been show that the eiCas9 itself can block transcription when recruited to early regions in the coding sequence, more robust repression can be achieved by fusing a transcriptional repression domain (for example RA B, SID or ERD) to the Cas9 and recruiting it to the promoter region of a gene. It is likely that targeting DNAsel hypersensitive regions of the promoter may yield more efficient gene repression or activation because these regions are more likely to be accessible to the Cas9 protein and are also more likely to harbor sites for endogenous transcription factors. Especially for gene repression, it is contemplated herein that blocking the binding site of an endogenous transcription factor would aid in downregulating gene expression. In another embodiment, an eiCas9 can be fused to a chromatin modifying protein. Altering chromatin status can result in decreased expression of the target gene.
[0601 ] In an embodiment, a gRNA molecule can be targeted to a known transcription response elements (e.g., promoters, enhancers, etc.), a known upstream activating sequences (UAS), and/or sequences of unknown or known function that are suspected of being able to control expression of the target DNA. [0602] CRISPR/Cas-mediated gene knockdown can be used to reduce expression of an unwanted allele or transcript. Contemplated herein are scenarios wherein permanent destruction of the gene is not ideal. In these scenarios, site-specific repression may be used to temporarily reduce or eliminate expression. It is also contemplated herein that the off-target effects of a Cas- repressor may be less severe than those of a Cas:nuclease as a nuclease can cleave any DNA sequence and cause mutations whereas a Cas-repressor may only have an effect if it targets the promoter region of an actively transcribed gene. However, while nuclease-mediated knockout is pennanent, repression may only persist as long as the Cas-repressor is present in the cells. Once the repressor is no longer present, it is likely that endogenous transcription factors and gene regulatory elements would restore expression to its natural state.
IV.4 Examples of gRNAs in Genome Editing Methods
[0603] gRNA molecules as described herein can be used with Cas9 molecules that generate a double strand break or a single strand break to alter the sequence of a target nucleic acid, e.g., a target position or target genetic signature. gRNA molecules useful in these methods are described below.
[0604] In an embodiment, the gRNA, e.g., a chimeric gRNA, is configured such that it comprises one or more of the following properties;
a) it can position, e.g., when targeting a Cas9 molecule that makes double strand breaks, a double strand break (i) within 50, 100, 150 or 200 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection;
b) it has a targeting domain of at least 17 nucleotides, e.g., a targeting domain of (i) 17, (ii) 18, or (iii) 20 nucleotides; and
c)
(i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S.
pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail and proximal domain, or a sequence that differs by no more than 1, 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom; (ii) there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilics, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1 , 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
(iii) there are at least 16, 19, 21 , 26, 31 , 32, 36, 41, 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S.
thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain; or, or a sequence that differs by no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; or
(v) the tail domain comprises 1 , 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain.
[0605] In an embodiment, the gRNA is configured such that it comprises properties: a and b(i).
[0606] In an embodiment, the gRNA is configured such that it comprises properties: a and b(iij.
[0607] In an embodiment, the gRNA is configured such that it comprises properties: a and b(iii).
[0608] In an embodiment, the gRNA is configured such that it comprises properties: a and c.
[0609] In an embodiment, the gRNA is configured such that in comprises properties: a, b, and c.
[0610] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(i). [0611 ] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(ii).
[0612] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(i).
[0613] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(ii).
[0614] In an embodiment, the gRNA, e.g., a chimeric gRNA, is configured such that it comprises one or more of the following properties;
a) it can position, e.g., when targeting a Cas9 molecule that makes single strand breaks, a single strand break (i) within 50, 100, 150 or 200 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection;
b) it has a targeting domain of at least 17 nucleotides, e.g., a targeting domain of (i) 1 7, (ii) 1 8, or (iii) 20 nucleotides; and
c)
(i) the proximal and tail domain, when taken together, comprise at least 15, 18, 20, 25, 30, 3 1 , 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S.
pyogenes, S. tkermophilus, S. aureus, or N. meningitidis tail and proximal domain, or a sequence that di ffers by no more than I , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
(ii) there are at least 1 5, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. tkermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
(iii) there are at least 16, 1 , 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides
1 2 1 i from the corresponding sequence of a naturally occurring S. pyogenes^ S.
thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1 , 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom;
iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain; or, a sequence that differs by no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; or
(v) the tail domain comprises 15, 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain.
[0615] In an embodiment, the gRNA is configured such that it comprises properties: a and b(i).
[0616] In an embodiment, the gRNA is configured such that it comprises properties: a and b(ii).
[0617] In an embodiment, the gRNA is configured such that it comprises properties: a and b(iii).
[0618] In an embodiment, the gRNA is configured such that it comprises properties: a and c.
[0619] In an embodiment, the gRNA is configured such that in comprises properties: a, b, and c.
[0620] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(i).
[0621] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(i), and c(ii).
[0622] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(i).
[0623] In an embodiment, the gRNA is configured such that in comprises properties: a(i), b(iii), and c(ii).
[0624] In an embodiment, the gRNA is used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at D 10, e.g., the D 10A mutation. [0625] In an embodiment, the gRNA is used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., a H840A.
[0626] In an embodiment, a pair of gRNAs, e.g., a pair of chimeric gRNAs, comprising a first and a second gRNA, is configured such that they comprises one or more of the following properties;
a) one or both of the gRNAs can position, e.g., when targeting a Cas9 molecule that makes single strand breaks, a single strand break within (i) 50, 100, 150 or 200 nucleotides of a target position, or (ii) sufficiently close that the target position is within the region of end resection;
b) one or both have a targeting domain of at least 17 nucleotides, e.g., a targeting domain of (i) 17 or, (ii) 18 nucleotides;
c) for one or both: <
(i) the proximal and tail domain, when taken together, comprise at least 1 , 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides, e.g., at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides from a naturally occurring S.
pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail and proximal domain, or a sequence that differs by no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
(ii) there are at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides 3' to the last nucleotide of the second complementarity domain, e.g., at least 15, 18, 20, 25, 30, 31 , 35, 40, 45, 49, 50, or 53 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRNA, or a sequence that differs by no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom;
(iii) there are at least 16, 19, 21 , 26, 31 , 32, 36, 41 , 46, 50, 51 , or 54 nucleotides 3' to the last nucleotide of the second complementarity domain that is complementary to its corresponding nucleotide of the first complementarity domain, e.g., at least 16, 19, 21, 26, 31, 32, 36, 41 , 46, 50, 51 , or 54 nucleotides from the corresponding sequence of a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis gRN A, or a sequence that differs by no more than 1 , 2, 3, 4, 5; 6, 7, 8, 9 or 10 nucleotides therefrom; iv) the tail domain is at least 10, 15, 20, 25, 30, 35 or 40 nucleotides in length, e.g., it comprises at least 10, 15, 20, 25, 30, 35 or 40 nucleotides from a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain; or, or a sequence that differs by no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides therefrom; or
(v) the tail domain comprises 1 , 20, 25, 30, 35, 40 nucleotides or all of the corresponding portions of a naturally occurring tail domain, e.g., a naturally occurring S. pyogenes, S. thermophilus, S. aureus, or N. meningitidis tail domain; d) the gRNAs are configured such that, when hybridized to target nucleic acid, they are separated by 0-50, 0- 100, 0-200, at least 10, at least 20, at least 30 or at least 50 nucleotides; e) the breaks made by the first gRNA and second gRNA are on different strands; and f) the PAMs are facing outwards.
[0627] In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(i).
[0628] In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(ii).
[0629] In an embodiment, one or both of the gRNAs is configured such that it comprises properties: a and b(iii).
[0630] In an embodiment, one or both of the gRNAs configured such that it comprises properties: a and c.
[0631] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a, b, and c.
[0632] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), and c(i).
[0633] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), and c(ii). [0634] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), c, and d.
[0635] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), c, and e.
[0636] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(i), c, d, and e.
[0637]In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), and c(i).
[0638] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), and c(ii).
[0639] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), c, and d.
[0640] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), c, and e.
[0641 ] In an embodiment, one or both of the gRNAs is configured such that in comprises properties: a(i), b(iii), c, d, and e.
[0642] In an embodiment, the gRNAs are used with a Cas9 nickase molecule having HNH activity, e.g., a Cas9 molecule having the'RuvC activity inactivated, e.g., a Cas9 molecule having a mutation at DIO, e.g., the DI OA mutation.
[0643] In an embodiment, the gRNAs are used with a Cas9 nickase molecule having RuvC activity, e.g., a Cas9 molecule having the HNH activity inactivated, e.g., a Cas9 molecule having a mutation at H840, e.g., a H840A.
V. Constructs/Components
[0644] The components, e.g., a Cas9 molecule or gRNA molecule, or both, can be delivered, formulated, or administered in a variety of forms, see, e.g., Table V- l a and Table V- l b. When a component is delivered encoded in DNA the DNA will typically include a control region, e.g., comprising a promoter, to effect expression. Useful promoters for Cas9 molecule sequences include CMV, EF- l a, MSCV, PGK, CAG control promoters. Useful promoters for gRNAs include H 1 , EF- 1 a and U6 promoters. Promoters with similar or dissimilar strengths can be selected to tune the expression of components. Sequences encoding a Cas9 molecule can comprise a nuclear localization signal (NLS), e.g., an SV40 NLS. In an embodiment, a promoter for a Cas9 molecule or a gRNA molecule can be, independently, inducible, tissue specific, or cell specific.
[0645] Table V-la' and Table V-lb provide examples of how the components can be formulated, delivered, or administered.
Table V-la
Figure imgf000127_0001
In an embodiment, the mRNA comprises one or more modifications, e.g., as described in Section X.
Protein DNA DNA In this embodiment a Cas9 molecule, typically an eaCas9 molecule, is provided as a protein. A gRNA is transcribed from DNA.
Protein RNA DNA In this embodiment an eaCas9 molecule is provided as a protein. A gRNA is provided as RNA. In an embodiment, the gRNA comprises one or more modifications, e.g., as described in Section X.
Table V-l b
Figure imgf000128_0001
embodiment, the gRNA comprises one or more modifications, e.g., as described in Section X. In an embodiment, the mRNA comprises one or more modifications, e.g., as described in section X.
Protein DNA Yes In this embodiment a Cas9 molecule, typically an eiCas9 molecule, is provided as a protein. A gRNA is provided encoded in DNA.
Protein RNA Yes In this embodiment a Cas9 molecule, typically an eiCas9 molecule, is provided as a protein. A gRNA is provided as RNA. In an embodiment, the gRNA comprises one or more modifications, e.g., as described in Section X.
DNA-based Delivery of a Cas9 molecule and or a gRNA molecule
[0646] DNA encoding Cas9 molecules (e.g., eaCas9 molecules or eiCas9 molecules) and/or gRNA molecules, can be administered to subjects or delivered into cells by art-known methods or as described herein. For example, Cas9-encoding and/or gRNA-encoding DNA can be delivered, e.g., by vectors (e.g., viral or non-viral vectors), non-vector based methods (e.g., using naked DNA or DNA complexes), or a combination thereof.
[0647] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a vector (e.g., viral vector/virus or plasmid).
[0648] A vector can comprise a sequence that encodes a Cas9 molecule and/or a gRNA molecule. A vector can also comprise a sequence encoding a signal peptide (e.g., for nuclear local ization, nucleolar local ization, mitochondrial localization), fused, e.g., to a Cas9 molecule sequence. For example, a vector can comprise a nuclear localization sequence (e.g., from SV40) fused to the sequence encoding the Cas9 molecule.
[0649] One or more regulatory/control elements, e.g., a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, internal ribosome entry sites (IRES), a 2A sequence, and a splice acceptor or donor can be included in the vectors. In some embodiments, the promoter is recognized by RNA polymerase II (e.g., a CMV promoter). In other embodiments, the promoter is recognized by RNA polymerase 111 (e.g., a U6 promoter). In some embodiments, the promoter is a regulated promoter (e.g., inducible promoter). In other embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue specific promoter. In some embodiments, the promoter is a viral promoter. In other embodiments, the promoter is a non-viral promoter.
[0650] In some embodiments, the vector or delivery vehicle is a viral vector (e.g., for generation of recombinant viruses). In some embodiments, the virus is a DNA virus (e.g., dsDNA or ssDNA vims). In other embodiments, the vims is an RNA vims (e.g., an ssRNA vims).
Exemplary viral vectors/viruses include, e.g., retroviruses, lentivimses, adenovirus, adeno- associated virus (AAV), vaccinia vimses, poxviruses, and heipes simplex viruses.
[0651] In some embodiments, the vims infects dividing cells. In other embodiments, the vims infects non-dividing cells. In some embodiments, the vims infects both dividing and non- dividing cells. In some embodiments, the vims can integrate into the host genome. In some embodiments, the vims is engineered to have reduced immunity, e.g., in human. In some embodiments, the vims is replication-competent. In other embodiments, the vims is replication- defective, e.g., having one or more coding regions for the genes necessary for additional rounds of virion replication and/or packaging replaced with other genes or deleted. In some
embodiments, the virus causes transient expression of the Cas9 molecule and/or the gRN A molecule. In other embodiments, the vims causes long-lasting, e.g., at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, or permanent expression, of the Cas9 molecule and/or the gRN A molecule. The packaging capacity of the vimses may vary, e.g., from at least about 4 kb to at least about 30 kb, e.g., at least about 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, or 50 kb.
[0652] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant retrovirus. In some embodiments, the retrovirus (e.g., Moloney murine leukemia vims) comprises a reverse transcriptase, e.g., that allows integration into the host genome. In some embodiments, the retrovirus is replication-competent. In other embodiments, the retrovirus is replication-defective, e.g., having one of more coding regions for the genes necessary for additional rounds of virion replication and packaging replaced with other genes, or deleted. [0653] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant lentivirus. For example, the lentivirus is replication-defective, e.g., does not comprise one or more genes required for viral replication.
[0654] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant adenovirus. In some embodiments, the adenovirus is engineered to have reduced immunity in human.
[0655] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a recombinant AAV. In some embodiments, the AAV can incorporate its genome into that of a host cell, e.g., a target cell as described herein. In some embodiments, the AAV is a self- complementary adeno-associated virus (scAAV), e.g., a scAAV that packages both strands which anneal together to form double stranded DNA. AAV serotypes that may be used in the disclosed methods include, e.g., AAV l , AAV2, modified AAV2 (e.g., modifications at Y444F, Y500F, Y730F and/or S662V), AAV3, modified AAV3 (e.g., modifications at Y705F, Y73 1 F and/or. T492V), AAV4, AAV5, AAV6, modified AAV6 (e.g., modifications at S663V and/or T492V), AAV8. AAV 8.2, AAV9, AAV rh 10, and pseudotyped AAV, such as AAV2/8, AAV2/5 and AAV2/6 can also be used in the disclosed methods.
[0656] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a hybrid virus, e.g., a hybrid of one or more of the viruses described herein.
[0657] A Packaging cell is used to form a virus particle that is capable of infecting a host or target cell. Such a cell includes a 293 cell, which can package adenovirus, and a ψ2 cell or a PA317 cell, which can package retrovirus. A viral vector used in gene therapy is usually generated by a producer cell line that packages a nucleic acid vector into a viral particle. The vector typically contains the minimal viral sequences required for packaging and subsequent integration into a host or target cell (if applicable), with other viral sequences being replaced by an expression cassette encoding the protein to be expressed. For example, an AAV vector used in gene therapy typically only possesses inverted terminal repeat (ITR) sequences from the AAV genome which are required for packaging and gene expression in the host or target cell. The missing viral functions are supplied in trans by the packaging cell line. Henceforth, the viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
[0658] In an embodiment, the viral vector has the ability of cell type and/or tissue type recognition. For example, the viral vector can be pseudotyped with a different/alternative viral envelope glycoprotein; engineered with a cell type-specific receptor (e.g., geneticmodification of the viral envelope glycoproteins to incorporate targeting ligands such as a peptide ligand, a single chain antibodie, a growth factor); and/or engineered to have a molecular bridge with dual specificities with one end recognizing a viral glycoprotein and the other end recognizing a moiety of the target cell surface (e.g., ligand-receptor, monoclonal antibody, avidin-biotin and chemical conjugation).
[0659] In an embodiment, the viral vector achieves cell type specific expression. For example, a tissue-specific promoter can be constructed to restrict expression of the transgene (Cas 9 and gRNA) in only the target cell. The specificity of the vector can also be mediated by microRNA- dependent control of transgene expression. In an embodiment, the viral vector has increased efficiency of fusion of the viral vector and a target cell membrane. For example, a fusion protein such as fusion-competent hemagglutin (HA) can be incorporated to increase viral uptake into cells. In an embodiment, the viral vector has the ability of nuclear localization. For example, aviruse that requires the breakdown of the cell wall (during cell division) and therefore will not infect a non-diving cell can be altered to incorporate a nuclear localization peptide in the matrix protein of the virus thereby enabling the transduction of non-proliferating cells.
[0660] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a non- vector based method (e.g., using naked DNA or DNA complexes). For example, the DNA can be delivered, e.g., by organically modified silica or silicate (Ormosil), electroporation, gene gun, sonoporation, magnetofection, lipid-mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphates, or a combination thereof.
[0661] In some embodiments, the Cas9- and/or gRNA-encoding DNA is delivered by a combination of a vector and a non-vector based method. For example, a virosome comprises a liposome combined with an inactivated virus (e.g., HIV or influenza virus), which can result in more efficient gene transfer, e.g., in a respiratory epithelial cell than either a viral or a liposomal method alone.
[0662] In an embodiment, the delivery vehicle is a non-viral vector. In an embodiment, the non- viral vector is an inorganic nanoparticle (e.g., attached to the payload to the surface of the nanoparticle). Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., Fe lvln02), or silica. The outer surface of the nanoparticle can be conjugated with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment (e.g., conjugation or entrapment) of payload. In an embodiment, the non-viral vector is an organic nanoparticle (e.g., entrapment of the payload inside the nanoparticle). Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG) and protamine and nucleic acid complex coated with lipid coating.
[0663] Exemplary lipids for gene transfer are shown in Table XII-2.
[0664] Exemplary polymers for gene transfer are shown below in Table XII-3.
[0665] In an embodiment, the vehicle has targeting modifications to increase target cell update of nanoparticles and liposomes, e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars, and cell penetrating peptides. In an embodiment, the vehicle uses fusogenic and endosome-destabilizing peptides/polymers. In an embodiment, the vehicle undergoes acid-triggered conformational changes (e.g., to accelerate endosomal escape of the cargo). In an embodiment, a stimuli-cleavable polymer is used, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.
[0666] In an embodiment, the delivery vehicle is a biological non-viral delivery vehicle. In an embodiment, the vehicle is an attenuated bacterium (e.g., naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis and expressing the transgene (e.g., Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli), bacteria having nutritional and tissue-specific tropism to target specific tissues, bacteria having modified surface proteins to alter target tissue specificity). In an embodiment, the vehicle is a genetically modified bacteriophage (e.g., engineered phages having large packaging capacity, less immunogenic, containing mammalian plasmid maintenance sequences and having incorporated targeting ligands). In an embodiment, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of the "empty" particles followed by ex vivo assembly of the virus with the desired cargo). The vehicle can also be engineered to incorporate targeting ligands to alter target tissue specificity. In an embodiment, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the subject (e.g., tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), or secretory exosomes - subject (i.e., patient) derived membrane-bound nanovescicle (30 - 100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need of for targeting ligands).
[0667] In an embodiment, one or more nucleic acid molecules (e.g., DNA molecules) other than the components of a Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component described herein, are delivered. In an embodiment, the nucleic acid molecule is delivered at the same time as one or more of the components of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered before or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered by a different means than one or more of the components of the Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, e.g., an integration-deficient lentivirus, and the Cas9 molecule component and/or the gRNA molecule component can be delivered by electroporation, e.g., such that the toxicity caused by nucleic acids (e.g., DNAs) can be reduced. In an embodiment, the nucleic acid molecule encodes a therapeutic protein, e.g., a protein described herein. In an embodiment, the nucleic acid molecule encodes an RNA molecule, e.g., an RNA molecule described herein. Delivery of RNA encoding a Cas9 molecule
[0668] RNA encoding Cas9 molecules (e.g., eaCas9 molecules, eiCas9 molecules or eiCas9 fusion proteins) and/or gRNA molecules, can be delivered into cells, e.g., target cells described herein, by art-known methods or as^described herein. For example, Cas9-encoding and/or gRNA-encoding RNA can be delivered, e.g., by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof.
Delivery Cas9 molecule protein
[0669] Cas9 molecules (e.g., eaCas9 molecules, eiCas9 molecules or eiCas9 fusion proteins) can be delivered into cells by art-known methods or as described herein. For example, Cas9 protein molecules can be delivered, e.g., by microinjection, electroporation, lipid-mediated transfection, peptide-mediated delivery, or a combination thereof. Delivery can be accompanied by DNA encoding a gRNA or by a gRNA.
Route of Administration
[0670] Systemic modes of administration include oral and parenteral routes. Parenteral routes include, by way of example, intravenous, intrarterial, intraosseous, intramuscular, intradermal, subcutaneous, intranasal and intraperitoneal routes. Components administered systemically may be modified or formulated to target the components to the eye.
[0671 ] Local modes of administration include, by way of example, , intrathecal,
intracerebroventricular, intraparenchymal (e.g., localized intraparenchymal delivery to the striatum (e.g., into the caudate or into the putamen)), cerebral cortex, precentral gyrus, hippocampus (e.g., into the dentate gyrus or CA3 region), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, hypothalamus, tectum, tegmentum or substantia nigra intraocular, intraorbital, subconjuctival, intravitreal, subretinal or transscleral routes. In an embodiment, significantly smaller amounts of the components (compared with systemic approaches) may exert an effect when administered locally (for example, intraparenchymal or intravitreal) compared to when administered systemically (for example, intravenously). Local modes of administration can reduce or eliminate the incidence of potentially toxic side effects that may occur when therapeutically effective amounts of a component are administered systemically.
[0672] In an embodiment, components described herein are delivered by intraparenchymal injection into discrete regions of the brain, including, e.g., regions comprising medium spiny neurons, or regions comprising cortical neurons. Injections may be made directly into more than one region of the brain.
[0673] In an embodiment, components described herein are delivered by subretinally, e.g., by subretinal injection. Subretinal injections may be made directly into the macular, e.g., submacular injection.
[0674] In an embodiment, components described herein are delivered by intravitreal injection. Intravitreal injection has a relatively low risk of retinal detachment risk. In an embodiment, a nanoparticle or viral vector, e.g., AAV vector, e.g., an AAV2 vector, e.g., a modified AAV2 vector, is delivered intravitreally.
[0675] In an embodiment, a nanoparticle or viral vector, e.g., AAV vector, delivery is via intraparenchymal injection.
[0676] Methods for administration of agents to the eye are known in the medical arts and can be used to administer components described herein. Exemplary methods include intraocular injection (e.g., retrobulbar, subretinal, submacular, intravitreal and intrachoridal), iontophoresis, eye drops, and intraocular implantation (e.g., intravitreal, sub-Tenons and sub-conjunctival).
[0677] Administration may be provided as a periodic bolus (for example, subretinally, intravenously or intravitreally) or as continuous infusion from an internal reservoir (for example, from an implant disposed at an intra- or extra-ocular location (see, U.S. Pat. Nos. 5,443,505 and 5,766,242)) or from an external reservoir (for example, from an intravenous bag). Components may be administered locally, for example, by continuous release from a sustained release drug delivery device immobilized to an inner wall of the eye or via targeted transscleral controlled release into the choroid (see, for example, PCT/USOO/00207, PCT US02/14279, Ambati et al. (2000) INVEST. OPHTHALMOL. VIS. SCI.41 : 1 181- 1 185, and Ambati et al. (2000) INVEST. OPHTHALMOL. VIS. SCI.41 : 1 186-1 191 ). A variety of devices suitable for administering components locally to the inside of the eye are known in the art. See, for example, U.S. Pat. Nos. 6,25 1 ,090, 6,299,895, 6,416,777, 6,413,540, and PCT USOO/28187.
[0678] In addition, components may be formulated to permit release over a prolonged period of time. A release system can include a matrix of a biodegradable material or a material which releases the incorporated components by diffusion. The components can be homogeneously or heterogeneously distributed within the release system. A variety of release systems may be useful, however, the choice of the appropriate system will depend upon rate of release required by a particular application. Both non-degradable and degradable release systems can be used. Suitable release systems include polymers and polymeric matrices, non-polymeric matrices, or inorganic and organic excipients and diluents such as, but not limited to, calcium carbonate and sugar (for example, trehalose). Release systems may be natural or synthetic. However, synthetic release systems are preferred because generally they are more reliable, more reproducible and produce more defined release profiles. The release system material can be selected so that components having different molecular weights are released by diffusion through or degradation of the material.
[0679] Representative synthetic, biodegradable polymers include, for example: polyamides such as poly( amino acids) and poly(peptides); polyesters such as poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), and poly(caprolactone); poly(anhydrides); polyorthoesters;
polycarbonates; and chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. Representative synthetic, non- degradable polymers include, for example: polyethers such as poly(ethylene oxide),
poly(ethylene glycol), and poly(tetramethylene oxide); vinyl polymers-polyacrylates and polymethacrylates such as methyl, ethyl, other alkyl, hydroxyethyl methacrylate, acrylic and methacrylic acids, and others such as poly(vinyl alcohol), poly(vinyl pyrolidone), and poly(vinyl acetate); poly(urethanes); cellulose and its derivatives such as alkyl, hydroxyalkyl, ethers, esters, nitrocellulose, and various cellulose acetates; polysiloxanes; and any chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), copolymers and mixtures thereof. [0680]Poly(lactide-co-glycolide) microsphere can also be used for intraocular injection.
Typically the microspheres are composed of a polymer of lactic acid and glycolic acid, which are structured to form hollow spheres. The spheres can be approximately 15-30 microns in diameter and can be loaded with components described herein.
Bi-Modal or Differential Delivery of Components
[0681 ] Separate delivery of the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component, and more particularly, delivery of the components by differing modes, can enhance performance, e.g., by improving tissue specificity and safety.
[0682] In an embodiment, the Cas9 molecule and the gRNA molecule are delivered by different modes, or as sometimes referred to herein as differential modes. Different or differential modes, as used herein, refer modes of delivery that confer different pharmacodynamic or
pharmacokinetic properties on the subject component molecule, e.g., a Cas9 molecule, gRNA molecule, or template nucleic acid. For example, the modes of delivery can result in different tissue distribution, different half-life, or different temporal distribution, e.g., in a selected compartment, tissue, or organ.
[0683] Some modes of delivery, e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result- in more persistent expression of and presence of a component.
V I. PAYLOADS
[0684] Cas9 molecules, typically eiCas9 molecules and gRNA molecules, e.g., an eiCas9 molecule/gRNA molecule complex, can be used to deliver a wide variety of payloads. In an embodiment, the payload is delivered to target nucleic acids or to chromatin, or other components, near or associated with a target nucleic acid.
[0685] While not wishing to be bound by theory, it is believed that the sequence specificity of the gRNA molecule of an eiCas9 molecule/gRNA molecule complex contributes to a specific interaction with the target sequence, thereby effecting the delivery of a payload associated with, e.g., covalently or noncovalently coupled to, the Cas9 molecule/gRNA molecule complex. [0686] In an embodiment, the payload is covalently or non-covalently coupled to a Cas9, e.g., an eiCas9 molecule. In an embodiment, the payload is covalently or non-covalently coupled to a gRNA molecule. In an embodiment, the payload is linked to a Cas9 molecule, or gRNA molecule, by a linker, e.g., a linker which comprises a bond cleavable under physiological conditions. In other embodiments the bond is not cleavable or is only poorly cleavable, under physiological conditions. In an embodiment, "covalently coupled" means as part of a fusion protein containing a Cas9 molecule.
Delivery of Multiple Payloads
[0687] In an embodiment, a first payload molecule is delivered by a first Cas9 molecule and a second payload molecule is delivered by a second Cas9 molecule. In an embodiment, the first and second payloads are the same. In an embodiment, first and second Cas9 molecules are the same, e.g. are from the same species, have the same PAM, and/or have the same sequence. In an embodiment, first and second Cas9 molecules are different, e.g. are from different species, have the different PAMs, and/or have different sequences. Examples of configurations are provided in Table VI- 1 . Typically the Cas9 molecules of Table VI- 1 are eiCas9 molecules. In other embodiments a Cas9 molecule is selected such that payload delivery and cleavage are both effected. In an embodiment, multiple payloads, e.g., two payloads, is delivered with a single Cas9 molecule.
Table VI-1 : Configurations for delivery of payloads by more than one Cas9
molecule/gRNA molecule complex
Figure imgf000139_0001
second Cas9 molecule are guided by different gRNA molecules.
C I C2 P I PI In this embodiment, the Cas9
molecules are different but each delivers the same payload. In an embodiment, the first and second Cas9 molecule are guided by different gRNA molecules.
C I C2 PI P2 In this embodiment, the Cas9.
molecules are different as are the payloads. In an embodiment, the first and second Cas9 molecule are guided by different gRNA
molecules.
[0688] In an embodiment, two different drugs are delivered. In an embodiment, a first payload, e.g., a drug, coupled by a first linker to a first Cas9 molecule and a second payload, e.g., a drug, coupled by a second linker to a second Cas9 molecule are delivered. In an embodiment, the first and second payloads are the same, and, in an embodiment, are coupled to the respective Cas9 molecule by different linkers, e.g., having different release kinetics. In an embodiment, the first and second payloads are different, and, in an embodiment, are coupled to the respective Cas9 molecule by the same linker. In an embodiment, the first and second payload interact. E.g., the first and second payloads form a complex, e.g., a dimeric or multimeric complex, e.g., a dimeric protein. In an embodiment, the first payload can activate the second payload, e.g., the first payload can modify, e.g., cleave or phosphorylate, the second payload. In an embodiment the first payload interacts with the second payload to modify, e.g., increase or decrease, an activity of the second payload.
[0689] A payload can be delivered in vitro, ex vivo, or in vivo.
Classes of Payloads
[0690] A payload can comprise a large molecule or biologies (e.g., antibody molecules), a fusion protein, an amino acid sequence fused, as a fusion partner, to a Cas9 molecule, e.g., an eiCas9 molecule, an enzyme, a small molecules (e.g., HDAC and other chromatin modifiers/inhibitors, exon skipping molecules, transcription inhibitors), a microsatellite extension inhibitor, a carbohydrate, and DNA degraders (e.g., in an infectious disease or "foreign" DNA setting), a nucleic acid, e.g., a DNA, RNA, mRNA, siRNA, RNAi, or an antisense oligonucleotide.
[0691 ] Table VI-2 provides exemplary classes of payloads. Table Vl-2
Exemplary Classes of Payloads
Large Molecules
Small Molecules
Polymers
Biologies
Proteins and polypeptides, e.g., antibodies, enzymes, structural peptides,
ligands, receptors, fusion proteins, fusion partners (as a fusion protein with
a Cas9, e.g., and eiCas9)
Carbohydrates
HDAC and other chromatin modifiers/inhibitors
Exon skipping molecules,
Transcription inhibitors
Microsatellite extension inhibitors
Entities that degrade DNA
Large Molecules
[0692] In an embodiment a payload comprises a polymer, e.g., a biological polymer, e.g., a protein, nucleic acid, or carbohydrate.
[0693] In an embodiment the payload comprises a protein, biologic, or other large molecule (i.e., a molecule having a molecular weight of at least, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 kD). In an embodiment a payload comprises a polymer, e.g., a biological polymer, e.g., a protein, nucleic acid, or carbohydrate. The polymer can be a naturally occurring or non-naturally occurring polymer. In an embodiment, the payload is a natural product. For example, the natural product can be a large molecule or a small molecule. Polypeptides, Proteins
[0694] In an embodiment the payload comprises a protein or polypeptide, e.g., a protein or polypeptide covalently or non-covalently coupled to a Cas9 molecule.
[0695] In an embodiment, the protein or polypeptide is dimeric or multimeric, and each subunit is delivered by a Cas9 molecule. In an embodiment, a first protein and second protein are delivered by one or more Cas9 molecules, e.g., each by a separate Cas9 molecule or both by the same Cas9 molecule.
[0696] In an embodiment, the protein or polypeptide is linked to a Cas9 molecule by a linker, e.g., a linker which comprises a bond cleavable under physiological conditions. In an embodiment, a linker is a linker from Section XI herein. In an embodiment, the bond is not cleavable under physiological conditions.
Specific Binding Ligands, Antibodies
[0697] In an embodiment the payload comprises a ligand, e.g., a protein, having specific affinity for a counter ligand. In an embodiment, the ligand can be a receptor (or the ligand for a receptor), or an antibody.
[0698] In an embodiment a payload comprises an antibody molecule. Exemplary antibody molecules include, e.g., proteins or polypeptides that include at least one immunoglobulin variable domain. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (de Wildt et al„ Eur J Immunol. 1996; 26(3):629-639)). For example, antigen-binding fragments of antibodies can include, e.g., (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH I domains; (i i) a F(ab')2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH I domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., ( 1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) that retains functionality. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). See, e.g., US patents 5,260,203, 4,946,778, and 4,881 , 175; Bird et al. (1 88) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof)- Antibodies may be from any source, but primate (human and non-human primate) and primatized are preferred. In some embodiments, the antibody is a human antibody or humanized antibody.
[0699] In an embodiment, the antibody molecule is a single-domain antibody (e.g., an sdAb, e.g., a nanobody), e.g., an antibody fragment consisting of a single monomelic variable antibody domain. In an embodiment, the molecular weight of the single-domain antibody is about 12-15 kDa. For example, the single-domain antibody can be engineered from heavy-chain antibodies found in camelids (e.g., VHH fragments). Cartilaginous fishes also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained. An alternative approach is to split the dimeric variable domains from common immunoglobulin G (IgG), e.g., from humans or mice, into monomers. Single-domain antibodies derived from either heavy or light chain can be obtained to bind specifically to target epitopes. For example, a single-domain antibody can be a peptide chain of about 1 10 amino acids long, comprising one variable domain ( VH) of a heavy-chain antibody, or of a common IgG.
[0700] Single-domain antibodies can have similar affinity to antigens as whole antibodies. They can also be more heat-resistant and/or stable towards detergents and high concentrations of urea. Those, e.g., derived from camelid and fish antibodies can be less lipophilic and more soluble in water, owing to their complementarity determining region 3 (CDR3), which forms an extended loop covering the lipophilic site that normally binds to a light chain. In an embodiment, the single-domain antibody does not show complement system triggered cytotoxicity, e.g., because they lack an Fc region. Single-domain antibodies, e.g., camelid and Fish derived sdAbs, can bind to hidden antigens that may not be accessible to whole antibodies, for example to the active sites of enzymes. This property can result from their extended CDR3 loop, which is able to penetrate such sites.
[0701] A single-domain antibody can be obtained by immunization of, e.g., dromedaries, camels, llamas, alpacas or sharks with the desired antigen and subsequent isolation of the mRNA coding for heavy-chain antibodies. By reverse transcription and polymerase chain reaction, a gene library of single-domain antibodies containing several million clones is produced.
Screening techniques like phage display and ribosome display help to identify the clones binding the antigen.
[0702] A different method uses gene libraries from animals that have not been immunized beforehand. Such naive libraries usually contain only antibodies with low affinity to the desired antigen, making it necessary to apply affinity maturation by random mutagenesis as an additional step.
[0703] When the most potent clones have been identified, their DNA sequence can be optimized, for example to improve their stability towards enzymes. Another goal is humanization to prevent immunological reactions of the human organism against the antibody. Humanization is unproblematic because of the homology between, e.g., camelid VHH and human VH fragments. The final step is the translation of the optimized single-domain antibody in E. coli,
Saccharomyces cerevisiae 'or other suitable organisms.
[0704] Alternatively, single-domain antibodies can be made from common murine or human IgG with four chains. The process is similar, comprising gene libraries from immunized or naive donors and display techniques for identification of the most specific antigens. Monomerization is usually accomplished by replacing lipophilic by hydrophilic amino acids. If affinity can be retained, the single-domain antibodies can likewise be produced in E. coli, S. cerevisiae or other organisms.
[0705] In an embodiment, a payload comprises a transcription activator protein or domain, e.g., a VP 16 protein or domain, or a transcription repressor protein or domain. Fusion Proteins and Fusion Partners
[0706] In an embodiment the payload comprises a fusion protein. Exemplary fusion proteins include a first and second fusion partner, which can possess different functional properties or which can be derived from different proteins. In an embodiment, the fusion protein can comprise a first fusion partner that binds a nucleic acid and a second fusion partner that that comprises an enzymatic activity or that promotes or inhibits gene expression. In an embodiment, the payload itself is a fusion protein. In an embodiment, the payload is fused to a Cas9 molecule.
[0707] For example, the fusion protein can contain a segment that adds stability and/or deliverability to the fused protein. In some embodiments, the fusion protein can be a protein described herein (e.g., a receptor) fused to an immunoglobulin fragment (e.g., Fc fragment), transferring, or a plasma protein, e.g., albumin. The fusion protein can also contain a segment that adds toxicity to the fused protein (e.g. conveyed by toxins, enzymes or cytokines). Fusion proteins can also be used to enable delivery and/or targeting routes (e.g., by HIV- 1 TAT protein). Other examples include, e.g., fusions that allow for mutivalency, such as streptavidin fusions, or fusions of two active components (e.g., with or without a cleavable linker in between).
[0708] In an embodiment, the protein or polypeptide is a fusion partner with a Cas9 molecule, e.g., an eiCas9 molecule.
[0709] In an embodiment, a payload comprises fusion partner with a Cas9 molecule comprising a transcription activator protein or domain, e.g., a VP16 protein or domain, or a transcription repressor protein or domain.
Enzymes
[0710] In an embodiment a payload comprises an enzyme. Exemplary enzymes include, e.g., oxidoreductases (e.g., catalyze oxidation/reduction reactions), transferases (e.g., transfer a functional group (e.g. a methyl or phosphate group)), hydrolases (e.g., catalyze the hydrolysis of various bonds), lyases (e.g., cleave various bonds by means other than hydrolysis and oxidation), isomerases (catalyze isomerization changes within a single molecule), and ligases (e.g., join two molecules with covalent bonds). In an embodiment an enzymes mediates or is associated with one or more functions in the cell nucleus, e.g., DNA synthesis, transcription, epigenetic modification of DNA and histones, RNA post-transcriptional modification, cell cycle control, DNA damage repair, or genomic instability.
Small Molecules
[0711] In an embodiment a payload comprises a small molecule compounds.
[0712] In an embodiment a small molecule is a regulator of a biological process. For example, a small molecule can bind to a second molecule, e.g., biopolymer, e.g., a carbohydrate, protein, polypeptide, or a nucleic acid, and in an embodiment, alter one or more of the structure, distribution, activity, or function of the second molecule. In some embodiments, the size of the small molecule is on the order of 10~9 m. In some embodiments, the molecular weight of the small molecule is, e.g., between 200 amu and 500 amu, between 300 amu and 700 amu, between 500 amu and 700 amu, between 700 amu and 900 amu, or between 500 amu and 900 amu.
[0713] Exemplary small molecules include histone deacetylase (HDAC) inhibitors (e.g., suberoylanilide hydroxamic acid (SAHA), or romidepsin), histone methyltransferase inhibitors (, DNA methyltransferase inhibitors (e.g., azacitidine (or 5-azacitidine), decitabine (or 5-aza-2'- deoxycytidine), or DNA replication inhibitors. Small molecules can also include, e.g., small nucleic acid molecules ( 1 -4 bases depending upon the base, e.g., that would be under 2 kD) and peptides.
[0714] Exemplary classes of small molecules that may be used as payloads include, but are not limited to, 5-alpha Reductase Inhibitor, 5-alpha Reductase Inhibitors, 5-Lipoxygenase Inhibitor, 5-Lipoxygenase Inhibitors, Acetyl Aldehyde Dehydrogenase Inhibitors, Acetylcholine Release Inhibitor, Acetylcholine Release Inhibitors, Acetylcholine Releasing Agent, Acidifying Activity, Actinomycin, Actively Acquired Immunity, Adenosine Deaminase, Adenosine Receptor Agonist, Adenosine Receptor Agonists, Adenovirus Vaccines, Adrenal Steroid Synthesis Inhibitor, Adrenal Steroid Synthesis Inhibitors, Adrenergic Agonists, Adrenergic alpha-Agonists, Adrenergic alpha-Antagonists, Adrenergic alpha2-Agonists, Adrenergic beta-Agonists,
Adrenergic beta- Antagonists, Adrenergic beta 1 -Antagonists, Adrenergic beta2- Agonists, Adrenergic beta2-Antagonists, Adrenergic beta3-Agonists, Adrenergic Receptor Agonist, Adrenocorticotropic Hormone, Adrenocorticotropic Hormone, Aldehyde Dehydrogenase Inhibitor, Aldosterone Antagonist, Aldosterone Antagonists, Alkylating Activity, Alkylating Drug, Allergens, Allogeneic Cord Blood Hematopoietic Progenitor Cell Therapy, Allogeneic Cultured Cell Scaffold, Allylamine Antifungal, Allylamine, alpha Glucosidase Inhibitors, alpha- Adrenergic Agonist, alpha-Adrenergic Blocker, alpha-Glucosidase Inhibitor, alpha-Glucosidases, Aluminum Complex, Alveolar Surface Tension Reduction, Amide Local Anesthetic, Amides, Amino Acid Hypertonic Solution, Amino Acid, Amino Acids, Aminoglycoside Antibacterial, Aminoglycosides, Aminoketone, Aminosalicylate, Aminosalicylic Acids, Ammonium Ion Binding Activity, AMPA Receptor Antagonists, Amphenicol-class Antibacterial, Amphenicols, Amphetamine Anorectic, Amphetamines, Amylin Agonists, Amylin Analog, Androgen Receptor Agonists, Androgen Receptor Antagonists, Androgen Receptor Inhibitor, Androgen,
Androstanes, Angiotensin 2 Receptor Antagonists, Angiotensin 2 Receptor Blocker, Angiotensin 2 Type 1 Receptor Antagonists, Angiotensin Converting Enzyme Inhibitor, Angiotensin- converting Enzyme Inhibitors, Ant Venoms, Anthracycline Topoisomerase Inhibitor,
Anthracyclines, Anti-anginal, Anti-coagulant, Anti-epileptic Agent, Anti-IgE, Anti-inhibitor Coagulant Complex, Antiarrhythmic, Antibodies, Monoclonal, Antibody-Surface Protein Interactions, Anticholinergic, Antidiarrheal, Antidiuretic Hormone Antagonists, Antidote for Acetaminophen Overdose, Antidote, Antiemetic, Antifibrinolytic Agent, Antigen Neutralization, Antigens, Bacterial, Antigens, Dermatophagoides, Antigens, Fungal, Antihelminthic,
Antihistamine, Antimalarial, Antimetabolite Immunosuppressant, Antimetabolite,
Antimycobacterial, Antiparasitic, Antiprotozoal, Antirheumatic Agent, Antiseptic, Antitoxins, Antivenin, Antivenins, Appetite Suppression, Aptamers, Nucleotide, Aromatase Inhibitor, Aromatase Inhibitors, Aromatic Amino Acid Decarboxylation Inhibitor, Arteriolar Vasodilation, Arteriolar Vasodilator, Asparaginase, Asparagine-specific Enzyme, Atypical Antipsychotic, Autologous Cellular Immunotherapy, Autologous Cultured Cell, Autonomic Ganglionic Blocker, Azole Antifungal, Azoles, B Lymphocyte Stimulator-directed Antibody Interactions, B
Lymphocyte Stimulator-specific Inhibitor, Bacterial Neurotoxin Neutralization, Bacterial Proteins, Barbiturate, Barbiturates, BCG Vaccine, Bee Venoms, Benzodiazepine Antagonist, Benzodiazepine, Benzodiazepines, Benzothiazole, Benzothiazoles, Benzylamine Antifungal, Benzylamines, beta Lactamase Inhibitor, beta Lactamase Inhibitors, beta-Adrenergic Agonist, beta-Adrenergic Blocker, beta2-Adrenergic Agonist, beta3-Adrenergic Agonist, Biguanide, Biguanides, Bile Acid Sequestrant, Bile Acid, Bile Acids and Salts, Bile-acid Binding Activity, Bismuth, Bismuth, Bisphosphonate, Blood Coagulation Factor, Blood Coagulation Factors, Blood Viscosity Reducer, Bovine Intestinal Adenosine Deaminase, Bradykinin B2 Receptor Antagonist, Bradykinin B2 Receptor Antagonists, Calcineurin Inhibitor Immunosuppressant, Calcineurin Inhibitors, Calcitonin, Calcitonin, Calcium Channel Antagonists, Calcium Channel Blocker, Calcium Chelating Activity, Calcium, Calcium, Calcium-sensing Receptor Agonist, Calculi Dissolution Agent, Cannabinoid, Cannabinoids, Carbamoyl Phosphate Synthetase 1 Activator, Carbamoyl Phosphate Synthetase 1 Activators, Carbapenems, Carbon Radioisotopes, Carbonic Anhydrase Inhibitor, Carbonic Anhydrase Inhibitors, Cardiac Glycoside, Cardiac Glycosides, Carnitine Analog, Carnitine, Caseins, Catechol O-Methyltransferase Inhibitors, Catechol-O-Methyltransferase Inhibitor, Catecholamine Synthesis Inhibitor, Catecholamine Synthesis Inhibitors, Catecholamine, Catecholamine-depleting Sympatholytic, Catecholamines, Cations, Divalent, CCR5 Co-receptor Antagonist, CD20-directed Antibody Interactions, CD20- directed Cytolytic Antibody, CD20-directed Radiotherapeutic Antibody, CD25-directed Cytotoxin, CD3 Blocker Immunosuppressant, CD3 Receptor Antagonists, CD3-directed Antibody Interactions, CD30-directed Antibody Interactions, CD30-directed Immunoconjugate, CD52-directed Antibody Interactions, CD52-directed Cytolytic Antibody, CD80-directed Antibody Interactions, CD86-directed Antibody Interactions, Cell Death Inducer, Cell-mediated Immunity, Cells, Allogeneic, Cells, Cultured, Allogeneic, Cells, Cultured, Autologous, Cells, Epidermal, Central alpha-2 Adrenergic Agonist, Central Nervous System Depressant, Central Nervous System Depression, Central Nervous System Stimulant, Central Nervous System Stimulation, Centrally-mediated Muscle Relaxation, Cephalosporin Antibacterial,
Cephalosporins, Chemokine Co-receptor 5 Antagonists, Chloride Channel Activation
Potentiators, Chloride Channel Activator, Chloride Channel Activators, Cholecalciferol, Cholecystokinin Analog, Cholecystokinin, Cholinergic Agonists, Cholinergic Antagonists, Cholinergic Muscarinic Agonist, Cholinergic Muscarinic Agonists, Cholinergic Muscarinic Antagonist, Cholinergic Muscarinic Antagonists, Cholinergic Nicotinic Agonist, Cholinergic Receptor. Agonist, Cholinesterase Inhibitor, Cholinesterase Inhibitors, Cholinesterase
Reactivator, Cholinesterase Reactivators, Chondrocytes, Collagen, Collagen-specific Enzyme, Collagenases, Competitive Opioid Antagonists, Complement Inhibitor, Complement Inhibitors, Contrast Agent for Ultrasound Imaging, Copper Absoiption Inhibitor, Copper, Copper- containing Intrauterine Device, Corticosteroid Hormone Receptor Agonists, Corticosteroid, CTLA-4-directed Antibody Interactions, CTLA-4-directed Blocking Antibody, Cyclooxygenase Inhibitors, Cysteine Depleting Agent, Cystic Fibrosis Transmembrane Conductance Regulator Potentiator, Cystine Disulfide Reduction, Cytochrome P450 1A2 Inhibitors, Cytochrome P450 2B6- Inducers, Cytochrome P450 2C19 Inducers, Cytochrome P450 2C 19 Inhibitors, Cytochrome P450 2C8 Inducers, Cytochrome P450 2C8 Inhibitors, Cytochrome P450 2C9 Inducers,
Cytochrome P450 2C9 Inhibitors, Cytochrome P450 2D6 Inhibitor, Cytochrome P450 2D6 Inhibitors, Cytochrome P450 3A Inducers, Cytochrome P450 3A Inhibitors, Cytochrome P450 3A4 Inducers, Cytochrome P450 3A4 Inhibitors, Cytochrome P450 3A5 Inhibitors,
Cytomegalovirus Nucleoside Analog DNA Polymerase Inhibitor, Cytoprotective Agent, Dander, Decarboxylase Inhibitor, Decarboxylase Inhibitors, Decreased Autonomic Ganglionic Activity, Decreased B Lymphocyte Activation, Decreased Blood Pressure, Decreased Cell Wall Integrity, Decreased Cell Wall Synthesis & Repair, Decreased Central Nervous System Disorganized Electrical Activity, Decreased Central Nervous System Organized Electrical Activity, Decreased Cholesterol Absorption, Decreased Coagulation Factor Activity, Decreased Copper Ion
Absorption, Decreased Cytokine Activity, Decreased DNA Replication, Decreased Embryonic Implantation, Decreased Fibrinolysis, Decreased GnRH Secretion, Decreased Histamine Release, Decreased IgE Activity, Decreased Immunologic Activity, Decreased Immunologically Active Molecule Activity, Decreased Leukotriene Production, Decreased Mitosis, Decreased
Parasympathetic Acetylcholine Activity, Decreased Platelet Aggregation, Decreased Platelet Production, Decreased Prostaglandin Production, Decreased Protein Synthesis, Decreased Renal > K+ Excretion, Decreased Respiratory Secretion Viscosity, Decreased RNA Replication,
Decreased Sebaceous Gland Activity, Decreased Sperm Motility, Decreased Striated Muscle Contraction, Decreased Striated Muscle Tone, Decreased Sympathetic Activity, Decreased Tracheobronchial Stretch Receptor Activity, Decreased Vascular Permeability, Demulcent Activity, Demulcent, Deoxyribonuclease I, Deoxyuridine, Depigmenting Activity, Depigmenting Agent, Depolarizing Neuromuscular Blocker, Diagnostic Dye, Dietary Cholesterol Absorption Inhibitor, Dietary Proteins, Digestive/GI System Activity Alteration, Digoxin Binding Activity, Dihydrofolate Reductase Inhibitor Antibacterial, Dihydrofolate Reductase Inhibitor Antimalarial, Dihydrofolate Reductase Inhibitors, Dihydroorotate Dehydrogenase Inhibitors, Dihydropyridine Calcium Channel Blocker, Dihydropyridines, Dipeptidase Inhibitors, Dipeptidyl Peptidase 4 Inhibitor, Dipeptidyl Peptidase 4 Inhibitors, Diphosphonates, Diphtheria Toxin, Direct Thrombin Inhibitor, DNA Polymerase Inhibitors, DOPA Decarboxylase Inhibitors, Dopamine Agonists, Dopamine D2 Antagonists, Dopamine Uptake Inhibitors, Dopamine-2 Receptor Antagonist, Dopaminergic Agonist, Dyes, Echinocandin Antifungal, Egg Proteins, Dietary, Emesis
Suppression, Endogenous Antigen Neutralization, Endoglycosidase, Endothelin Receptor Antagonist, Endothelin Receptor Antagonists, Enzyme Precursors, Epidermal Growth Factor Receptor Antagonist, Ergocalciferols, Ergolines, Ergot Alkaloids, Ergot Derivative, Ergot- derived Dopamine Receptor Agonist, Ergotamine Derivative, Ergotamines, Erythropoiesis- stimulating Agent, Erythropoietin, Ester Local Anesthetic, Esters, Estradiol Congeners,
Estradiol, Estrogen Agonist/Antagonist, Estrogen Receptor Agonists, Estrogen Receptor Antagonist, Estrogen Receptor Antagonists, Estrogen, Estrogens, Conjugated (USP), Factor VIII Activator, Factor VIII, Factor Xa Inhibitor, Factor Xa Inhibitors, Fatty Acids, Omega-3,
Feathers, Fibroblast Growth Factor 7, Fibroblasts, Fish Proteins, Dietary, Folate Analog
Metabolic Inhibitor, Folate Analog, Folic Acid Metabolism Inhibitors, Folic Acid, Food
Additives, Free Radical Scavenging Activity, Fruit Proteins, Full Opioid Agonists, Fungal Proteins, Fur, Fusion Protein Inhibitors, GABA A Agonists, GABA B Agonists, Gadolinium- based Contrast Agent, gamma-Aminobutyric Acid A Receptor Agonist, gamma-Aminobutyric Acid-ergic Agonist, General Anesthesia, General Anesthetic, Genitourinary Arterial
Vasodilation, GI Motility Alteration, Glinide, GLP-1 Receptor Agonist, Glucagon-Like Peptide 1 , Glucagon-like Peptide- 1 (GLP- 1) Agonists, Glucosylceramidase, Glucosylceramide Synthase Inhibitor, Glucosylceramide Synthase Inhibitors, Glycerol, Glycopeptide Antibacterial,
Glycopeptides, Glycosaminoglycan, Glycosaminoglycans, Glycoside Hydrolases, Gonadotropin Releasing Hormone Antagonist, Gonadotropin Releasing Hormone Receptor Agonist,
Gonadotropin Releasing Hormone Receptor Agonists, Gonadotropin Releasing Hormone Receptor Antagonists, Gonadotropin, Gonadotropins, Grain Proteins, Granulocyte Colony- Stimulating Factor, Granulocyte-Macrophage Colony-Stimulating Factor, Growth Hormone Receptor Antagonist, Growth Hormone Receptor Antagonists, Growth Hormone Releasing Factor Analog, Guanylate Cyclase Activators, Guanylate Cyclase Stimulators, Guanylate Cyclase-C Agonist, HEALTHCARE/PHARMACEUTICAL INDUSTRY MENU, Home, News, DailyMed Announcements, Get RSS News & Updates, Search, Advanced Search, Browse Drug Classes, Labels Archives, Tablet/Capsule ID Tool, FDA Guidances & Information, NLM SPL Resources, Download Data, All Drug Labels, All Index Files, All Mapping Files, SPL Image Guidelines, Presentations & Articles, Application Development Support, Resources, Web Services, Mapping Files, Help, SWITCH TO CONSUMER/PATIENT MENU, HCV NS3/4A Protease Inhibitors, Hedgehog Pathway Inhibitor, Helicobacter pylori Diagnostic, Hematologic Activity Alteration, Hematopoietic Stem Cell Mobilizer, Hematopoietic Stem Cells, Heparin Binding Activity, Heparin Reversal Agent, Heparin, Heparin, Low-Molecular-Weight, Hepatitis B Virus Nucleoside Analog Reverse Transcriptase Inhibitor, Hepatitis C Virus NS3/4A Protease Inhibitor, HER 1 Antagonists, HER2 Receptor Antagonist, HER2/Neu/cerbB2 Antagonists, Herpes Simplex Virus Nucleoside Analog DNA Polymerase Inhibitor, Herpes Zoster Virus Nucleoside Analog DNA Polymerase Inhibitor, Herpesvirus Nucleoside Analog DNA
Polymerase Inhibitor, Histamine H I Receptor Antagonists, Histamine H2 Receptor Antagonists, Histamine Receptor Antagonists, Histamine- 1 Receptor Antagonist, Histamine-1 Receptor Inhibitor, Histamine-2 Receptor Antagonist, Histone Deacetylase Inhibitor, Histone Deacetylase Inhibitors, HIV Integrase Inhibitors, HIV Protease Inhibitors, HMG-CoA Reductase Inhibitor, House Dust, Human alpha- 1 Proteinase Inhibitor, Human Antihemophilic Factor, Human Blood Coagulation Factor, Human C I Esterase Inhibitor, Human Immunodeficiency Virus 1 Fusion Inhibitor, Human Immunodeficiency Virus 1 Non-Nucleoside Analog Reverse Transcriptase Inhibitor, Human Immunodeficiency Virus Integrase Strand Transfer Inhibitor, Human
Immunodeficiency Virus Nucleoside Analog Reverse Transcriptase Inhibitor, Human
Immunoglobulin G, Human Immunoglobulin, Human Platelet-derived Growth Factor, Human Serum Albumin, Hydrolytic Lysosomal Glucocerebroside-specific Enzyme, Hydrolytic
Lysosomal Glycogen-specific Enzyme, Hydrolytic Lysosomal Glycosaminoglycan-specific Enzyme, Hydrolytic Lysosomal Neutral Glycosphingolipid-specific Enzyme,
Hydroxymethylglutaryl-CoA Reductase Inhibitors, Hydroxyphenyl-Pyruvate Dioxygenase Inhibitor, Hydroxyphenylpyruvate Dioxygenase Inhibitors, IgE-directed Antibody Interactions, Immunoconjugates, Immunoglobulin G, Immunoglobulins, Inactivated Salmonella Typhi Vaccine, Increased Acetylcholine Activity, Increased Blood Pressure, Increased Calcium-sensing Receptor Sensitivity, Increased Cellular Death, Increased Coagulation Activity, Increased Coagulation Factor Activity, Increased Coagulation Factor IX Activity, Increased Coagulation Factor VIII Activity, Increased Coagulation Factor VIII Concentration, Increased Coagulation Factor X Activity, Increased Cytokine Activity, Increased Cytokine Production, Increased Diuresis at Loop of Henle, Increased Diuresis, Increased Dopamine Activity, Increased Epithelial Proliferation, Increased Erythroid Cell Production, Increased Fibrin Polymerization Activity, Increased GHRH Activity, Increased Glutathione Concentration, Increased
Hematopoietic Stem Cell Mobilization, Increased Histamine Release, Increased IgG Production, Increased Immunologically Active Molecule Activity, Increased Intravascular Volume,
Increased Large Intestinal Motility, Increased Lymphocyte Activation, Increased Lymphocyte Cell Production, Increased Macrophage Proliferation, Increased Medullary Respiratory Drive, Increased Megakaryocyte Maturation, Increased Myeloid Cell Production, Increased
Norepinephrine Activity, Increased Oncotic Pressure, Increased Platelet Aggregation, Increased Platelet Production, Increased Prostaglandin Activity, Increased Prothrombin Activity, Increased Sympathetic Activity, Increased T Lymphocyte Activation, Increased T Lymphocyte
Destruction, Increased Thrombolysis, Increased Uterine Smooth Muscle Contraction or Tone, Influenza A M2 Protein Inhibitor, Inhalation Diagnostic Agent, Inhibit Ovum Fertilization, Insect Proteins, Insulin Analog, Insulin, Insulin, Integrin Receptor Antagonist, Integrin Receptor Antagonists, Interferon Alfa-2a, Interferon Alfa-2b, Interferon alpha, Interferon gamma, Interferon Inducers, Interferon-alpha, Interferon-beta, Interferon-gamma, Interleukin 1 Receptor Antagonists, Interleukin 2 Receptor Antagonists, Interleukin 2 Receptor-directed Antibody Interactions, Interleukin 6 Receptor Antagonists, Interleukin- 1 Receptor Antagonist, Interleukin- 2 Receptor Blocking Antibody, Interleukin-2, Interleukin-6 Receptor Antagonist, Intestinal Lipase Inhibitor, Iodine, Iron Chelating Activity, Iron Chelator, Iron, Irrigation, Kallikrein Inhibitors, eratinocytes, Ketolide Antibacterial, etolides, Kinase Inhibitor, 1-Thyroxine, 1- Triiodothyronine, Lead Chelating Activity, Lead Chelator, Leukocyte Growth Factor,
Leukotriene Receptor Antagonist, Leukotriene Receptor Antagonists, Lincosamide Antibacterial, Lincosamides, Lipase Inhibitors, Lipid-based Polyene Antifungal, Lipopeptide Antibacterial, Lipopeptides, Live Attenuated Bacillus Calmette-Guerin Immunotherapy, Live Attenuated Bacillus Calmette-Guerin Vaccine, Live Attenuated Mumps Virus Vaccine, Live Human Adenovirus Type 4 Vaccine, Live Human Adenovirus Type 7 Vaccine, Live Rotavirus Vaccine, Local Anesthesia, Local Anesthetic, Loop Diuretic, Low Molecular Weight Heparin,
Lymphocyte Function Alteration, Lymphocyte Growth Factor, M2 Protein Inhibitors, Macrolide Antibacterial, Macrolide Antimicrobial, Macrolide, Macrolides, Magnesium Ion Exchange Activity, Magnetic Resonance Contrast Activity, Mast Cell Stabilizer, Meat Proteins,
Megakaryocyte Growth Factor, Melanin Synthesis Inhibitor, Melanin Synthesis Inhibitors, Melatonin Receptor Agonist, Melatonin Receptor Agonists, Metal Chelating Activity, Metal Chelator, Methylated Sulfonamide Antibacterial, Methylated Sulfonamides, Methylating Activity, Methylating Agent, Methylxanthine, Microtubule Inhibition, Microtubule Inhibitor, Milk Proteins, Monoamine Oxidase Inhibitor, Monoamine Oxidase Inhibitors, Monoamine Oxidase Type B Inhibitor, Monoamine Oxidase-B Inhibitors, Monobactam Antibacterial, Monobactams, Mood Stabilizer, mTOR Inhibitor Immunosuppressant, mTOR Inhibitors, Mucocutaneous Epithelial Cell Growth Factor, Mucolytic, Mumps Vaccine, Muscle Relaxant, N- Calcium Channel Receptor Antagonists, N-methyl-D-aspartate Receptor Antagonist, N- substituted Glycines, N-type Calcium Channel Antagonist, Natriuretic Peptide, Natriuretic Peptides, Neuraminidase Inhibitor, Neuraminidase Inhibitors, Neurokinin 1 Antagonists, Neuromuscular Depolarizing Blockade, Neuromuscular Nondepolarizing Blockade, Nicotine, Nicotinic Acid, Nicotinic Acids, Nitrate Vasodilator, Nitrates, Nitrofuran Antibacterial,
Nitrofurans, Nitrogen Binding Agent, Nitrogen Mustard Compounds, Nitroimidazole
Antimicrobial, Nitroimidazoles, NMDA Receptor Antagonists, Non-narcotic Antitussive, Non- Nucleoside Analog, Non-Nucleoside Reverse Transcriptase Inhibitors, Non-Standardized Animal Dander Allergenic Extract, Non-Standardized Animal Hair Allergenic Extract, Non- Standardized Animal Skin Allergenic Extract, Non-Standardized Bacterial Allergenic Extract, Non-Standardized Chemical Allergen, Non-Standardized Feather Allergenic Extract, Non- Standardized Food Allergenic Extract, Non-Standardized Fungal Allergenic Extract, Non- Standardized House Dust Allergenic Extract, Non-Standardized Insect Allergenic Extract, Non- Standardized Insect Venom Allergenic Extract, Non-Standardized Plant Allergenic Extract, Non- Standardized Plant Fiber Allergenic Extract, Non-Standardized Pollen Allergenic Extract, Noncompetitive AMPA Glutamate Receptor Antagonist, Nondepolarizing Neuromuscular Blocker, Nonergot Dopamine Agonist, Nonsteroidal Anti-inflammatory Compounds,
Nonsteroidal Anti-inflammatory Drug, Norepinephrine Reuptake Inhibitor, Norepinephrine Uptake Inhibitors, Nucleic Acid Synthesis Inhibitors, Nucleoside Analog Antifungal, Nucleoside Analog Antiviral, Nucleoside Analog, Nucleoside Metabolic Inhibitor, Nucleoside Reverse Transcriptase Inhibitors, Nut Proteins, Oligonucleotides, Omega-3 Fatty Acid, Opioid Agonist, Opioid Agonist/Antagonist, Opioid Agonists, Opioid Antagonist, Opioid Antagonists, Organic Anion Transporting Polypeptide 1 B 1 Inhibitors, Organic Anion Transporting Polypeptide 1 B3 Inhibitors, Organic Anion Transporting Polypeptide 2B 1 Inhibitors, Organic Cation Transporter 2 Inhibitors, Organometallic Compounds, Osmotic Activity, Osmotic Diuretic, Osmotic
Laxative, Oxazolidinone Antibacterial, Oxazolidinones, Oxytocic, Oxytocin, P-Glycoprotein Inhibitors, P-Glycoprotein Interactions, P2Y 12 Platelet Inhibitor, P2Y 12 Receptor Antagonists, Paramagnetic Contrast Agent, Parathyroid Hormone Analog, Parathyroid Hormone, Parenteral Iron Replacement, Partial Cholinergic Nicotinic Agonist, Partial Cholinergic Nicotinic Agonists, Partial Opioid Agonist, Partial Opioid Agonist/Antagonist, Partial Opioid Agonists, Passively Acquired Immunity, Pediculicide, peginterferon alfa-2a, peginterferon alfa-2b, Penem
Antibacterial, Penicillin-class Antibacterial, Penicillins, Peripheral Blood Mononuclear Cells, Peroxisome Prolif'erator Receptor alpha Agonist, Peroxisome Proliferator Receptor gamma Agonist, Peroxisome Proliferator-activated Receptor Activity, Peroxisome Proliferator-activated Receptor alpha Agonists, Phenothiazine, Phenothiazines, Phenylalanine Hydroxylase Activator, Phenylalanine Hydroxylase Activators, Phosphate Binder, Phosphate Chelating Activity, Phosphodiesterase 3 Inhibitor, Phosphodiesterase 3 Inhibitors, Phosphodiesterase 5 Inhibitor, Phosphodiesterase 5 Inhibitors, Photoabsorption, Photoactivated Radical Generator,
Photoenhancer, Photosensitizing Activity, Plant Proteins, Plasma Volume Expander, Platelet Aggregation Inhibitor, Platelet-Derived Growth Factor, Platelet-reducing Agent, Platinum-based Drug, Platinum-containing Compounds, Pleuromutilin Antibacterial, pleuromutilin, Pollen, Polyene Antifungal, Polyene Antimicrobial, Polyenes, Polymyxin-class Antibacterial,
Polymyxins, Porphyrin Precursor, Porphyrinogens, Positron Emitting Activity, Potassium Channel Antagonists, Potassium Channel Opener, Potassium Channel Openers, Potassium Compounds, Potassium Salt, Potassium-sparing Diuretic, Poultry Proteins, PPAR alpha, PPAR gamma, Progestational Hormone Receptor Antagonists, Progesterone Congeners, Progesterone, Progesterone, Progestin Antagonist, Progestin, Progestin-containing Intrauterine Device, Prostacycline Vasodilator, Prostacycline, Prostaglandin Analog, Prostaglandin El Agonist, Prostaglandin E l Analog, Prostaglandin Receptor Agonists, Prostaglandins E, Synthetic, Prostaglandins I, Prostaglandins, Protease Inhibitor, Proteasome Inhibitor, Proteasome Inhibitors, Protein C, Protein Kinase Inhibitors, Protein Synthesis Inhibitors, Proton Pump Inhibitor, Proton Pump Inhibitors, Provitamin D2 Compound, Psoralen, Psoralens, Purine Antimetabolite, Purines, Pyrethrins, Pyrethroid, Pyrimidine Synthesis Inhibitor, Pyrophosphate Analog DNA Polymerase Inhibitor, Pyrophosphate Analog, Quaternary Ammonium Compounds, Quinolone
Antimicrobial, Quinolones, Radioactive Diagnostic Agent, Radioactive Therapeutic Agent, Radioactive Tracers, Radiographic Contrast Agent, Radiopharmaceutical Activity, RANK Ligand Blocking Activity, RANK Ligand Inhibitor, Receptor Tyrosine Kinase Inhibitors, Recombinant Antithrombin, Recombinant Fusion Proteins, Recombinant Human
Deoxyribonuclease 1 , Recombinant Human Growth Hormone, Recombinant Human Growth Hormones, Recombinant Human Interferon beta, Recombinant Proteins, Reducing and
Complexing Thiol, Reduction Activity, Renal Dehydropeptidase Inhibitor, Renin Inhibitor, Renin Inhibitors, Respiratory Stimulant, Respiratory Syncytial Virus Anti-F Protein Monoclonal Antibody, Retinoid, Retinoids, Reversed Anticoagulation Activity, Rifamycin Antibacterial, Rifamycin Antimycobacterial, Rifamycins, RNA Synthetase Inhibitor Antibacterial, RNA Synthetase Inhibitors, Rotavirus Vaccines, Salivary Proteins and Peptides, Sclerosing Activity, Sclerosing Agent, Seed Storage Proteins, Selective Estrogen Receptor Modulators, Selective T Cell Costimulation Blocker, Selective T Cell Costimulation Modulator, Serotonin lb Receptor Agonists, Serotonin Id Receptor Agonists, Serotonin 2c Receptor Agonists, Serotonin 3 Receptor Antagonists, Serotonin 4 Receptor Antagonists, Serotonin and Norepinephrine Reuptake Inhibitor, Serotonin Reuptake Inhibitor, Serotonin Uptake Inhibitors, Serotonin- lb and Serotonin- I d Receptor Agonist, Serotonin-2c Receptor Agonist, Serotonin-3 Receptor
Antagonist, Serotonin-4 Receptor Antagonist, Serum Albumin, Shellfish Proteins, Sigma- 1 Agonist, Sigma- 1 Receptor Agonists, Silk, Skeletal Muscle Relaxant, Skin Barrier Activity, Skin Test Antigen, Smoothened Receptor Antagonists, Sodium-Glucose Cotransporter 2 Inhibitor, Sodium-Glucose Transporter 2 Inhibitors, Soluble Guanylate Cyclase Stimulator, Somatostatin Analog, Somatostatin Receptor Agonists, Sphingosine 1 -phosphate Receptor Modulator, Sphingosine 1 -Phosphate Receptor Modulators, Standardized Animal Hair Allergenic Extract, Standardized Animal Skin Allergenic Extract, Standardized Chemical Allergen, Standardized Insect Allergenic Extract, Standardized Insect Venom Allergenic Extract, Standardized Pollen Allergenic Extract, Starch, Stimulant Laxative, Stimulation Large Intestine Fluid/Electrolyte Secretion, Streptogramin- Antibacterial, Streptogramins, Substance P/Neurokinin- 1 Receptor Antagonist, Sucrose-specific Enzyme, Sulfonamide Antibacterial, Sulfonamide Antimicrobial, Sulfonamides, Sulfone, Sulfones, Sulfonylurea Compounds, Sulfonylurea, Surfactant Activity, Surfactant, Sympathomimetic Amine Anorectic, Sympathomimetic-like Agent, T Lymphocyte Costimulation Activity Blockade, Tetracycline-class Antibacterial, Tetracycline-class
Antimicrobial, Tetracycline-class Drug, Tetracyclines, Thalidomide Analog, Thiazide Diuretic, Thiazide-like Diuretic, Thiazides, Thiazolidinedione, Thiazolidinediones, Thrombin Inhibitors, Thrombolytic Agent, Thrombopoiesis Stimulating Agent, Thrombopoietin Receptor Agonists, Thrombopoietin Receptor Interactions, Thrombopoietin, Thyroid Hormone Synthesis Inhibitor, Thyroid Hormone Synthesis Inhibitors, Thyroid Stimulating Hormone, Thyrotropin, Thyroxine, Tissue Scaffolds, Topoisomerase Inhibitor, Topoisomerase Inhibitors, Transglutaminases, Tricyclic Antidepressant, Triiodothyronine, Trypsin Inhibitors, Tuberculosis Skin Test, Tumor Necrosis Factor alpha Receptor Blocking Activity, Tumor Necrosis Factor Blocker, Tumor Necrosis Factor Receptor Blocking Activity, Typical Antipsychotic, Ultrasound Contrast Activity, Uncompetitive N-methyl-D-aspartate Receptor Antagonist, Uncompetitive NMDA Receptor Antagonists, Unfractionated Heparin, Urate Oxidase, Urea, Urease Inhibitor, Urease Inhibitors, Uric Acid-specific Enzyme, Vaccines, Attenuated, Vaccines, Inactivated, Vaccines, Live, Unattenuated, Vaccines, Typhoid, Vascular Endothelial Growth Factor Receptor Inhibitors, Vascular Endothelial Growth Factor-directed Antibody Interactions, Vascular Endothelial Growth Factor-directed Antibody, Vascular Sclerosing Activity, Vasodilation, Vasodilator, Vasopressin Analog, Vasopressin Antagonist, Vasopressins, Vegetable Proteins, Venom
Neutralization, Venous Vasodilation, Vi polysaccharide vaccine, typhoid, Vinca Alkaloid, Vinca Alkaloids, Virus Neutralization, Virus-specific Hyperimmune Globulins, Vitamin A, Vitamin A, Vitamin B 12, Vitamin B Complex Compounds, Vitamin B Complex Member, Vitamin B 12, Vitamin D Analog, Vitamin D, Vitamin D, Vitamin D2 Analog, Vitamin D3 Analog, Vitamin K Antagonist, Vitamin K Inhibitors, Vitamin K, Vitamin K, von Willebrand Factor, Warfarin Reversal Agent, Wasp Venoms, X-Ray Contrast Activity, Xanthine Oxidase Inhibitor, Xanthine Oxidase Inhibitors, Xanthines, or combinations thereof.
Microsatellitc extension inhibitors
[0715] In an embodiment a payload comprises a microsatellite extension inhibitor. In an embodiment, the microsatellite extension inhibitor is a DNA mismatch repair protein.
Exemplary DNA mismatch repair proteins that can be delivered by the molecules and methods described herein include, e.g., MSH2, MSH3, MSH6, MLH 1 , MLH3, PMS 1 , PMS2. Signal generators, radionuclides, reporter molecules, diagnostic probes
[0716] In an embodiment a payload comprises a molecule that generates a signal. Such payloads are useful, e.g., in research, therapeutic (e.g., cancer therapy) and diagnostic applications. In an embodiment, the signal comprises: an electromagnetic emission, e.g., in the infrared, visible, or ultraviolet range; a particle, e.g., a product of radioactive decay, e.g., an alpha, beta, or gamma particle; a detectable substrate, e.g., a colored substrate; a reaction product, e.g., the product of an enzymatic reaction; or a ligand detectable by a specific binding agent, e.g., an antibody; or a dye. In an embodiment the signal comprises a fluorescent emission, e.g., by a fluorescent protein. Exemplary fluorescent proteins include, Blue UV Proteins (e.g., TagBFP, mTagBFP, Azurite, EBFP2, m alamal , Si ius, Sapphire, T-Sapphire), Cyan Proteins (e.g., ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP, mTFPl), Green Proteins (e.g., EGFP, Emerald, Supeifolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen), Yellow Proteins (e.g., EYFP, Citrine, Venus, SYFP2, TagYFP), Orange Proteins (e.g., Monomeric Kusabira-Orange, ΙΏΚΟΚ, mK02, mOrange, mOrange2), Red Proteins (mRaspberry, mCherry, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mRuby2), Far-Red Proteins (e.g., mPlum, HcRed- Tandem, m ate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP), Long Stokes Shift Proteins (e.g., mKeima Red, LSS-mKatel , LSS-mKate2, mBeRFP), Photoactivatible Proteins (e.g., PA- GFP, PAmCherryl , PATagRFP), Photoconvertible Proteins (e.g., Kaede (green), Kaede (red), KikGR l (green), KikGR l (red), PS-CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2 (red), PSmOrange), Photoswitchable Proteins (e.g., Dronpa).
[0717] In an embodiment, a signal producing moiety is provided as the fusion partner of a Cas9 molecule, e.g., an eiCas9 molecule.
[0718] Signal generators or reporters, useful, e.g., for labelingr polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., indium (l uIn), iodine (13 1 I or 125I), yttrium (90Y), lutetium (l77Lu), actinium (225Ac), bismuth (2l 2Bi or 2l 3Bi), sulfur (35S), carbon ( C), tritium ( H), rhodium ( Rh), technetium ( mTc), praseodymium, or phosphorous ( ~P) or a positron-emitting radionuclide, e.g., carbon- 1 1 ("C), potassium-40 (40K), nitrogen- 13 (13N), oxygen- 15 (l 50), fluorine- 18 (l8F), and iodine- 121 ( 1211)), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, beta- galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups (which can be detected by a marked avidin, e.g., a molecule containing a streptavidin moiety and a fluorescent marker or an enzymatic activity that can be detected by optical or calorimetric methods), and predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.
[071 ] In an embodiment, a payload comprises a radionuclide. The radionuclide can be incorporated into the gRNA molecule, the Cas9 molecule, or into a payload molecule.
Exemplary radionuclides include, e.g., beta emitters, alpha emitters or gamma emitters. In an embodiment the radionuclide is iodine, e.g., I 31I or 125I, yttrium, e.g., 90Y, lutetium, e.g., l 77Lu, Actinium, e.g., Ac, bismuth, e.g., " "Bi or " Bi), sulfur, e.g., S), carbon, e.g., C, tritium, H), rhodium, e.g., 188Rh, technetium, e.g., 99Tc, praseodymium, or phosphorous, e.g., 32P.
Modulators of DNA and Chromatin Structure
[0720] In an embodiment a payload comprises an endogenous or exogenous modulator of DNA structure. A modulator, as is typical of payloads, can be delivered in vitro, ex vivo, or in vivo.
[0721] In an embodiment, the payload comprises a modulator of an epigenetic state or characteristic of DNA. In an embodiment an epigenetic state or characteristic can be altered to treat a disorder, or to influence the developmental or other state of a cell.
[0722] In an embodiment, the epigenetic state or characteristic comprises DNA methylation. For example, the payloads described herein can modulate the addition of methyl groups to DNA, e.g., to convert cytosine to 5-methylcytosine, e.g., at CpG sites.
[0723] Aberrant DNA methylation patterns (e.g., hypermethylation and hypomethylation compared to normal tissue) are associated with various diseases and conditions, e.g., cancer. The modulators described herein can be used to reactivate transcriptionally silenced genes or to inhibit transcriptionally hyperactive genes, e.g., to treat diseases, e.g., cancer.
[0724] DNA methylation can affect gene transcription. Genes with high levels of 5- methylcytosine, e.g., in their promoter region, can be transcriptionally less active or silent. Thus, methods described herein can be used to target and suppress transcriptional activity, e.g., of genes described herein.
[0725] In some embodiments, the modulator promotes maintenance of DNA methylation. For example, the modulators can have DNA methyltransferase (DNMT) activity or modulate DNMT activity, e.g\, to maintain DNA methylation or reduce passive DNA demethylation, e.g., after DNA replication.
[0726] In some embodiments, the modulator promotes de novo DNA methylation. For example, the modulators described herein can have de novo DNA methyltransferase (DNMT) (e.g., DNMT3a, DNMT3b, DNMT3L) activity or modulate de novo DNMT (e.g., DNMT3a,
DNMT3b, DNMT3L) activity, e.g., to produce DNA methylation patterns, e.g., early in development.
[0727] Epigenetic changes in DNA (e.g., methylation), can be evaluated by art-known methods or as described herein. Exemplary methods for detecting DNA methylation include, e.g., Methylation-Specific PCR (MSP), whole genome bisulfite sequencing (BS-Seq), HELP (Hpall tiny fragment Enrichment by Ligation-mediated PCR) assay, ChlP-on-chip assays, restriction landmark genomic scanning. Methylated DNA immunoprecipitation (MeDIP), pyrosequencing of bisulfite treated DNA, molecular break light assay for DNA adenine methyltransferase activity, methyl sensitive Southern Blotting, separation of native DNA into methylated and unmethylated fractions using MethylCpG Binding Proteins (MBPs) and fusion proteins containing just the Methyl Binding Domain (MBD).
[0728] In an embodiment, the modulator cleaves DNA. For example, a modulator can catalyze the hydrolytic cleavage of phosphodiester linkages in the DNA backbone. In some
embodiments, the modulator (e.g., DNase I) cleaves DNA preferentially at phosphodiester linkages adjacent to a pyrimidine nucleotide, yielding 5'-phosphate-terminated polynucleotides with a free hydroxyl group on position 3'. In other embodiments, the modulator (e.g., DNase II) hydrolyzes deoxyribonucleotide linkages in DNA, yielding products with 3'-phosphates. In some embodiments, the modulator comprises endodeoxyribonuclease activity. In other embodiments, the modulator comprises exodeoxyribonuclease activity (e.g., having 3' to 5' or 5' to 3' exodeoxyribonuclease activity). In some embodiments, the modulator recognizes a specific DNA sequence (e.g., a restriction enzyme). In other embodiments, the modulator does not cleave DNA in a sequence-specific manner. A modulator can cleave single- stranded DNA (e.g., having nickase activity), double-stranded DNA, or both.
[0729] In an embodiment, modulator affects, e.g., alters or preserves, tertiary or quaternary DNA structure. For example, the modulators described herein can modulate tertiary structure, e.g., handedness (right or left), length of the helix turn, number of base pairs per turn, and/or difference in size between the major and minor grooves. In some embodiments, the modulator mediates the formation of B-DNA, A-DNA, and/or Z-DNA. The modulators described herein can also modulate quaternary structure, e.g., the interaction of DNA with other molecules (DNA or non-DNA molecules, e.g., histones), e.g., in the form of chromatin. In some embodiments, the modulator that mediate or modify tertiary or quatemary DNA structure comprises DNA helicases activity or modulates DNA helicase activity.
[0730] In some embodiments, the modulator promotes or inhibits DNA damage response and/or repair. For example, a modulator can promote one or more DNA damage response and repair mechanisms, e.g., direct reversal, base excision repair (BER), nucleotide excision repair (NER) (e.g., global genomic repair (GG-NER), transcription-coupled repair (TC-NER)), mismatch repair (MMR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), homologous recombination, and/or translesion synthesis (TLS). In some embodiments, a modulator promotes the step of damage recognition. In other embodiments, a modulator promotes the step of DNA repair.
[0731 ] Aberrant DNA damage repair is associated with various diseases and conditions, e.g., aging, hereditary DNA repair disorders, and cancer. For example, DNA repair gene mutations that can increase cancer risk include, e.g., BRCA 1 and BRCA2 (e.g., involved in homologous recombination repair (HRR) of double-strand breaks and daughter strand gaps, e.g., in breast and ovarian cancer); ATM (e.g., different mutations reduce HRR, single strand annealing (SSA), NHEJ or homology-directed DSBR (HDR), e.g., in leukemia, lymphoma, and breast cancer), NBS (e.g., involved in NHEJ, e.g., in lymphoid malignancies); MRE1 1 (e.g., involved in HRR, e.g., in breast cancer); BLM (e.g., involved in HRR, e.g., iri leukemia, lymphoma, colon, breast, skin, auditory canal, tongue, esophagus, stomach, tonsil, larynx, lung, and uterus cancer); WRN (e.g., involved in HRR, NHEJ, long-patch BER, e.g., in soft tissue sarcomas, colorectal, skin, thyroid, and pancreatic cancer); RECQ4 (RECQL4) (e.g., involved in HRR, e.g., causing Rothmund-Thomson syndrome (RTS), RAPAD1L1NO syndrome or Bailer Gerold syndrome, cutaneous carcinomas, including basal cell carcinoma, squamous cell carcinoma, and Bowen's disease); FANCA, FANCB, FANCC, FANCD1 , FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, and FANCN (e.g., involved in HRR and TLS, e.g., in leukemia, liver tumors, solid tumors in many locations), XPC and XPE(DDB2) (e.g., involved in
NER(GGR type), e.g., in skin cancer (melanoma and non-melanoma)); XPA, XPB, XPD, XPF, and XPG (e.g., involved in NER (both GGR type and TCR type), e.g., in skin cancer (melanoma and non-melanoma) and central nervous system); XPV(POLH) (e.g., involved in TLS, e.g., in skin cancer (melanoma and non-melanoma)); hMSH2, hMSH6, hMLH l, and hPMS2 (involved in MMR, e.g., in colorectal, endometrial and ovarian cancer); MUTYH (e.g., involved in BER of A mispaired with 80H-dG, as well as mispairs with G, FapydG and C, e.g., in colon cancer)
[0732] Modulators can be used to treat a disease or condition associated with aberrant DNA damage repair, e.g., by modulating one or more DNA damage repair mechanisms described herein.
[0733] In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in direct reversal, e.g., methyl guanine methyl transferase (MGMT).
[0734] In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in BER, e.g., DNA glycosylase, AP endonuclease, DNA polymerase, DNA ligase.
[0735] In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in GG-NER, e.g., XPC, HR23b, CAK, TFIIH, XPA, RPA, XPG, XPF, ERCC 1 , TFIIH, PCNA, RFC, ADN Pol, and Ligase I.
[0736] In some embodiments, the modulator is selected from ,or modulates, one or more proteins involved in TC-NER, e.g., CSB, XPA, RPA, XPG, XPF, ERCC 1 , CSA-CNS, TFIIH, CAK, PCNA, RFC, Ligase I, and RNA Polymerase II.
[0737] In some embodiments, the modulator is selected from, or modulates, one or more DNA mismatch repair proteins.
[0738] In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in NHEJ, e.g., Ku70/80, DNA-PKcs, DNA Ligase IV, XRCC4, XLF, Artemis, DNA polymerase mu, DNA polymerase lambda, PNKP, Aprataxin, and APLF. [0739] In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in homologous recombination, e.g., as described herein.
[0740] In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in TLS, e.g., DNA polymerase eta, iota, kappa, zeta, and PCNA.
[0741] In an embodiment, a modulator can modulate global response to DNA damage, e.g., DNA damage checkpoints and/or transcriptional responses to DNA damage. For example, DNA damage checkpoints can occur at the G l/S and G2/ boundaries. An intra-S checkpoint can also exist. Checkpoint activation can be modulated by two master kinases, ATM and ATR. ATM can respond to DNA double-strand breaks and disruptions in chromatin structure and ATR can respond to stalled replication forks. These kinases can phosphorylate downstream targets in a signal transduction cascade, e.g., leading to cell cycle arrest. A class of checkpoint mediator proteins (e.g., BRCA 1 , MDC1 , and 53BP1 ), which transmit the checkpoint activation signal to downstream proteins, can be modulated. Exemplary downstream proteins that can be modulated include, e.g., p53, p21 , and cyclin/cyclin-dependent kinase complexes.
[0742] In some embodiments, the modulator modulates nuclear DNA damage response and repair. In other embodiments, the modulator modulates mitochondrial DNA damage response and repair.
[0743] In some embodiments, the modulator promotes or inhibits DNA replication. For example, a modulator can promote or inhibit one or more stages of DNA replication, e.g., initiation (e.g., assembly of pre-replicative complex and/or initiation complex), elongation (e.g., formation of replication fork), and termination (e.g., formation of replication fork barrier). In some embodiments, the modulator is selected from, or modulates, one or more proteins involved in initiation, e.g., the origin recognition complex (ORC), CDC6, CDT1 , minichromosome maintenance proteins (e.g., MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, and MCM 10), CDC45, CD , DDK, CDC 101 , CDC 1 02, CDC 103, and CDC 105. In some embodiments, the modulator is selected from, or modulates ,one or more proteins involved in elongation, e.g., DNA hel icases, DNA polymerase, PCNA, CDC45-MCM-GI S helicase complex, and
Replication Factor C complex.
[0744] In some embodiments, the modulator is selected, from or modulates, one or more proteins involved in termination, e.g., type II topoisomerase and telomerase. In some embodiments, the modulator is selected from, or modulates, one or more replication checkpoint proteins, e.g., ATM, ATR, ATRIP, TOPBP1 , RAD9, HUS 1 , Rad l , and CH 1.
[0745] In some embodiments, the payload comprises a modulator of nuclear DNA replication. In other embodiments, the modulator promotes or inhibits mitochondrial DNA replication.
[0746] Defects in DNA replication can be associated with various diseases and conditions, e.g., cancer and neurological diseases (e.g., Alzheimer's disease). Defects in mitochondrial DNA replication can also be associated with diseases and conditions, e.g., mtDNA depletion syndromes (e.g., Alpers or early infantile hepatocerebral syndromes) and mtDNA deletion disorders (e.g., progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)). A modulator can be used to treat a disease or condition associated with aberrant DNA replication, e.g., by modulating DNA replication as described herein.
[0747] Exemplary endogenous or exogenous modulators of DNA structure are described herein, e.g., in Table VI-3.
Tabic VI-3
Figure imgf000163_0001
DNAH 17-AS 1 DNAH 17 antisense RNA 1
DNAI 1 dynein, axonemal, intermediate chain 1
DNAI2 dynein, axonemal, intermediate chain 2
DNAJB8-AS 1 DNAJB8 antisense RNA 1
DNAJC3-AS 1 DNAJC3 antisense RNA 1 (head to head)
DNAJC9-AS 1 DNAJC9 antisense RNA 1
DNAJC25- DN A JC25-GNG 10 readthrough
GNG 10
DNAJC27-AS 1 DNAJC27 antisense RNA 1
DNAL1 dynein, axonemal, light chain 1
DNAL4 dynein, axonemal, light chain 4
DNALI 1 dynein, axonemal, light intermediate chain 1
DNASE 1 deoxy ibonuclease 1
DNASE 1 L1 deoxyribonuclease I-like 1
DNASE1 L2 deoxyribonuclease I-like 2
DNASE1L3 deoxyribonuclease I-like 3
DNASE2 deoxyribonuclease II, lysosomal
DNASE2B deoxyribonuclease II beta
CD226 CD226 molecule
FAM 120A family with sequence similarity 120A
GAK cyclin G associated kinase
GCFC2 GC-rich sequence DNA-binding factor 2
MCM 10 minichromosome maintenance complex component 10
PR DC protein kinase, DNA-activated, catalytic polypeptide
SACS spastic ataxia of Chai evoix-Saguenay (sacsin)
SCNN 1 D sodium channel, non-voltage-gated 1, delta subunit
SPATS2L spermatogenesis associated, serine-rich 2-like
MT7SDNA mitochondrially encoded 7S DNA
DCLRE 1 A DNA cross-link repair 1 A
DCLRE 1 B DNA cross-link repair I B
DCLRE 1 C DNA cross-link repair 1 C
DDIT3 DNA-damage-inducible transcript 3
DDIT4 DNA-damage-inducible transcript 4
DD1T4L DNA-damage-inducible transcript 4-like
DFFA DNA fragmentation factor, 45kDa, alpha polypeptide
DFFB DNA fragmentation factor, 40kDa, beta polypeptide (caspase-activated
DNase)
DMAP 1 DNA methyltransferase 1 associated protein 1
DMC 1 DNA meiotic recombinase 1 DNMT 1 DNA (cytosine-5-)-methyltransferase 1
DNMT3A DNA (cytosine-5-)-methyltransferase 3 alpha
DNMT3B DNA (cytosine-5-)-methyltransferase 3 beta
DNMT3L DNA (cytosine-5-)-methyltransferase 3-like
DNTT DNA nucleotidylexotransferase
DRAM 1 DNA-damage regulated autophagy modulator 1
DRAM2 DNA-damage regulated autophagy modulator 2
DSCC 1 DNA replication and sister chromatid cohesion 1
ZBP 1 Z-DNA binding protein 1
SON SON DNA binding protein
TARDBP TAR DNA binding protein
BMF Bcl2 modifying factor
CENPBD 1 CENPB DNA-binding domains containing 1
UNG uracil-DNA glycosylase
PDRG 1 p53 and DNA-damage regulated 1
TDG thymine-DNA glycosylase
TDP 1 tyrosyl-DNA phosphodiesterase 1
TDP2 tyrosyl-DNA phosphodiesterase 2
AHDC1 AT hook, DNA binding motif, containing 1
GMNN geminin, DNA replication inhibitor
PRIM 1 primase, DNA, polypeptide 1 (49kDa)
PRIM2 primase, DNA, polypeptide 2 (58kDa)
HELB helicase (DNA) B
LIG 1 ligase I, DNA, ATP-dependent
SUMF 1 sulfatase modifying factor 1
SUMF2 sulfatase modifying factor 2
LIG4 ligase IV, DNA, ATP-dependent
LIG3 ligase III, DNA, ATP-dependent
MDC 1 mediator of DNA-damage checkpoint 1
MMS22L MMS22-like, DNA repair protein
POLA l polymerase (DNA directed), alpha 1, catalytic subunit
POLA2 polymerase (DNA directed), alpha 2, accessory subunit
POLB polymerase (DNA directed), beta
POLD 1 polymerase (DNA directed), delta 1 , catalytic subunit
POLD2 polymerase (DNA directed), delta 2, accessory subunit
POLD3 polymerase (DNA-directed), delta 3, accessory subunit
POLD4 polymerase (DNA-directed), delta 4, accessory subunit
POLDIP2 polymerase (DNA-directed), delta interacting protein 2 -r
POLDIP3 polymerase (DNA-directed), delta interacting protein 3 POLE polymerase (DNA directed), epsilon, catalytic subunit
POLE2 polymerase (DNA directed), epsilon 2, accessory subunit
POLE3 polymerase (DNA directed), epsilon 3, accessory subunit
POLE4 polymerase (DNA-directed), epsilon 4, accessory subunit
POLG polymerase (DNA directed), gamma
POLG2 polymerase (DNA directed), gamma 2, accessory subunit
POLH polymerase (DNA directed), eta
POLI polymerase (DNA directed) iota
POLK polymerase (DNA directed) kappa
POLL polymerase (DNA directed), lambda
POLM polymerase (DNA directed), mu
POLN polymerase (DNA directed) nu
POLO polymerase (DNA directed), theta
ID 1 inhibitor of DNA binding 1 , dominant negative helix-loop-helix protein
ID2 inhibitor of DNA binding 2, dominant negative helix-loop-helix protein
ID3 inhibitor of DNA binding 3, dominant negative helix-loop-helix protein
ID4 inhibitor of DNA binding 4, dominant negative helix-loop-helix protein
OGG 1 8-oxoguanine DNA glycosylase
MSANTD 1 Myb/SANT-like DNA-binding domain containing 1
MSANTD2 Myb/SANT-like DNA-binding domain containing 2
MSANTD3 Myb/SANT-like DNA-binding domain containing 3
MSANTD4 Myb/SANT-like DNA-binding domain containing 4 with coiled-coils
P1F 1 PIF1 5'-to-3' DNA helicase
TONSL tonsoku-like, DNA repair protein
MPG N-methylpurine-DNA glycosylase
TOPI topoisomerase (DNA) I
TOP 1 MT topoisomerase (DNA) I, mitochondrial
TOP2A topoisomerase (DNA) II alpha 170kDa
TOP2B topoisomerase (DNA) II beta 180kDa
TOP3A topoisomerase (DNA) III alpha
TOP3B topoisomerase (DNA) III beta
TOPBP 1 topoisomerase (DNA) II binding protein 1
DDB 1 damage-specific DNA binding protein 1 , 127kDa
DDB2 damage-specific DNA binding protein 2, 48kDa
SSBP 1 single-stranded DNA binding protein 1 , mitochondrial
SSBP2 single-stranded DNA binding protein 2
SSBP3 single stranded DNA binding protein 3
SSBP4 single stranded DNA binding protein 4
GADD45A growth arrest and DNA-damage-inducible, alpha GADD45B growth arrest and DNA-damage-inducible, beta
GADD45G growth arrest and DNA-damage-inducible, gamma
GADD45GIP 1 growth arrest and DNA-damage-inducible, gamma interacting protein 1
MGMT O-6-methylguanine-DNA methyltransferase
REV 1 REV 1 , polymerase (DNA directed)
RECOL RecQ protein-like (DNA helicase Ql -like)
CCDC6 coiled-coil. domain containing 6
KLRK 1 killer cell lectin-like receptor subfamily K, member 1
N6AMT1 N-6 adenine-specific DNA methyltransferase 1 (putative)
N6AMT2 N-6 adenine-specific DNA methyltransferase 2 (putative)
POLR2A polymerase (RNA) 11 (DNA directed) polypeptide A, 220kDa
POLR2B polymerase (RNA) II (DNA directed) polypeptide B, 140kDa
POLR2C polymerase (RNA) II (DNA directed) polypeptide C, 33kDa
POLR2D polymerase (RNA) II (DNA directed) polypeptide D
POLR2E polymerase (RNA) II (DNA directed) polypeptide E, 25kDa
POLR2F polymerase (RNA) II (DNA directed) polypeptide F
POLR2G polymerase (RNA) II (DNA directed) polypeptide G
POLR2H polymerase (RNA) II (DNA directed) polypeptide H
POLR2I polymerase (RNA) II (DNA directed) polypeptide I, 14.5kDa
POLR2J polymerase (RNA) II (DNA directed) polypeptide J, 13.3kDa
POLR2J2 polymerase (RNA) II (DNA directed) polypeptide J2
POLR2J3 polymerase (RNA) 11 (DNA directed) polypeptide J3
POLR2 polymerase (RNA) II (DNA directed) polypeptide , 7.0kDa
POLR2L polymerase (RNA) II (DNA directed) polypeptide L, 7.6kDa
POLR2M polymerase (RNA) II (DNA directed) polypeptide M
TRDMTl tRNA aspartic acid methyltransferase 1
CHD 1 chiOmodomain helicase DNA binding protein 1
CHD 1 L chromodomain helicase DNA binding protein 1 -like
CHD2 chromodomain helicase DNA binding protein 2
CHD3 chromodomain helicase DNA binding protein 3
CHD4 chiOmodomain helicase DNA binding protein 4
CHD5 chromodomain helicase DNA binding protein 5
CHD6 chj-omodomain helicase DNA binding protein 6
CHD7 chromodomain helicase DNA binding protein 7
CHD8 chi modomain helicase DNA binding protein 8
CHD9 chromodomain helicase DNA binding protein 9
LLN killin, p53-regulated DNA replication inhibitor
POLR3A polymerase (RNA) III (DNA directed) polypeptide A, 155kDa
POLR3B polymerase (RNA) III (DNA directed) polypeptide B POLR3C polymerase (RNA) III (DNA directed) polypeptide C (62kD)
POLR3D polymerase (RNA) III (DNA directed) polypeptide D, 44kDa
POLR3E polymerase (RNA) III (DNA directed) polypeptide E (80kD)
POLR3F polymerase (RNA) III (DNA directed) polypeptide F, 39 kDa
POLR3G polymerase (RNA) III (DNA directed) polypeptide G (32kD)
POLR3GL polymerase (RNA) III (DNA directed) polypeptide G (32kD)-like
POLR3H polymerase (RNA) III (DNA directed) polypeptide H (22.9kD)
POLR3K polymerase (RNA) III (DNA directed) polypeptide , 12.3 kDa
WDHD 1 WD repeat and HMG-box DNA binding protein 1
PGAP1 post-GPI attachment to proteins 1
PGAP2 post-GPI attachment to proteins 2
PGAP3 post-GPI attachment to proteins 3
REV3L REV3-like, polymerase (DNA directed), zeta, catalytic subunit
CDT 1 chromatin licensing and DNA replication factor 1
PANDAR promoter of CD N 1 A antisense DNA damage activated RNA
APEX 1 APEX nuclease (multifunctional DNA repair enzyme) 1
CHMP 1 A charged multivesicular body protein 1 A
CHMP 1 B charged multivesicular body protein I B
CHMP2A charged multivesicular body protein 2A
CHMP2B charged multivesicular body protein 2B
CHMP4A charged multivesicular body protein 4A
CHMP4B charged multivesicular body protein 4B
CHMP4C charged multivesicular body protein 4C
CHMP5 charged multivesicular body protein 5
CHMP6 ■ charged multivesicular body protein 6
POLRMT polymerase (RNA) mitochondrial (DNA directed)
SPIDR scaffolding protein involved in DNA repair
MCIDAS multiciliate differentiation and DNA. synthesis associated cell cycle protein
PAPD7 PAP associated domain containing 7
RFX8 RFX family member 8, lacking RFX DNA binding domain
DE DEK oncogene
NUB 1 negative regulator of ubiquitin-like proteins 1
PAXBP1 PAX3 and PAX7 binding protein 1
RAMPI receptor (G protein-coupled) activity modifying protein 1
RAMP2 receptor (G protein-coupled) activity modifying protein 2
RAMP3 receptor (G protein-coupled) activity modifying protein 3
RC3H2 ring finger and CCCH-type domains 2
ARHGAP35 Rho GTPase activating protein 35
SMUG 1 single-strand-selective monofunctional uracil-DNA glycosylase 1 CXXC 1 CXXC finger protein 1
FAM50A family with sequence similarity 50, member A
FANCG Fanconi anemia, complementation group G
GL13 GLI family zinc finger 3
GTF2H5 general transcription factor IIH, polypeptide 5
LAGE3 L antigen family, member 3
MYCNOS MYCN opposite strand/antisense RNA
NFRKB nuclear factor related to kappaB binding protein
RAD51D RAD51 paralog D
RFX2 regulatory factor X, 2 (influences HLA class II expression)
RFXANK regulatory factor X-associated ankyrin-containing protein
RRP1 ribosomal RNA processing 1
SPRTN SprT-like N-terminal domain
XRCC4 X-ray repair complementing defective repair in Chinese hamster cells 4
CDK 1 1A cyclin-dependent kinase 1 1A
CDK1 1B cyclin-dependent kinase 1 IB
LURAP 1 L leucine rich adaptor protein 1-like
MAD2L2 MAD2 mitotic arrest deficient-like 2 (yeast)
PRDM2 PR domain containing 2, with ZNF domain
NABP2 nucleic acid binding protein 2
NABP 1 nucleic acid binding protein 1
PPP1R 15A protein phosphatase 1 , regulatory subunit 15A
TATDN 1 TatD DNase domain containing 1
TATDN2 TatD DNase domain containing 2
TATDN3 TatD DNase domain containing 3
CEBPB CCAAT/enhancer binding protein (C/EBP), beta
INIP EMTS3 and NABP interacting protein
I TS3 integrator complex subunit 3
SDIM 1 stress responsive DNAJB4 interacting membrane protein 1
DHX9 DEAH (Asp-Glu-Ala-His) (SEQ ID NO: 39) box helicase 9
SATB l SATB homeobox 1
FEN 1 flap structure-specific endonuclease 1
HCST hematopoietic cell signal transducer
TYROBP TYRO protein tyrosine kinase binding protein
AFA ankyloblepharon filiforme adnatum
C9orfl69 chromosome 9 open reading frame 169
TSP02 translocator protein 2
TCIRG 1 T-cell, immune regulator 1 , ATPase, H+ transporting, lysosomal VO subunit A3 C l orf61 chromosome 1 open reading frame 61
HLA-DOA major histocompatibility complex, class II, DO alpha
SPINK 13 serine peptidase inhibitor, Kazal type 13 (putative)
[0748] In some embodiments, the payload comprises a modulator of an epigenetic state or characteristic of a component of chromatin, e.g., a chromatin associated protein, e.g., a histone. For example, the epigenetic state or characteristic can comprise histone acetylation,
deacetylation, methylation (e.g., mono, di, or tri-methylation), demethylation, phosphorylation, dephosphorylation, ubiquitination (e.g., mono or polyubiquitination), deubiquitination, sumoylation, ADP-ribosylation, deimination, or a combination thereof.
[0749] In some embodiments, the modulator is selected from, or modulates, one or more histone modifying enzymes. In an embodiment, the histone modifying enzyme is a histone
methyltransferase (HMT). In some embodiments, the histone modifying enzyme is a histone demethyltransferase (HDMT). In some embodiments, the histone modification enzyme is a histone acetyltransferase (HAT). In some embodiments, the histone modifying enzyme is a histone deacetylase (HDAC). In some embodiments, the histone modification enzyme is a kinase. In some embodiments, the histone modifying enzyme is a phosphatase. In some embodiments, the histone modifying enzyme is ubiquitin-activating enzymes (Els), ubiquitin- conjugating enzymes (E2s), or ubiquitin ligases (E3s). In some embodiments, the histone modi fying enzyme is a deubiquitinating (DUB) enzyme.
[0750] In some embodiments, histone modifications involved in regulation of gene transcription are modulated. For example, mono-methylation of H3K4, H3K9, H3K27, H3K79, H4K20, H2B 5, di-methylation of H3K79, tri-methylation of H3K4, H3K79, H3K36, and acetylation of H3K9, H3K 14, H3K27, can be associated with transcription activation. As another example, di- methylation of H3K9, H3K27, and tri-methylation of H3K9, H3K27, H3K79, H2BK5 can be associated with transcription repression. In some embodiments, the modulator modulates trimethylation of H3 lysine 4 (H3K4Me3) and/or trimethylation of H3 lysine 36 (H3K36Me3), e.g., in active genes. In other embodiments, the modulator modulates trimethylation of H3 lysine 27 (H3K27Me3), di- and tri-methylation of H3 lysine 9 (H3K9Me2/3), and/or trimethylation of H4 lysine 20 (H4K20Me3), e.g., in repressed genes. In some embodiments, the modulator modulates both activating (e.g., H3K4Me3) and repressing (e.g., H3K27Me3) marks, e.g., in stem cells.
[0751] In some embodiments, histone modifications involved in DNA damage response and repair are modulated. For example, the modulators described herein can modulate
phosphorylation of H2AX at Serine 139 and/or acetylation of H3 lysine 56 (H3K56Ac).
[0752] Aben ant histone modifications are associated with various diseases and conditions, e.g., cancer, cardiovascular disease, and neurodegenerative disorder. The modulators described herein can be used to treat a disease or condition described herein, e.g., by modulating one or more histone modifications, as described herein.
[0753] Epigenetic changes in histones can be evaluated by art-known methods or as described herein. Exemplary methods for detecting histone modifications include, e.g., chromatin immunoprecipitation (ChIP) using antibodies against modified histones, e.g., followed by quantitative PCR.
[0754] Exemplary endogenous or exogenous modulators of chromatin structure are described herein, e.g., in Table Vl-4.
Table VI-4
Figure imgf000171_0001
KMT1F
KMT2A lysine (K)-specific methyltransferase TRX1,HRX, ALL- 1 , NM_005933
2A HTRX1, CXXC7,
MLL1A
KMT2B lysine (K)-specific methyltransferase IAA0304, MLL2, NM_014727
2B TRX2, HRX2, WBP7,
MLL1B, MLL4
KMT2C lysine (K)-specific methyltransferase KIAA1506, HALR
2C
KMT2D lysine (K)-specific methyltransferase ALR, MLL4,
2D CAGL114
KMT2E lysine (K)-specific methyltransferase HDCMC04P
2E
SETD 1 A SET domain containing 1A IAA0339, Setl, NM_014712
KMT2F
SETD1B SET domain containing IB KIAA1076, SetlB, XM_037523
MT2G
ASH 1 L ashl (absent, small, or homeotic)-like huASHl, ASH1, NM_018489
(Drosophila) ASH 1 LI, KMT2H
SETD2 SET domain containing 2 HYPB,HIF-1, NM_014159
IAA1732, FLJ23184,
MT3A
NSDl nuclear receptor binding SET domain ARA267, FLJ22263, NM_172349 protein 1 KMT3B
SMYD2 SET and MYND domain containing HSKM-B, ZMYND14, NM_020197
2 KMT3C
SMYD1 ■ SET and MYND domain containing BOP, ZMYND22, XM_097915
1 KMT3D
SMYD3 SET and MYND domain containing KMT3E NM_022743
3
DOT1L DOTl-likehistone H3K79 IAA1814, DOT1, NM_032482 meth y 1 tran sf era se KMT4
SETD8 SET domain containing (lysine SET8, SET07, PR- NM_020382 methyltransferase) 8 Set7, KMT5A
SUV420H suppressor of variegation 4-20 CGI-85, MT5B NM_017635 1 homolog 1 (Drosophila)
SUV420H suppressor of variegation 4-20 MGC2705, KMT5C NM_032701
2 homolog 2 (Drosophila)
EZH2 enhancer of zeste homolog 2 EZH1, ENX-1, MT6,
(Drosophila) KMT6A EZH1 enhancer of zeste homolog 1 KIAA0388, MT6B NM_001991 (Drosophila)
SETD7 SET domain containing (lysine KIAA1717,SET7, NM_030648 methyltransferase) 7 SET7/9, Set9, KMT7
PRDM2 PR domain containing 2, with ZNF RIZ, RIZ1, RIZ2, NM_012231 domain MT8, MTB-ZF,
HUMHOXY1
HAT1 histone acetyltransferase 1 KAT1 NM_003642
KAT2A K(lysine) acetyltransferase 2 A GCN5,PCAF-b NM_021078
KAT2B K(lysine) acetyltransferase 2B P/CAF, GCN5, NM_003884
GCN5L
CREBBP CREB binding protein RTS, CBP, KAT3A NM_004380
EP300 El A binding protein p300 p300, AT3B NM_001429
TAFl TAFl RNA polymerase II, TATA NSCL2, TAFII250, NM_004606 box binding protein (TBP)-associated AT4, DYT3TAF1
factor, 250kDa
KAT5 K(lysine) acetyltransferase 5 TIP60, PLIP, cPLA2, NM_006388
HTATIP1,ESA1,
ZC2HC5
KAT6A K(lysine) acetyltransferase 6A MOZ, ZC2HC6A NM_006766
KAT6B K(lysine) acetyltransferase 6B querkopf, qkf, Morf, NM_012330
MOZ2, ZC2HC6B
KAT7 K(lysine) acetyltransferase 7 HBOA, HBOl, NM_007067
ZC2HC7
KAT8 (lysine) acetyltransferase 8 MOF, FLJ 14040, NM_032188 hMOF, ZC2HC8
ELP3 elongator acetyltransferase complex FLJ 10422, KAT9 NM_018091 subunit 3
GTF3C4 general transcription factor IIIC, TFIIIC90, KAT12
polypeptide 4, 90kDa
NCOA1 nuclear receptor coactivator 1 SRC1,F-SRC-1, NM_147223
NCoA-1, KAT13A,
RIP 160, bHLHe74
NCOA3 nuclear receptor coactivator 3 RAC3, AIB1, ACTR, NM_006534 p/CIP, TRAM-1,
CAGH16, TNRC16,
KAT13B, bHLHe42,
SRC-3, SRC3
NCOA2 nuclear receptor coactivator 2 TIF2, GRIP1, NCoA-2,
AT13C, bHLHe75 CLOCK clock circadian regulator KIAA0334, KAT13D, NM_004898 bHLHe8
KDM1A lysine (K)-specific demethylase 1 A KIAA0601.BHC110, NM_015013
LSD1
KDM1B lysine (K)-specific demethylase IB FLJ34109, FLJ33898, NM_ 153042 dJ298J15.2,
bA204B7.3, FLJ43328,
LSD2
KDM2A lysine (K)-specific demethylase 2A K1AA1004, FBL11, NM_012308
LILINA,
DKFZP434M1735,
FBL7, FDO0115,
CXXC8, JHDM1A
KDM2B lysine (K)-specific demethylase 2B PCCX2, CXXC2, NM_032590
FbllO, JHDM1B
KDM3A lysine (K)-specific demethylase 3A TSGA, KIAA0742, ' NM_018433
JHMD2A
KDM3B lysine (K)-specific demethylase 3B KIAA1082, NET22 NM_016604
KDM4A lysine (K)-specific demethylase 4A KIAA0677, JHDM3A, NM_014663
TDRD14A
KDM4B lysine (K)-specific demethylase 4B KIAA0876, TDRD14B NM_01501
KDM4C lysine (K)-specific demethylase 4C GASC1, KIAA0780, NM_015061
TDRD14C
KDM4D lysine (K)-specific demethylase 4D FLJ 10251 NM_018039
KDM4E ■ lysine (K)-speci ic demethylase 4E JMJD2E NM_001161630
KDM5A lysine (K)-specific demethylase 5A NM_005056
KDM5B lysine (K)-specific demethylase 5B RBBP2H1A, PLU-1, NM_006618
CT31
KDM5C lysine (K)-specific demethylase 5C DXS1272E, XE169 NM_004187
KDM5D lysine (K)-specific demethylase 5D ΚΓΑΑ0234 NM_004653
KDM6A lysine (K)-specific demethylase 6A NM_021140
KDM6B lysine (K)-specific demethylase 6B KIAA0346 XM_043272
JHDM1D jumonji C domain containing histone KIAA1718 NM_030647 demethylase 1 homolog D (S.
cerevisiae)
PHF8 PHD finger protein 8 ZNF422, KIAA1111, NM_015107
JHDM1F
PHF2 PHD finger protein 2 KIAA0662, JHDM1E, NM_005392
CENP-35
KDM8 lysine (K)-specific demethylase 8 FLJ 13798 NM_024773 Modulators of Gene Expression
[0755] In an embodiment a payload comprises a modulator of gene expression. A modulator of gene expression can be delivered in vitro, ex vivo, or in vivo.
[0756] In an embodiment, the payload comprises a transcription factor. Transcription factors can bind to specific DNA sequences (e.g., an enhancer or promoter region) adjacent to the genes that they regulate. For example, transcription factors can stabilize or inhibit the binding of RNA polymerase to DNA, catalyze the acetylation or deacetylation of histone proteins (e.g., directly or by recruiting other proteins with such catalytic activity), or recruit coactivator or corepressor proteins to the transcription factor DNA complex. Modulators of gene expression also include, e.g., any proteins that interact with transcription factors directly or indirectly.
[0757] In an embodiment, the transcription factor is a general transcription factor, e.g., is ubiquitous and interacts with the core promoter region surrounding the transcription start site(s) of many, most or all class IT genes. Exemplary general transcription factors include, e.g., TFI1A, TF1IB, TFIID, TFIIE, TFIIF, and TFIIH. In an embodiment, the transcription factor is an upstream transcription factor, e.g., binds upstream of the initiation site to stimulate or repress transcription. In an embodiment, the transcription factor is a specific transcription factor, e.g., a transcription factor dependent on a recognition sequence present in the proximity of the gene. Exemplary specific transcription factors include, e.g., SP1, AP- 1 , C EBP, heat shock factor, ATF/CREB, -Myc, OCT- 1 , and NF- 1 .
[0758] In an embodiment, the transcription factor is constitutively active, e.g., a general transcription factor, SP 1 , NF- 1 , or CCAAT. In other embodiments, the transcription factor is conditionally active, e.g. it requires activation, e.g., developmental (e.g., GATA, HNF, PIT-1 , MyoD, Myf5, Hox, Winged Helix), signal-dependent (e.g., extracellular ligand (endocrine or paracrine)-dependent, intracellular ligand (autocrine)-dependent (e.g., SREBP, p53, orphan nuclear receptors), cell membrane receptor-dependent (e.g., resident nuclear factors (e.g., CREB, AP- 1 , Mef2) or latent cytoplasmic factors (e.g., STAT, R-SMAD, NF-κΒ, Notch, TUBBY, NFAT). [0759] Other exemplary transcription factors are described herein, e.g., in Tables VI-5 and V I-6. (Table VI-5 Transcription Factors, is provided in Annex VI-5)
Table VI-6: Selected Transcription Factors with Anotations
Figure imgf000176_0001
angiogenesis, enhanced survival of tumors and immunosuppression.
Thyroid hormone Involved in widely diverse physiological functions, including control receptor(25) of embryonic development, cell differentiation and homeostasis zf-C2HC(6) Highly transcribed in the developing nervous system. Exemplary diseases include Duane Radial Ray Syndrome.
Androgen Exemplary functions include diverse physiological functions, receptorf 1 ) including control of embryonic development, cell differentiation and homeostasis. Exemplary diseases include X-linked spinal, bulbar muscular atrophy and prostate cancer.
CG- 1 C2) Exemplary functions include calcium signaling by direct binding of calmodulin.
CTF/NF 4) Exemplary functions include both viral DNA replication and
regulation of gene expression. Exemplary diseases include leukemia, juvenile myelomonbcytic.
Fork headf49) Involvement in early developmental decisions of cell fates during embryogenesis. Exemplary diseases include lymphedema-distichiasis, developmental verbal dyspraxia, autoimmune diseases.
Homeobox(205) Exemplary functions include involvement in a wide range of critical activities during development. Exemplary diseases include limb malformations, eye disorders, and abnormal head, face, and tooth development. Additionally, increased or decreased activity of certain homeobox genes has been associated with several forms of cancer.
MYB(25 Exemplary functions include regulator of proliferation, differentiation and cell fate. Exemplary diseases include cancer (e.g., oncogenic disease).
Oestrogen Control of embryonic development, cell differentiation and receptor( 1 ) homeostasis. Exemplary diseases include estrogen resistance, familial breast cancer, migrane, myocardial infaction.
POU 21 ) Wide variety of functions, related to the function of the
neuroendocrine system and the development of an organism.
Exemplary diseases include non-syndromic deafness.
RHDH O") Exemplary diseases include autoimmune arthritis, asthma, septic shock, lung fibrosis, glomerulonephritis, atherosclerosis, and AIDS.
T-box( 17)
TSC22(4)
zf-GATA( 14) AP-2(5)
COE(4)
CUT(7)
GCM(2)
HSFC8)
NDT80/PhoGfl
Other nuclear - receptor(2)
PPAR
receptor(3)
ROR receptor(4)
TEA (4)
Tub(5)
zf-LITAF-like(2)
ARID05)
COUP(3)
DMf7)
GCR(l)
ΗΤΗΓ2)
NF-ΥΑΠ)
OthersO)
Progesterone
receptor( 1 )
RuntO)
TF bZIP(46)
ZBTB(48)
zf- IZ(7)
bHLHil06
CP27
E2FC1 n
GTF2 5
IRF(9)
NF-YB/CC2)
P53G
Proxl(2)
SAND(8)
TF Otx(3)
zf-BED(5)
zf-NF-Xl(2) C/EBP( 10)
CSD 8)
Ecdystd
receptor(2)
HMGC50
MBDf9)
NrfK l )
PAX(9)
Retinoic acid
receptor(7)
SRF(6)
ΤΗΑΡΠ 2)
zf-C2H2(634)
CRX Exemplary diseases include dominant cone-rod dystrophy. Repair mutation.
FOCX2 Exemplary diseases include lymphedema-distichiasis. Repair
mutation.
FO P2 Exemplaiy diseases include developmental verbal dyspraxia. Repair mutation.
FOXP3 Exemplary diseases include autoimmune diseases. Repair mutation.
GAT4 Exemplary diseases include congenital heart defects. Repair mutation.
HNF1 through Exemplary diseases include mature onset diabetes of the young HNF6 (MODY), hepatic adenomas and renal cysts. Repair mutation.
LHX3 Exemplaiy diseases include Pituitary disease. Repair mutation.
MECP2 Exemplary diseases include Rett syndrome. Repair mutation.
MEF2A Exemplary diseases include Coronary arteiy disease. Repair mutation.
NARA2 Exemplary diseases include Parkinson disease. Repair mutation.
NF-KB Exemplary diseases include autoimmune arthritis, asthma, septic Activation shock, lung Fibrosis, glomerulonephritis, atherosclerosis, and AIDS.
Repair mutation.
NF-KB Inhibition Exemplaiy diseases include apoptosis, inappropriate immune cell development, and delayed cell growth. Repair mutation. NIKX2-5 Exemplary diseases include cardiac malformations and
atrioventricular conduction abnormalities.
NOTCH 1 Exemplary diseases include aortic valve abnormalities.
Modulators of alternative splicing
[0760] In an embodiment, the modulator of gene expression modulates splicing. For example, a modulator can modulate exon skipping or cassette exon, mutually exclusive exons, alternative donor site, alternative acceptor site, intron retention, or a combination thereof. In some ' embodiments, the modulator is selected from or modulates one or more general or alternative splicing factors, e.g., ASF1. In some embodiments, the modulator modulates alternative splicing (e.g., influences splice site selection) in a concentration-dependent manner.
Modulators of post-transcriptional modification
[0761] In an embodiment, the modulator of gene expression modulates post-transcriptional modification. For example, the modulators described herein can promote or inhibit 5' capping, 3' polyadenylation, and RNA splicing. In an embodiment, the modulator is selected from, or modulates, one or more factors involved in 5' capping, e.g., phosphatase and guanosyl transferase. In an embodiment, the modulator is selected from, or modulates, one or more factors involved in 3' polyadenylation, e.g., polyadenylate polymerase, cleavage and
polyadenylation specificity factor (CPSF), and poly(A) binding proteins. In an embodiment, the modulator is selected from, or modulates, one or more factors involved in RNA splicing, e.g., general or alternative splicing factors.
[0762] Exemplary endogenous or exogenous modulators of post-transcriptional modification are described herein, e.g., in Table VI-7.
Table VI-7
Figure imgf000180_0001
PAN: PolyA nuclease
CPSF: cleavage/polyadenylation specificity factor CstF: cleavage stimulation factor
PAP: polyadenylate polymerase
PABP: polyadenylate binding protein
PAB2: polyadenylate binding protein 2
CFI: cleavage factor I
CFII: cleavage factor Π
Capping/Mcthylation of 5'end
RNA triposphatase
RNA gluanyltransferase
RNA mehyl transferase
SAM synthase
ubiquitin-conjugating enzyme E2R 1
Splicing
SR proteins SFRS 1 - SFR 1 1 which, when bound to exons, tend to promote
hnRNP proteins: coded by the following genes:HNRNPA0, HNRNPA 1 , HNRNPA 1L1 ,
HNRNPA 1L2, HNRNPA3, HNRNPA2B 1 ,
HNRNPAB, HNRNPB 1 , HNRNPC, HNRNPCL1 ,
HNRNPD, HNRPDL, HNRNPF, HNRNPH 1 ,
HNRNPH2, HNRNPH3, HNRNPK, HNRNPL,
HNRPLL, HNRNPM, HNRNPR, HNRNPU,
HNRNPUL1 , HNRNPUL2, HNRNPUL3
Editing protein
ADAR
Nuclear export proteins
Mex67
Mtr2
Nab2
DEAD-box helicase ("DEAD" disclosed as SEQ ID
. NO: 40)
TRANSLATION
Initiation
eIF4A, eIF4B, elF4E, and eIF4G: Eukaryotic initiation factors
GEF: Guanine exchange factor
GCN2, PKR, HRI and PERK: Kinases involved in phosphorylating some of the initiation factors
Elongation
eEFl and eEF2: elongation factors
GCN: kinase Termination
eRF3: translation termination factor
POS^ r-TRANSLATIONAL CONTROL
mRNA Degradation
ARE-specific binding proteins
EXRN 1 : exonuclease
DCP1 , DCP2: Decapping enzymes
RCK/p54, CPEB, eIF4E: Translation repression microRNAs and siRNAs: Probably regulate 30% of all genes
DICER
Ago proteins
Nonsense-mediated mRNA decay proteins
UPF3A
UPF3B
eIF4A3
MLN51
Y 14/MAGOH
MG- 1
SMG-5
SMG-6
SMG-7
mRNA Modification
Enzymes carry the following functions
Phosphorylation
N-linked glycosylation
Acetylation
Amidation
Hydroxylation
Methylation
O-linked glycosylation
Ubiquitylation
Inhibitors
[0763] In an embodiment a payload comprises an inhibitor of a payload described above, e.g., an inhibitor of an enzyme transcription factor. In an embodiment a payload comprises an inhibitor of any of the aforementioned payload molecules, processes, activities or mechanisms. In an embodiment, the inhibitor is an antibody molecule (e.g., a full antibody or antigen binding fragment thereof) specific for one of the payload molecules described herein. In an embodiment the inhibitor is a small molecule compound. In some embodiments, the inhibitor is a nucleic acid (e.g., siRNA, shRNA, ribozyme, antisense-oligonucleotide, and aptamer). For example, the payload is an inhibitor of a target, e.g., a trasnscription factor, a post-translational modification enzyme, a post-transcriptional modification enzyme, etc., or a nucleic acid sequence encoding any of the foregoing.
Oithologs
[0764] If a non-human gene or protein is recited herein it is understood that the invention also comprises the human counterpart or ortholog and uses thereof.
VI1A. TARGETS: CELLS
[0765] Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., an animal cell or a plant cell), e.g., to deliver a payload, or edit a target nucleic acid, in a wide variety of cells. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid. Delivery or editing can be performed in vitro, ex vivo, or in vivo.
[0766] In some embodiments, a cell is manipulated by editing (e.g., introducing a mutation or correcting) one or more target genes, e.g., as described herein. In other embodiments, a cell is manipulated by delivering a payload comprising one or more modulators (e.g., as described herein) to the cell, e.g., to a target sequence in the genome of the cell. In some embodiments, the expression of one or more target genes (e.g., one or more target genes described herein) is modulated, e.g., in vivo. In some embodiments, the expression of one or more target genes (e.g., one or more target genes described herein) is modulated, e.g., ex vivo. '
[0767] In some embodiments, the cells are manipulated (e.g., converted or differentiated) from one cell type to another. In some embodiments, a pancreatic cell is manipulated into a beta islet cell. In some embodiments, a fibroblast is manipulated into an iPS cell. In some embodiments, a preadipocyte is manipulated into a brown fat cell. Other exemplary cells include, e.g., muscle cells, neural cells, leukocytes, and lymphocytes. [0768] In some embodiments, the cell is a diseased or mutant-bearing cell. Such cells can be manipulated to treat the disease, e.g., to correct a mutation, or to alter the phenotyope of the cell, e.g., to inhibit the growth of a cancer cell. For examples, a cell is associated with one or more diseases or conditions describe herein. In some embodiments, the cell is a cancer stem cell. For example, cancer stem cells can be manipulated by modulating the expression of one or more genes selected from: TWIST (TF), ΗΓΡ- 1 a, HER2/neu, Snail (TF), or Wnt.
[0769] In some embodiments, the manipulated cell is a normal cell.
[0770] In some embodiments, the manipulated cell is a stem cell or progenitor cell (e.g., iPS, embryonic, hematopoietic, adipose, germline, lung, or neural stem or progenitor cells).
[0771 ] In some embodiments, the manipulated cells are suitable for producing a recombinant biological product. For example, the cells can be CHO cells or fibroblasts. In an embodiment, a manipulated cell is a cell that has been engineered to express a protein.
[0772] In some embodiments, the cell being manipulated is selected from fibroblasts, monocytic precursors, B cells, exocrine cells, pancreatic progenitors, endocrine progenitors, hepatoblasts, myoblasts, or preadipocytes. In some embodiments, the cell is manipulated (e.g., converted or differentiated) into muscle cells, erythroid-megakaryocytic cells, eosinophils, iPS cells, macrophages, T cells, islet beta-cells, neurons, cardiomyocytes, blood cells, endocrine progenitors, exocrine progenitors, ductal cells, acinar cells, alpha cells, beta cells, delta cells, PP cells, hepatocytes, cholangiocytes, or brown adipocytes.
[0773] In some embodiments, the cell is a muscle cell, erythroid-megakaryocytic cell, eosinophil, iPS cell, macrophage, T cell, islet beta-cell, neuron, cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or white or brown adipocyte.
[0774] The Cas9 and gRNA molecules described herein can be delivered to a target cell. In an embodiment, the target cell is a normal cell.
[0775] In an embodiment, the target cell is a stem cell or progenitor cell (e.g., iPS, embryonic, hematopoietic, adipose, germline, lung, or neural stem or progenitor cells).
[0776] In an embodiment, the target cell is a CHO cell. [0777] In an embodiment, the target cell is a fibroblast, monocytic precursor, B cells exocrine cell, pancreatic progenitor, endocrine progenitor, hepatoblast, myoblast, or preadipocyte.
[0778] In an embodiment, the target cell is a muscle cell, erythroid-megakaryocytic cell, eosinophil, iPS cell, macrophage, T cell, islet beta-cell, neurons (e.g., a neuron in the brain, e.g., a neuron in the striatum (e.g., a medium spiny neuron), cerebral cortex, precentral gyrus, hippocampus (e.g., a neuron in the dentate gyrus or the CA3 region of the hippocampus), temporal cortex, amygdala, frontal cortex, thalamus, cerebellum, medulla, putamen, hypothalamus, tectum, tegmentum or substantia nigra), cardiomyocyte, blood cell, endocrine progenitor, exocrine progenitor, ductal cell, acinar cell, alpha cell, beta cell, delta cell, PP cell, hepatocyte, cholangiocyte, or brown adipocyte.
[0779] In an embodiment, the target cell is manipulated ex vivo by editing (e.g., introducing a mutation or correcting) one or more target genes and/or modulating the expression of one or more target genes, and administered to the subject.
[0780] Exemplary cells that can be manipulated and exemplary genes that can be modulated are described in Table VII-8.
TableVII-8
Figure imgf000185_0001
in vivo. Multiplex.
B cells Macrophages Deliver Cas9-activators to C/EBPot target activation of
transcription factors
required for differentiation
in vivo.
B cells T cells, Delivery Cas9-repressors Pax5 macrophages OR deliver Cas9
endonuclease to ablate
Pax5
Exocrine Islet β-cells Deliver Cas9-activators to Pdx l cells target activation of Ngn3 transcription factors MafA required for differentiation
in vivo. Multiplex.
Fibroblasts Neurons Deliver Cas9-activators to A sel l target activation of Bm2 transcription factors Mytl l required for differentiation
in vivo. Multiplex.
fibroblasts cardiomyocytes Deliver Cas9-activators to Gata4 target activation of Mef2c transcription factors Tbx5 required for differentiation
in vivo. Multiplex.
Fibroblasts Blood cells Deliver Cas9-activators to Oct4 target activation of
transcription factors
required for differentiation
in vivo.
Fibroblasts cardiomyocytes Deliver Cas9-activators to Oct4 target activation of Sox2 transcription factors lf4 required for differentiation
in vivo. Multiplex.
Pancreatic Endocrine Deliver Cas9-activators to Ngn3 progenitor progenitor target activation of
transcription factors
required for differentiation
in vivo. Pancreatic .Exocrine Deliver Cas9-activators to P48 progenitor progenitor target activation of
transcription factors
required for differentiation
in vivo.
Pancreatic Duct Deliver Cas9-activators to Hnf6/OC- l progenitor target activation of
transcription factors
required for differentiation
in vivo.
Pancreatic acinar Deliver Cas9-activators to Ptfla progenitor target activation of Rpbjl transcription factors
required for differentiation
in vivo. Multiplex.
Endocrine a cell Deliver Cas9-activators to Foxa2 progenitor target activation of Nkx2.2
(to make transcription factors Pax6 glucagon) required for differentiation Arx
in vivo. Multiplex.
Endocrine β cell Deliver Cas9-activators to Mafa progenitor target activation of Pdx l
(to make transcription factors Hlxb9 insulin) required for differentiation Pax4
in vivo. Multiplex. Pax6
Isl l
Nkx2.2 Nkx6.1
Endocrine δ cell Deliver Cas9-activators to Pax4 progenitor target activation of Pax6
(to make transcription factors
somatostatin) required for differentiation
in vivo. Multiplex.
Endocrine PP cell Deliver Cas9-activators to Nkx2.2 progenitor target activation of
(to make transcription factors
pancreatic required for differentiation
polypeptide) in vivo.
Hepatoblast hepatocyte Deliver Cas9-activators to Hnf4
target activation of transcription factors
required for differentiation
in vivo.
Hepatoblast Cholangiocyte Deliver Cas9-activators to Hnf6/OC- l target activation of
transcription factors
required for differentiation
in vivo.
Myoblasts Brown adipocyte Deliver Cas9-activators to PRDM 16 target activation of C/EBP transcription factors PGCl CC required for differentiation PPARy in vivo. Multiplex.
preadipocytes Brown adipocyte Deliver Cas9-activators to PRDM 16 target activation of C EBP transcription factors
required for differentiation
in vivo. Multiplex.
Table VII-9: Exemplary cells for manipulation
Pancreatic cells, e.g., beta cells
Muscle cells
Adipocytes
Pre-adipocytes
Neural cells
Blood cells
Leukocytes
Lymphocyes
B cells
T cells
Table VII-10: Exemplaiy stem cells for manipulation
embryonic stem cells
non-embryonic stem cells
hematopoietic stem cells
adipose stem cells
germline stem cells
lung stem cells
neural stem cells Table VII-11 : Exemplaiy cancer cells for manipulation
lung cancer cells
breast cancer cells
skin cancer cells
brain cancer cells,
pancreatic cancer cells
hematopoietic cancer cells
liver cancer cells
kidney cancer cells
ovarian cancer cells
Table VTI-12: Exemplaiy non-human cells for manipulation
Table VII- 12 Non-human cells for manipulation
Plant cells, e.g., crop cells, e.g., corn, wheat,
soybean, citrus or vegetable cells
Animal cells, e.g., a cow, pig, horse, goat, dog or cat
cell
[0781] Exemplary endogenous or exogenous modulators of cancer stem cells (CSCs) are described herein, e.g., in Table VII-13:
Table VII-13
• TWIST 1 (TF)
• HIF- la (TF)
• HER2/neu
• Snail (TF)
• Wnt
• TGFP
• FGF
• EGF
• HGF
• STAT3 (TF)
• Notch
• P63 (TF) • PI3K)/AKT
• Hedgehog
• NF-κΒ (TF)
• ATF2 (TF)
• miR-200 and miR-34
• P53 (TF)
• E-cadherin
• Transcription factors that inhibit E-cadherin directly .
• ZEB 1
• ZEB2
• E47
• KLF8
• Transcription factors that inhibit E-cadherin directly
• TCF4
• SIX 1
• FOXC2
• G-CSF and CD34 in A ML
• PML and FOXO in CML
• CD133 in glioblastoma multiforme, osteosarcoma, Ewing's sarcoma,
endometrial, hepatocellular, colon and lung carcinomas and ovarian
and pancreatic adenocarcinoma
• CD44 in head and neck cancer, prostate, gastric and colorectal
carcinoma stem cells
• CD34 in leukemia
• CD38 in leukemia
β IL3Ra in leukemia
• EpCAM in colon carcinoma and pancreatic adenocarcinoma stem
cells
• ALDH in melanoma, colorectal, breast, prostate and squamous cell
carcinomas, pancreatic adenocarcinoma, and osteosarcoma
■ MAP2 in melanoma
t a6-integrin in glioblastoma
• SSEA-1 in gliobalstoma
• CD24 in breast cancer and other tumors
[0782] Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., a cell described herein), e.g., to deliver a payload, or edit a target nucleic acid, e.g., to increase cell engraftment, e.g., to achieve stable engraftment of cells into a native microenvironment. The engrafting cells, the cells in the native microenvironment, or both, can be manipulated. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid.
[0783] For example, increased efficiency of engraftment of cells can be achieved by: increasing the expression of one or more of the genes described herein, e.g., homing genes, adhesion genes, survival genes, proliferative genes, immune evasion genes, and/or cell protection genes, and/or decreasing the expression of one or more of the genes described herein, e.g., quiescence genes, death/apoptosis genes, and/or immune recognition genes.
[0784] In an embodiment, the gene encodes a homing receptor or an adhesion molecule, e.g., that is involved in directing cell migration towards a tissue in association with a tissue-expressed ligand or region rich in soluble cytokine. In an embodiment, the homing receptor or adhesion molecule is expressed on leukocytes, e.g., lymphocytes or hematopoietic stem cells. In an embodiment, the tissue is bone marrow, e.g., extracellular matrix or stromal cells. In an embodiment, the homing receptor or adhesion molecule is C-X-C chemokine receptor type 4 (CXCR4, also known as fusin or CD184). For example, the expression of CXCR4 on hematopoietic stem cells is upregulated. In an embodiment, the ligand is stromal-derived-factor- 1 (SDF- 1 , also known as CXCL12). In an embodiment, the homing receptor or adhesion molecule is CD34. In an embodiment, the ligand is addressin (also known as mucosal vascular addressin cell adhesion molecule 1 (MAdCAM- 1 )).
[0785] In an embodiment, the gene encodes a receptor, e.g., expressed on a stem cell or progenitor cell, that binds to a ligand, e.g., a chemokine or cytokine. For example, the receptor can be associated with sternness of the cell and/or attracting the cell to a desired
microenvironment. In an embodiment, the receptor is expressed on a hematopoietic stem cell. a In an embodiment, the receptor is expressed on a neural stem cell. In an embodiment, the receptor is mast/stem cell growth factor receptor (SCFR, also known as proto-oncogene c-Kit or tyrosine-protein kinase Kit or CD 1 17). In an embodiment, the ligand is stem cell factor (SCF, also known as steel factor or c-kit ligand). In an embodiment, the receptor is myeloproliferative leukemia virus oncogene (MPL, also known as CD 1 10). In an embodiment, the ligand is thrombopoietin (TPO). [0786] In an embodiment, the gene encodes a marker, e.g., that promotes survival or proliferation of the cells expressing that marker, or allows the cells expressing that marker to evade an immune response or to be protected from an adverse environment, e.g., that leads to cell death. For example, cells expressing CD47 (also known as integrin associated protein (IAP) can avoid phagocytosis, e.g., during cell migration. As another example, cells that express BCL2 can be protected from apoptosis. In an embodiment, the cell is a blood cell, e.g., an erythrocyte or leukocyte. In an embodiment, the cell is a hematopoietic stem cell or progenitor cell.
[0787] In an embodiment, the expression of one or more of CXCR4, SDF1, CD 1 17, MPL, CD47, or BCL2, in a stem cell or progenitor cell, e.g., a hematopoietic stem cell or progenitor cell, is upregulated.
[0788] Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., a cell described herein), e.g., to deliver a payload, or edit a target nucleic acid, e.g., to manipulate (e.g., dictate) the fate of a targeted cell, e.g., to better target specific cell type of interest and/or as a suicide mechanism. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and/or an eaCas9
molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid. Exemplaiy genes that can be modulated include, e.g., one or more of chemotherapy resistance genes, chemotherapy sensitivity genes, antibiotic resistance genes, antibiotic sensitivity genes, and cell surface receptor genes, e.g., as described herein.
[0789] In an embodiment, a chemotherapy resistance gene, a chemotherapy sensitivity gene, an antibiotic resistance gene, and/or an antibiotic sensitivity gene is modulated, e.g., such that modified or undesirable cells (e.g., modified or undesirable hematopoietic stem cells (HSCs), e.g., in bone marrow) can be reduced or removed, e.g., by chemotherapeutic or antibiotic treatment.
[0790] For example, genes or gene products that modulate (e.g., increase) chemotherapy resistance or antibiotic resistance can be delivered into the cells. Cells modified by the chemotherapy or antibiotic resistance gene or gene product can have a higher (e.g., at least about 2, 3, 4, 5, 6, 7, 8. 9, 10, 25, 50, 75, or 100 fold higher) survival rate than cells without such ' modification after chemotherapeutic or antibiotic treatment. In an embodiment, the chemotherapeutic or antibiotic'treatment is performed in vivo. In an embodiment, the chemotherapeutic or antibiotic treatment is performed in vilro or ex vivo. In an embodiment, the chemotherapy resistance gene is a gene encoding 06-alkylguanine DNA alkyltransferase (MGMT). In an embodiment, the chemotherapy comprises temozolomide.
[0791] As another example, genes or gene products that modulate (e.g., increase) chemotherapy sensitivity or antibiotic sensitivity can be delivered into the cells. The genes or gene products that confer chemotherapy sensitivity or antibiotic sensitivity can be used as suicide, signals, e.g., causing apoptosis of the cells. Cells modified by the chemotherapy or antibiotic sensitivity gene or gene product can have a lower (e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, or 100 fold lower) survival rate than cells without such modification after chemotherapeutic or antibiotic treatment. In an embodiment, the chemotherapeutic or antibiotic treatment is performed in vivo. In an embodiment, the chemotherapeutic or antibiotic treatment is performed in vilro or ex vivo.
[0792] The method described herein can be used to select or enrich cells that have a modified or desired phenotype, e.g., chemotherapy resistance and/or antibiotic resistance. The method described herein can also be used to remove or reduce the number of cells that have a modified or undesired phenotype, e.g., chemotherapy sensitivity and/or antibiotic sensitivity. For example, cells that exhibit an undesired effect, e.g., an off-target effect or a cancer phenotype, e.g., caused by editing of a nucleic acid in an undesired genomic location or cell type, can be removed.
[0793] In an embodiment, a cell surface receptor gene is modulated (e.g., the expression of the cell surface receptor is increased or decreased), such that a therapeutic agent (e.g., a therapeutic antibody) can be used to target a cell (e.g., to kill the cell) that has increased or decreased expression of the cell surface receptor. In an embodiment, the cell surface receptor is CD20. In some embodiments, the therapeutic antibody is Rituximab.
[0794] In an embodiment, the cell surface receptor is selected from, e.g., CD52, VEGFR, CD30, EGFR, CD33, or ErbB2. In an embodiment, the therapeutic antibody is selected from, e.g., Alemtuzumab, Rituximab, Cetuximab, Panitumumab, Gentuzaumab, and Trasruzumab. In an embodiment, the cell surface receptor is CD52 and the therapeutic antibody is Alemtuzumab. In an embodiment, the gene encodes VEGF and the therapeutic antibody is Rituximab. In an embodiment, the cell surface receptor is EGFR and the therapeutic antibody is Cetuximab or Panitumumab. In an embodiment, the cell surface receptor is CD33 and the therapeutic antibody is Gentuzaumab. In an embodiment, the cell surface receptor is ErbB2 and the therapeutic antibody is Trastuzumab.
[0795] In an embodiment, the expression or activity of the Cas9 molecule and/or the gRNA molecule is induced or repressed, e.g., when the cell is treated with a drug, e.g., an antibiotic, e.g., in vivo. For example, the induction or repression of the expression or activity of the Cas9 molecule and/or the gRNA molecule can be used to reduce toxicity and/or off-target effects, e.g., in certain tissues. In an embodiment, the expression of the Cas9 molecule, the gRNA molecule, or both, is driven by an inducible promoter. In an embodiment, binding of a drug (e.g., an antibiotic) to the Cas9 molecule and/or the gRNA molecule activates or inhibits the activity of the Cas9 molecule and/or the gRNA molecule. In an embodiment, the drug (e.g., antibiotic) is administered locally. In an embodiment, the cell treated with the drug (e.g., antibiotic) is located in the eye, ear, nose, mouth, or skin.
[0796] Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., a cell described herein), e.g., to deliver a payload, or edit a target nucleic acid, e.g., in directed enzyme prodrug therapy (DEPT). Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid.
[0797] Directed enzyme prodrug therapy (DEPT) uses enzymes artificially introduced into the body to convert prodrugs, which have no or poor biological activity, to the active form in the desired location within the body. For example, directed enzyme prodrug therapy can be used to reduce the systemic toxicity of a drug, by achieving high levels of the active drug only at the desired site.
[0798] In an embodiment, an enzyme required for prodrug conversion or a gene encoding such an enzyme is delivered to a target cell, e.g., a cancer cell. For example, the enzymes or genes can be delivered by a method described herein. In an embodiment, the gene encoding the enzyme required for prodrug conversion is delivered by a viral vector.
[0799] Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate a cell (e.g., a cell described herein), e.g., to deliver a payload, or edit a target nucleic acid, e.g., to improve immunotherapy, e.g. cancer immunotherapy. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid. Exemplary genes that can be modulated include, e.g., one or more genes described herein, e.g., PD-L1 and/or PD-L2 genes.
VIIB. TARGETS: PATHWAYS AND GENES
[0800] Cas9 molecules and gRNA molecules, e.g., a Cas9 molecule/gRNA molecule complex, can be used to manipulate one, two, three or more, elements or a pathway, e.g., by targeting sequences that encode an RNA or protein of a pathway, or sequences that control the expression of an RNA or protein of a pathway. In an embodiment, an element of a first pathway and an element of a second pathway are manipulated. In an embodiment, manipulation comprises delivery of a payload to, or editing, a target nucleic acid. Typically an eiCas9 molecule/gRNA molecule complex is used to deliver a payload and an eaCas9 molecule/gRNA complex is used to edit or alter the structure of a target nucleic acid. Delivery or editing can be performed in vitro, ex vivo, or in vivo.
[0801] An element of a pathway can be up or down regulated, e.g., the expression of a gene encoding a protein of a pathway can be increased or decreased. The increase or decrease can be effected by delivery of a payload (e.g., a transcription factor or inhibitor of a transcription factor) or by editing a target nucleic acid (e.g., the use of a template nucleic acid to alter a sequence, e.g., correct or introduce a mutation, in e.g., a control or coding region).
[0802] Exemplary pathways comprise pathways associated with: cell proliferation; cell cycle; carbon metabolism; energy metabolism; glycolysis, anerobic respiration, anerobic respiration; transmembrane signal transduction, angiogenesis, DNA. replication or repair, or pain.
[0803] Exemplary pathways and genes are discussed herein. It will be understood that a pathway or gene can be associated with one or more aspect of cell or organismal function, e.g., a pathway or gene can be involved in both cancer and energy metabolism. Manipulation of a pathway or gene is not limited to the exemplary cell or organismal function listed below. In an embodiment a pathway is associated with one or more diseases or conditions. [0804] In an embodiment, the pathway is associated with cancer, e.g., associated with proliferation (e.g., RAF pathway), evading growth repressors, resisting cell death, enabling replicative immortality/aging, inducing angiogenesis, activating invasion and metastasis, energy metabolism and evading, cancer stem cells, cytokine-receptor interactions, or tumor suppressors. In some embodiments, the pathway is associated with cell cycle control. In some embodiments, the pathway is associated with angiogenesis.
[0805] Pathways and genes associated with cancer are described herein, e.g., include the following:
Table VII-14 Target Genes from Selected Pathways
Figure imgf000196_0001
FAK Proliferation Down
PKA Proliferation Down
RAC Proliferation Down
ALK Proliferation Mutated in a subset (2-7%) of lung
cancers
Rb Evading growth Up suppressors/ pro- apoptotic
P53 Evading growth Mutation in colon, lung, esophagus, Up suppressors/ pro- breast, liver, brain reticuloendothelial apoptotic tissues, and hemopoietic tissues
APC Evading growth Mutations found in colon and intestine
suppressors/ pro- apoptotic
CD 4/6 Evading growth Up suppressors/ pro- apoptotic
INK4B Evading growth Up suppressors/ pro- apoptotic
CD 2 Evading growth Up suppressors/ pro- apoptotic
WNT Evading growth Up suppressors/ pro- apoptotic
WAF 1 Evading growth Up suppressors/ pro- apoptotic
Frizzled Evading growth Up suppressors/ pro- apoptotic
VHL Evading growth Mutated in all clear cell renal Up suppressors/ pro- carcinomas
apoptotic
Fas ligand Resisting cell death/ Down anti-apoptotic
Fas receptor Resisting cell death/ Down anti-apoptotic
Caspase 8 Resisting cell death/ Down anti-apoptotic
Caspase 9 Resisting cell death/ Down anti-apoptotic
Bcl-2 Resisting cell death/ Correct mutation large deletion in Down anti-apoptotic follicular lymphoma, breast prostate
CLL, melanoma
Bcl-xL Resisting cell death/ Down anti-apoptotic
Bcl-w Resisting cell death/ Down anti-apoptotic
Mcl- 1 Resisting cell death/ Down anti-apoptotic
Bax Resisting cell death/ Down anti-apoptotic
Bak Resisting cell death/ Down anti-apoptotic
IGF- 1 Resisting cell death/ Down anti-apoptotic
Puma Resisting cell death/ Down anti-apoptotic
Bim Resisting cell death/ Down anti-apoptotic
Beclin- 1 Resisting cell death/ Down anti-apoptotic
TGF-b Enabling replicative
immortality/aging
Telomerase/TERT Enabling replicative Down immortality/aging
ATAD2 Enabling replicative
immortality/aging
DAF-2 Enabling replicative
immortality/aging
SRT Enabling replicative
immortality/aging
Eph-A/B Inducing angiogenesis Down
Robo Inducing angiogenesis Down
Neuropilin Inducing angiogenesis Down
Notch Inducing angiogenesis Down
Endostatin Inducing angiogenesis Down
Angiostatin Inducing angiogenesis Down FGF family Inducing angiogenesis Down
Extracellular Inducing angiogenesis Down matrix-degrading
proteases (e.g.,
MMP-2 & MMP-
9)
VEGF-A Inducing angiogenesis Down
TSP- 1 Inducing angiogenesis Down
VEGFR- 1 Inducing angiogenesis Down
VEGFR-2 Inducing angiogenesis Down
VEGFR-3 Inducing angiogenesis Down
NF2 Activating invasion and Down metastasis
L B 1 Activating invasion and Up- regulated in multiple cancer, Down metastasis including intestine
Snail Activating invasion and Down metastasis
Slug Activating invasion and Down metastasis
Twist Activating invasion and Down metastasis
Zeb l /2 Activating invasion and Down metastasis
CCLR5 Activating invasion and Down metastasis
cysteine cathepsin Activating invasion and Down protease family metastasis
Extracellular Activating invasion and Down matrix-degrading metastasis
proteases (e.g.,
MMP-2 & MMP-
9)
EGF Activating invasion and Down metastasis
CSF- 1 Activating invasion and
metastasis
PP2 Energy metaboli m Down elF4E Energy metabolism Down
RS Energy metabolism Down
PI 3CA Energy metabolism Mutated in many breast, bladder Down cancers and hepatocellular carcinoma
BAP 1 Energy metabolism Mutated in renal cell carcinoma Down
TWIST (TF) Cancer Stem Cells Down
H IF- l cc Cancer Stem Cells Over expressed in renal cell carcinoma Down
HER2/neu Cancer Stem Cells Down
Snail (TF) Cancer Stem Cells Down
Wnt Cancer Stem Cells Down
EPCAM Cancer Stem Cells Overexpressed in breast, colon, uterus Down and other cancers
EGF Cytokine-receptor Down interactions
TGFa Cytokine-receptor Down interactions
PDGF Cytokine-receptor Down
IGF- 1 interactions
ILTLG
FLT3LG Cytokine-receptor Down interactions
HGF Cytokine-receptor Down interactions
FGF Cytokine-receptor Down interactions
EGFR Cytokine-receptor Mutated in lung cancer (40% of all Down interactions Asians and 10- 15% of all Caucasians)
ERBB2 Cytokine-receptor Down interactions
PDGFR Cytokine-receptor Down interactions
IGFR Cytokine-receptor Down interactions
c-KlT Cytokine-receptor Down interactions
FLT3 Cytokine-receptor Down interactions
MET Cytokine-receptor Down interactions
FGFR Cytokine-receptor Mutations in bladder cancer Down interactions
D A damage and genomic instability DNMT 1 Methyl transferases
DNMT2 Methyl transferases
DNMT3a Methyl transferases
DNMT3b Methyl transferases
H3K9Me3 Histone methylation
H3K27Me Histone methylation
Lsh Helicase activity
BLM Helicase activity Bloom's syndrome >Cancer Correct
WRN Helicase activity Werner's syndrome > Cancer Correct
RTS Helicase activity Rothmund-Thompson > Cancer Correct
XPA through XPG Nucleotide excision Xeroderma pigmentosa
repair
XPB Nucleotide excision Cockayne's syndrome
repair
XAB2 Nucleotide excision
repair
XPD Nucleotide excision Cockayne's syndrome
repair
TF1IH Nucleotide excision
repair
RFC Nucleotide excision
repair
PCNA Nucleotide excision
repair
LIG 1 Nucleotide excision
repair
Flap Nucleotide excision
endonueclease 1 repair
MNAT Nucleotide excision
repair
MMS 19 Nucleotide excision
repair
RAD23A Nucleotide excision
repair
RAD23B Nucleotide excision
repair
RPA 1 Nucleotide excision
repair
RPA2 Nucleotide excision
repair CCNH Nucleotide excision
repair
CD 7 Nucleotide excision
repair
CETN2 Nucleotide excision
repair
DDB 1 Nucleotide excision
repair
DDB2 Nucleotide excision
repair
ERCC 1 Nucleotide excision
repair
ATM Recombinational repair
NBN Recombinational repair
BRCA 1 Recombinational repair Breast, ovarian and pancreatic cancer Correct susceptibility or Up
BRCA2 Recombinational repair Breast cancer and ovarian Correct susceptibility or UP
RAD51 Recombinational repair
RAD52 Recombinational repair
WRN Recombinational repair
BLM Recombinational repair
FANCB Recombinational repair
MLH 1 Mismatch repair Multiple (including colon and uterus)
MLH2 Mismatch repair Multiple (including colon and uterus)
MSH2 Mismatch repair
MSH3 Mismatch repair
MSH4 Mismatch repair
MSH5 Mismatch repair
MSH6 Mismatch repair Multiple (including colon and uterus)
PMS 1 Mismatch repair
PMS2 Mismatch repair Multiple (including colon and uterus)
PMS2L3 Mismatch repair
Aging
DAF-2
lGF- 1
SRT 1 Table VII-15
Genes Mutated in Common Cancers
Bladder FGFR3, RBI, HRAS, KRAS, TP53, TSC1, FGFR3
BRCA, BRCA 2, BARD1, BRIP1, CHEK2, MRE11A, NBN,
Breast and Ovarian PALB2, PTEN, RAD50, RAD50, RAD51C, RAD51D, PPMDD,
TP53, BRIP1 , RAD54L, SLC22A1L, PIK3CA, RB1CC1,
Cervical FGFR3
PT53, STK11, PTEN, BMPR1A, SMAD, MLHl, MSH2,
Colon and Rectal
MSH6, PMS, EPCAM, AKT1, APC, MYH, PTPRJ, AXIN2
Endonietrial/Uterine MLH 1 , MSH2, MSH6, PMS, EPCAM
Esophageal DLEC1, TGFBR2, RNF6, LZT1S1, WWOX
Hepatocellular carcinoma PDGFRL, CTNNB1, TP53, MET, CASP8, PIK3CA
Renal VHL, PBRMQ, BAP1, SETD2, HIF1-
KRAS, EGFR, ALK,BRAF, ERBB2, FLCN, DIRC2, RNF139,
Lung
OGG1, PRCC, TFE, MET, PPP2R1B, RASSF1, SLC22A1L
BRAF, CDKA, CDKN2A, CDKN2B, CDKND, MCIR, TERT,
Melanoma
ATF1,CREB1,EWSR1
Non-Hodgkin Lymphoma C ASP 10, EGFR, IRF1, PIK3CA
Osteosarcoma CKEK2, LOJ18CR1, RBI
Ovarian PRKN, AKT1
KRAS, BRCA2, CDKN2A, MANF, PALB2, SMAD4, TP53,
Pancreatic
IPF1
MLHl, MSH2, MSH6, and PMS2, BRCA 1, HOXB13, CHEK2,
Prostate
ELAC2, EPHB2, SDR5A2, PRKAR1A, PMC1
Papillary and Follicular BRAF, NARAS, ERCI, FOXEl, GOLGA5, NCOA4, NKX2-1, Thyroid PMC1, tfET, TFG, TPR, TREM24, TRIM27, TRIM33
Erwing Sarcoma ERG, ETV 1 , ETV4, EWSR 1 , FLI 1
BRC, AMCR2, GMPS, JAK2, AF10, ARFGEF12, CEBPA,
Leukemia
FLT3, KIT, LPP, MLF1, NPM1, NSD1, NUP214, PICALM, RUNXl, SH3GL1, WHSC1L1, ETV6, RARA, BCR,
ARHGAP26, NFl, PTPN11, GATAl
[0806] Any of the following cancer associated genes provided in Table VII- 16 can be targeted.
[0807] Table VII- 16 Exemplary Target Genes Associated With Cancer:
Table VII-16:
ABL1, ABL2, ACSL3, AF15Q14, AF1Q, AF3p21, AF5q31, AKAP9, A T1, AKT2, ALDH2, AL , AL017, APC, ARHGEF12, ARHH, ARID1A, ARID2, ARNT, ASPSCR1, ASXL1, ATF1, ATIC, ATM, ATRX, AXIN1, BAP1, BCL10, BCL11A, BCL11B, BCL2, BCL3, BCL5, BCL6, BCL7A, BCL9, BCOR, BCR, BHD, BIRC3, BLM, BMPRIA, BRAF, BRCAl, BRCA2, BRD3, BRD4, BRIPI, BTG1, BUB1B, C12ori9, C15ort21, C15orf55, C16orl75, C2orf44, CAMTA1, CANT1, CARD11, CARS, CBFA2T1, CBFA2T3, C.BFB, CBL, CBLB, CBLC, CCDC6, CCNB1IP1, CCND1, CCND2, CCND3, CCNE1, CD273, CD274, CD74, CD79A, CD79B, CDH1, CDH11, CDK12, CDK4, CDK6, CD N2A, CD N2a(pl4), CD N2C, CDX2, CEBPA, CEPl, CHCHD7, CHEK2, CHIC2, CHNl, CIC, Cin A, CLTC, CLTCL1, CMKOR1, CNOT3, COL1 Al, COPEB, COX6C, CREB1, CREB3L1, CREB3L2, CREBBP, CRLF2, CRTC3, CTNNB1, CYLD, D10S170, DAXX, DDB2, DDIT3, DDX10, DDX5, DDX6, DEK, D1CER1, DNM2, DNMT3A, DUX4, EBFI, ECT2L, EGFR, E1F4A2, ELF4, ELK4, ELKS, ELL, ELN, EML4, EP300, EPS 15, ERBB2, ERCC2, ERCC3, ERCC4, ERCC5, ERG, ETVl, ETV4, ETV5, ETV6, EVIl, EWSR1, EXTl, EXT2, EZH2, EZR, FACL6, FAM22A, FAM22B, FAM46C, 1ANCA, EANCC, FANCD2, FANCE, FANCF, FANCG, FBXOl 1, FBXW7, FCGR2B, FEV, FGFR1, FGFRIOP, FGFR2, FGFR3, FTI, FIIIT, FIP1L1, FLU , FLJ27352, FLT3, FNBP1, FOXL2, FOXOIA, FOX03A, FOXP1, FSTL3, FUBP1, FUS, FVT1, GAS7, GATA1, GATA2, GATA3, GMPS, GNA11, GNAQ, GNAS, GOLGA5, GOPC, GPC3, GPHN, GRAF, H3F3A,
IICMOGT-1, IIEAB, HERPUD1, IIEY1, IIIPl, HIST1IT3B, IIIST1II4I, IILF, HLXB9, HMGA1, HMGA2, HNRNPA2BI, HOOK3, HOXA11, HOXA13, HOXA9, HOXC11, HOXC13, HOXD11, HOXD13, HRAS, IIRPT2, HSPCA, HSPCB, IDHl, IDH2, IGH@, IGK@, IGL@, IKZFl, IL2, TL21R, IL6ST, IL7R, IRF4, IRTA1, ITK, JAK1, JAK2, JAK3, JAZF1, JUN, KCNJ5, KDM5A, KDM5C, KDM6A, KDR, KIAA1549, KIF5B, KIT, KLF4, KLK2, KRAS, KTN1, LAF4, LASPl, LCK, LCP1, LCX, LHFP, LIFR, LMOl, LM02, LPP, LRIG3, LYL1, MADH4, MAF, MAFB, MALT1, MAML2, MAP2KL MAP2K2, ΜλΡ2Κ4, MAX, MDM2, MDM4, MDS1, MDS2, MECTl, MED12, MEN1, MET, MITF, MKL1, MLF1, MLIIl, MLL, MLL2, MLL3, MLLT1, MLLT10, MLLT2, MLLT3, MLLT4, MLLT6, MLLT7, MN1, MPL, MSF, MSH2, MSH6, MSI2, MSN, MTCP1, MUC1, MUTYH, MYB, MYC, MYCL1, MYCN, MYD88, MYH11, MYH9, MYST4, NACA, NBS1, NCOA1, NCOA2, NCOA4, NDRG1, NF1 , NF2, NFE2L2, NFIB, NFKB2, NIN, NKX2-1, NONO, NOTCH I, NOTCH2, Table VII-16:
NPMl, NR4A3, NRAS, NSDl, NT5C2, NTRKl, NTRK3, NUMAl, NUP214, NUP98, OLIG2, OMD, P2RY8, PAFAH1B2, PA LB 2, PAX3, PAX5, PAX7, PAX8, PBRM1, PBX1, PCM1, PCSK7,
PDE4DIP, PDGFB, PDGFRA, PDGFRB, PERI, PIIF6, PHOX2B, PICALM, PIK3CA, PIK3R1, PIM1, PLAG 1 , PML, PMS1, PMS2, PMX1, PNUTL1, POT1, POU2AF1, POU5F1, PPARG, PPP2R1A, PRCC, PRDM1, PRDM16, PRF1, PRKAR1 A, PRO1073, PSIP2, PTCH, PTEN, PTPN11 , RAB5EP, RACl, RAD51L1, RAFl, RALGDS, RANBP17, RAPIGDSI, RARA, RBI, RBM15, RECQL4, REL, RET, RNF43, ROS1, RPL10, RPL22, RPL5, RPN1, RUNDC2A, RUNX1, RUNXBP2, SBDS, SDC4, SDH5, SDHB, SDHC, SDHD, SEPT6, SET, SETBP1, SETD2, SF3B1, SFPQ, SFRS3, SH2B3, SH3GL1, SIL, SLC34A2, SLC45A3, SMARCA4, SMARCB1, SMARCE1, SMO, SOCS1, SOX2, SRGAP3, SRSF2, SSI 8, SS18L1, SSH3BP1, SSX1, SSX2, SSX4, STAT3, STK11, STL, SUFU, SIJZ12, SYK, TAF15, TALI, TAL2, TCEA1, TCF1, TCF12, TCF3, TCF7L2, TCL1A, TCL6, TERT, TET2, TFE3, TFEB, TFG, TFPT, TFRC, THRAP3, TIF1, TLX1, TLX 3, TMPRSS2, TNFAIP3, TNFRSF14, TNFRSF17, TNFRSF6, TOPI, TP53, TPM3, TPM4, TPR, TRA@, TRAF7, TRB@, TRD@, TRIM27, TRIM33, TRIP11 , TSC1 , TSC2, TSHR, TTL, U2AF1, USP6, VHL, VTUA, WAS, WHSC1, WHSC1L1, WIF1, WRN, WT1, WTX, WWTR1, XPA, XPC, XPOl, YWHAE, ZNF145, ZNF198, ZNF278, ZNF331 , ZNF384, ZNF521 , ZNF9, or ZRSR2
[0808] Exemplary pathways and genes associated with energy metabolism are provided in Table VII-17. Exemplary metabolic targets disclosed herein may be modulated using CRISPR/Cas9 as described herein. Modulation may be used to knockdown a gene of interest, correct a defect or mutation in the gene, or to activate a gene of interest.
Table VII-17
Figure imgf000206_0001
GL, gastric lipase Knock down
PL, pancreatic lipase Knock down
sPLA2, secretory phospholipase A2 Knock down
ACC, acetyl-CoA carboxylase Knock down
CPT, carnitine palmitoyl transferase Knock down
FAS, fatty-acid synthase Knock down
MTP, microsomal triglyceride-transfer protein Knock down
Insulin receptor Correct defects or activate
SU receptor/K+ ATP channel Activate with mutation a-glucosidase Knock down
PPARy Activate with mutation
Glycogen phosphorylase Knock down
Fructose- 1 , 6-bisphosphatase Knock down
glucose-6-phosphatase Knock down
PTP- 1B Knock down
SHIP-2 Knock down
GSK-3 Knock down
lkB kinase Knock down
PKCq Knock down
GLP 1 R Correct mutation
GIPR Correct mutation
GPR40 Correct mutation
GPR 1 19 Correct mutation
GPR41 Correct mutation
GPR43 Correct mutation
GPR 120 Correct mutation
GCGR Correct mutation
PAC 1 Correct mutation
VPAC2 Correct mutation
Y l Knock down
GHSR Knock down
CC AR Correct mutation b2 Correct mutation a2 Knock down
MT 1 Knock down
M3 Correct mutation
CB 1 Knock down
P2Y Correct mutation
H3 Inhibit MCH-R ] Coirect mutation
MCH-R2 Correct mutation
Ghrelin R Inhibit
FASN Inhibit
Bombesin-R3 Inhibit
CCK-A Receptor Correct mutation
Seratonin System Correct mutation
CBI Cannabinoid Receptors Inhibit
Dopaminergic System Correct mutation
Enterostatin Mutate to super agonist
CNTF Mutate to super agonist
CNTF-R Correct mutation
SOCS-3 Knock down
46a Knock down
PrPP Receptors Correct mutation
Amylin Mutate to super agonist
CRH System Mutate to super agonist
Galanin Receptors Knock down
Orexin Receptors Knock down
Noradrenalin System Mutate to super agonist
CART Mutate to super agonist
FATP4 Knock down
Pancreatic Lipase Knock down
ACRP30 Super agonist mutations
Thyroid Hormone Correct mutation
B-3 Adrenergic Receptor Correct mutation
UCPs Upregulate
PTP- 1 B Knock down
MC3 Correct mutation
ACC2 Knock down
Perilipin Knock down
HMGIC Knock down l l BHSD- 1 Knock down
Glucagon R Knock down
Glucocoricoid R Knock down
1 l beta-HSD I Knock down
PGC- 1 Correct mutation
DPPP-rV Knock down
GLP Mutate to super agonist GIP Mutate to super agonist
GLP-IR Correct mutation
AMP Kinase Correct mutation
IKK-b Knock down
PPARa/g Knock down
INS-R Knock down
SGLT Knock down- a-glucosidase Knock down
HMGCR Knock down
PCSK9 Knock down
ApoB- 100 Knock down
Leptin Mutate to super agonist
Leptin Receptor Mutate to constitutively active
receptor
MC4R Mutate to constitutively active
receptor
VOMC Mutate MSH region to super
agonist
AGRP Knock down
rVPY Receptors Introduce constitutively active
mutations
5HT2C Introduce constitutively active
mutations
GLP- 1 Mutate to super agonist
GLP- 1 Receptor Mutate to constitutively active
receptor
[0809] In an embodiment, the pathways and genes described herein, e.g., in Table VII- 17, are also associated with diabetes, obesity, and/or cholesterol and lipids.
[0810] Exemplary pathways and genes associated with the cell cycle are provided in Table VII- 18.
Table VII-18
Figure imgf000209_0001
MRE11 MLH1 FasR
NBS1 MSH6 Trail-L
RAD50 MSH2 Trail-R
53BP1 RFC TNF-Ct
P53 PCNA TNF-R1
CHKE MSH3 FADD
E2F1 MutS homolog TRADD
PML MutL homolog R1P1
FANCD2 Exonuclease MyD88
SMC1 DNA Polymerase delta IRAK
BLM1 (POLDl, POLD2,POLD3, NIL
BRCA1 and POLD4 -genes IKK
H2AX encoding subunits) NF-Κβ
ATR Topoisomerase 1 ΙκΒα
RPA Topoisomerase 2 IAP
ATRIP RNAseHl Caspase 3
RAD9 Ligase 1 Caspase 6
RAD1 DNA polymerase 1 Caspase 7
HUS DNA polymerase 3 Caspase 8
RAD17 Primase Caspase 10
RFC Helicase HDAC1
CHK1 Single-strand binding HDAC2
TLK1 proteins Cytochrome C
CDC25 Bxl-xL
STAT3
STAT5
DFF45
Vcl-2
ENDO-G
PI3K
Akt
Calpain
Bad
Bax
Ubiqiiitin-mediated proteolysis Hypoxia Cell Proliferation
HIF-loc MAPK
El HERC1 TRAF6 HIF-Ιβ MAPKK
E2 UBE2Q MEKK1 Refl MAPKKK
E3 UBE2R COP! HSP90 c-Met UBLE1A UBE2S PIFH2 VEGF HGF
UBLE1B UBE2U cIAP PAS ER S1/2
UBLEIC UBE2W PIAS ARNT ATK
UBE2A UBE2Z SYVN VHL PKCs
UBE2B AFC LLC N NHLRC1 HLF Paxilin
UBE2C UBE1 AIRE EPF FAK
UBE2A E6AP MGRN1 VDU2 Adducin
UBE2E UBE3B BRCA1 SUMORESUME PYK1
UBE2F Smurf FANCL SENP1 RB
UBE2G1 Itch MIDI Calcineurin A RBI
UBE2G2 HERC2 Cdc20 RACK1 Raf-1
UBE2I HERC3 Cdhl PTB A-Raf
UBE2J1 HERC4 Apcl Hur B-raf
UBE2J2 UBE4A Apc2 PHD2 MEK1/2
UBE2L3 UBE4B Apc3 SSAT2 ERK1/2
UBE2L6 CHIP Apc4 SSAT1 Ets
UBE2M CYC4 Apc5 GSK3 Elkl
UBE2N PPR19 Apc6 CBP SAP1
UBE20 UIP5 Apc7 FOX04 cPLA2
WWPI Mdm2 Apc8 FlH-1
WWP2 Parkin Apc9
TRIP 12 Trim32 Ape 10
NEED4 Trim37 Ape 11
ARF-BP1 SIAH-1 Ape 12
EDD1 PML
Cell survival Cell cycle arrest
SMADI P21
SMAD5 BAX
SAMD8 MDR
LEF1 DRAIL IGFBP3
TCF3 GADD45
TCF4
P300
HAT1
PI3
Akt
GF1
[0811] Exemplary cell cycle genes characterized by their function are provided in Table VII-19. Table VU-19
Figure imgf000212_0001
[0812] Exemplary pathways and genes associated with the angiogenesis are described provided in Table VII-20.
Table VII-20
Figure imgf000212_0002
VEGF VEGFR2 SHC E2F7
VEGFB VEGFR3 PI3
VEGFC Nrp l PIP3
VEGFD IP3
DAG
GRB2
SOS
Akt
P B
PKC
Ras
RAF1
DAG
eNOS
NO
ERK 1
ER 2
cPLA2
ME 1
MEK2
[0813] Exemplary pathways and genes associated with the mitochondrial function are provided in Table VII-25.
Table VII-25
Figure imgf000213_0001
Malate dehydrogenase Aminotransferase
Hydratase
Deacylase
Dehydrogenase
Carboxylase
Mutase
Fatty acid oxidation Leucine Oxidation Isoleucine disorders (enzyme Pathway oxidation pathway deficiencies) Aminotransferase Aminotransferase
OCTN2 Branched chain Branched chain
FATP1 -6 aminotransferase 2, aminotransferase 2,
CPT- 1 mitochondrial mitochondrial
CACT Isobutytyl-CoA 2-methylbutytyl-CoA
CPT-II dehydrogenase Dehydrogenase
SCAD (Branched Chain (Branched Chain
MCAD Keto Acid Keto Acid
VLCAD Dehydrogase Dehydrogenase
ETF-DH Complex) Complex)
Alpha-ETF Hydratase Hydratase
Beta-ETF HMG-CoA lyase 2-methyl-3-OH-
SCHAD butyryl-CoA
LCHAD dehydrogenase
MTP 3-Oxothiolase
LKAT
DECR 1
HMGCS2
HMGCL
Additional mitochondrial genes and related diseases caused by mutations
Mt-ND l Leber's hereditary optic neu ropathy
Mt-ND4 Leber's hereditary optic neuropathy
Mt-ND6 Leber's hereditary optic neuropathy
OPA 1 Autosomal dominant optic atrophy
C T2A Charcot-Marie-Toothhereditary neuropathy type 2A
mt-T Myoclonic epilepsy with ragged red fibres
Mitochondrial Related diseases
Respiratory chain
genes
NADH CoQ Alpers, Alzheimer's, Parkinsonism, Cardiomyopathy, Deficiency (Barth
Reductase and/or Lethal Infantile), Encephalopathy, Infantile CNS, Leber's, Leigh, Longevity, MELAS, MERRF, Myopathy ± CNS, PEO, Spinal cord
disorders
Succinate-CoQ Keams-Sayre, Leigh's, Myopathy (e.g., Infantile ± CNS), Paraganglioma,
Reductase Pheochromocytoma
CoQ-Cytochrome C Cardiomyopathy, Fatal infantile, GRACILE, Leber's, Myopathy (e.g., ±
Reductase CNS, PEO)
Cytochrome C Alper's, Ataxia, Deafness, Leber's, Leigh's, Myopathy (e.g., Infantile (e.g.,
Oxidase Fatal, Benign), Adult), Rhabdomyolysis, PEO, KSS, MNGEE, MERRF,
MELAS
ATP Synthase Cardiomyopathy, Encephalopathy, Leber's, Leigh, Multisystem, NARP
Complex I (NADH-Ubiquinone Oxidoreductase)
Nuclear encoded Mitochondral DNA Supernumerary Subunits involved proteins encoded proteins subunits in regulation of
NDUFAB 1 (SDAP): Complext I activity
NDUFS 1 : Childhood NDl Carrier of fatty acid NDUFS4 (AQDQ) encephalopathy; Most chain
common Complex 1 ND2 Functions:
mutations (3%) NDUFA 1 (MWFE) Increased Complex
ND3 I activity with
NDUFS2: Primarily expressed phosphorylation
Cardiomyopathy + ND4 in heart & skeletal
Encephalomyopathy muscle Disorders:
ND4L Multisystem
NDUFS3: Leigh Disorders: childhood
ND5 Encephalopathies encephalopathy
NDUFS7: Leigh with Complex I
ND6 NDUFA2: deficiency; Leigh
NDUFS8: Leigh Encephalopathy & syndrome
Cardiomyopathy
NDUFV 1 : Childhood
encephalopathy NDUFA9: Leigh
syndrome
NDUFV2:
Encephalopathy + NDUFA 10: Leigh
Cardiomyopathy syndrome
ELAC2: NDUFA 1 1
Cardiomyopathy,
Hypertrophic Disorder:
Encephalopathy &
Cardiomyopathy
NDUFA 12: Leigh syndrome
NDUFB9:
Hypotonia
NDUFS6: Lethal Infantile Mitochondrial Disease
Proteins involved in Other
Complex I assembly
NDUFA 13: Thyroid
• NDUFAF1 : carcinoma (Hurthle
Cardiomyopathy + cell)
Encephalomyopathy
• NDUFAF2 NDUFB3: Severe
(NDUFA 12L): lethal mitochondrial
Childhood complex I deficiency
encephalopathy;
Usually null MTHFR deficiency
mutations -
• NDUFAF3: Lethal MGME1 : PEO +
neonatal Myopathy
encephalopathy
• NDUFAF4:
Encephalopathy
• C6ORF66:
Encephalopathy
• C8orf38: Leigh
syndrome
• C20orf7: Lethal
neonatal
• NUBPL:
Encephalomyopathy
• ACAD9: Fatigue &
Exercise intolerance;
Most missense
mutations
• FOXRED 1 : Leigh
syndrome
• Ecsit
• A1F (A1FM 1 ; -
PDCD8) • Ind l
C omplex 1 (NADH-lJbit|uinoiu' Oxidoroductase)
Flavoprotein: FAD (SDHA; Fp) • Mutations cause Leigh syndrome with
Complex II deficiency
• Late onset neurodegenerative disorder)
Iron-Sulfur protein: SDHB (Ip) 0 Mutations cause Reduced tumor suppression
0 Neoplasms: Pheochromocytoma &
Paraganglioma
SDHC ; SDHD (cytochrome C subunits) - o mutations lead to paraganglioma
Complex H I ( Cytochrome reductase)
Cytochrome c l (CYC 1 )
Rieske FeS protein (UQCRFS 1 )
Ubiquinol-cytochrome c reductase core May mediate formation of complex between protein I (UQCRC 1 ; QCR; Subunit 1 ) cytochromes c and cl
Ubiquinol-cytochrome c reductase core Required for assembly of complex III
protein II (UQCRC2; QCR2; Subunit 2)
UQCRH (Subunit 6) May mediate formation of complex between
cytochromes c and c l
Ubiquinone-binding protein (UQBC; Redox-linked proton pumping
UQPC; UQCRB; UQBP; Subunit 7)
UOCRO (Subunit 8) Binds to ubiquinone
Ubiquinol-cytochrome C reductase Interacts with cytochrome c l
complex, 7.2-KD Subunit (UCRC;
UQCR 10; Subunit 9)
UQCR (UQCR 1 1 ; Subunit 10) function as iron-sulfur protein binding factor
Cleavage product of UQCRFS 1
(Cytochrome b-c l complex subunit 1 1)
Inner membrane proteins and related disorders
ABCB7: Ataxia + Anemia
ACADVL: Myopathy
ADC 3: SACR9
AGK: Sengers
ATP5A 1 : Encephalopathy, neonatal
ATP5E: Retardation + Neuropathy
BRP44L: Encephalopathy
c l 2orf62: Encephalocardiomyopathy
Cardiolipin: Barth
COX4I2: Pancreas + Anemia COX6B1: Encephalomyopathy
CPT2: Myopathy
C AT: Encephalomyopathy
CYC1 : Hyperglycemia & Encephalopathy
CYCS
CYP11A1
CYP11B1
CYP11B2
CYP24A1
CYP27A1: Cerebrotendinous Xanthomatosis
CYP27B1
DHODH
DNAJC19: Cardiac + Ataxia
FASTKD2: Encephalomyopathy
GPD2
HADHA: Multisystem; Myopathy
HADHB: Encephalomyopathy
HCCS: MIDAS
L2HGDH: Encephalopathy
MMAA
MPV17: Hepatocerebral
NDUFAl: Encephalopathy
NDUFA2: Leigh + Cardiac
NDUFA4: Leigh
NDUFA9: Leigh
NDUFA10: Leigh
NDUFAl 1: Encephalocardiomyopathy NDUFA12: Leigh
NDUFAl 3
NDUFB3: Lethal infantile
NDUFB9: Encephalopathy
NDUFV1: Encephalopathy
NDUFV2: Encephalopathy + Cardiac NDUFSl: Leukodystrophy
NDUFS2: Encephalopathy + Cardiac NDUFS3: Dystonia
NDUFS4: Encephalopathy
NDUFS6: Lethal infantile
NDUFS7: Encephalopathy
NDUFS8: CNS + Cardiac
OPA 1 : Optic atrophy
OPA3: Optic atrophy
PDSS1: Coenzyme Q10 deficiency
SDHA: Leigh; Cardiac; Paraganglioma SDHB: Paraganglioma
SDHC: Paraganglioma
SDHD: Paraganglioma
SLC25A earners
SLC25A 1 : Epileptic encephalopathy
SLC25A3: Cardiac; Exercise intolerance
SLC25A4: PEOA2
SLC25A 12: Hypomyelination
SLC25A 13: Citrullinemia
SLC25A 15: HHH
SLC25A 1 : Microcephaly
SLC25A20: Encephalocardiomyopathy
SLC25A22: Myoclonic epilepsy
SLC25A38: Anemia
Paraplegin: SPG7
TIMM8A: Deaf'-Dystonia-Dementia
UCP1
UCP2
UCP3
UQCRB: Hypoglycemia, Hepatic
UQCRC2: Episodic metabolic encephalopathy
UQCRQ: Encephalopathy
[0814] Pathways and genes associated with DNA damage and genomic instability include the following methyl transferases, histone methylation, helicase activity, nucleotide excision repair, recombinational repair, or mismatch repair provided in Table VII-21. See also Table VI-22.
Table VII-21
Figure imgf000219_0001
53BP1 BLM MBD2 DNA ligase 4
MDC1 H2AX XLF
SMC1 53BP1 Rad50
P53 P53 Artemis
Rad27
TdT
Base-Excision repair Nucleotide-Excision Homologous Mismatch repair
APE1 Repair Recombination PMS2
APE2 UvrA RecA MLH1
NEIL1 UvrB SSB MSH6
NEIL2 UvrC Mrell MSH2
NEIL3 XPC Rad50 RFC
XRCC1 Rad23B Nbsl PCNA
PNKP CEN2 CtIP , MSH3
Tdpl DDB1 RPA MutS
APTX XPE Rad51 MutL
DNA polymerase β CSA, Rad52 Exonuclease
DNA polymerase δ CSB Rad54 Topoisomerase 1
DNA polymerase's TFT1H BRCA1 Topoisomerase 2
PCNA XPB BRCA2 RNAseHl
FEN1 XPD Exol Ligase 1
RFC XPA BLM DNA polymerase 1
PAR 1 RPA Topllla DNA polymerase 3
Ligl XPG GEN1 · Primase
Lig3 ERCC1 Yenl Helicase
UNG XPF Slxl SSBs
MUTY DNA polymerase δ Slx4
SMUG DNA polymerase ε Mus8
MBD4 Emel
Dssl
Histone Methylation
ASH1L SETD4
DQT1L SETD5
EHMT1 SETD6
EHMT2 SETD7
EZH1 SETD8
EZH2 SETD9
MLL SETDB1
MLL2 SETDB2
MLL3 SETMAR MLL4 SMYD 1
MLL5 SMYD2
NSD 1 SMYD3
PRDM2 SMYD4
SET SMYD5
SETBP1 SUV39H 1
SETD 1 A SUV39H2
SETD 1 B SUV420H 1
SETD2 SUV420H2
SETD3 ■
Table VII-22
Selected Transcript ion FactorsTranscription factors
NI X2-5 Cardiac malformations and
atrioventricular conduction abnormalities
MECP2 Rett syndrome
HNF1 through Mature onset diabetes of the young HNF6 (MODY), hepatic adenomas and renal cysts
FOXP2 Developmental verbal dyspraxia
FOXP3 Autoimmune diseases
NOTCH 1 Aortic valve abnormalities
MEF2A Coronary artery disease
CRX Dominant cone-rod dystrophy
FOCX2 Lymphedema-distichiasis
NF-KB Autoimmune arthritis, asthma, septic Activation shock, lung fibrosis,
glomerulonephritis, atherosclerosis, and AIDS
NF-KB Inhibition Apoptosis, inappropriate immune cell development, and delayed cell growth
NARA2 Parkinson disease
LHX3 Pituitary disease
GAT4 Congenital heart defects
P53, APC Cancer
CTCF Epigenetics and cell growth regulation
EGR2 Congenital hypomyelinating
neuropathy (CHN) and Charcot-Marie- Tooth type 1 (CMT 1 )
STAT family Cancer and immunosuppression
NF-A T family Cancer and inflammation .
AP-1 family Cancer and inflammation
[0815] A gene including receptors and ionophores relevant to pain in this table can be targeted, by editing or payload delivery. Pathways and genes associated with pain are described herein, e.g., include the following those in Table VII-24.
Table VII-24
Figure imgf000222_0001
nociceptive central 5HT3 central inhibition antiemetic
nociceptive central 5HT4 central inhibition gastroproknetics nociceptive central 5HT5A central inhibition
nociceptive central 5HT5B central inhibition
nociceptive central 5HT6 central inhibition antidepressant (antagonists and agonists), anxiolytic (antagonists and agonists), nootropic (antagonists), anorectic (antagonists) nociceptive central 5HT7 central inhibition antidepressant (antagonists), anxiolytics (antagonists), nootropic (antagonists) nociceptive central CB 1 central inhibition
nociceptive central GABA central inhibition
nociceptive central GABAA-$ central inhibition
nociceptive central GABAB-R central inhibition
nociceptive central Glucine-R central inhibition
nociceptive central NE central inhibition
nociceptive central Opiod central inhibition
receptors
nociceptive central c-fos gene expression
nociceptive central c-jun gene expression
nociceptive central CREB gene expression
nociceptive central DREAM gene expression
nociceptive peripheral + channel membrane
excitability of
primary afferents
nociceptive peripheral Nav l .8 membrane
excitability of
primary afferents
nociceptive peripheral Nav l .9 membrane
excitability of
primary afferents
nociceptive peripheral CaMKIV peripheral
sensitization
nociceptive peripheral COX2 peripheral
sensitization
nociceptive peripheral cPLA2 peripheral
sensitization
nociceptive peripheral EP1 peripheral sensitization nociceptive peripheral EP3 peripheral sensitization nociceptive peripheral EP4 peripheral sensitization nociceptive peripheral ERK 1/2 peripheral sensitization nociceptive peripheral IL- 1 beta peripheral sensitization nociceptive peripheral JNK peripheral sensitization nociceptive peripheral Nav l .8 peripheral sensitization nociceptive peripheral NGF peripheral sensitization nociceptive peripheral p38 peripheral sensitization nociceptive peripheral PKA peripheral sensitization nociceptive peripheral P C peripheral isoforms sensitization nociceptive peripheral TNFalpha peripheral sensitization nociceptive peripheral TrkA peripheral sensitization nociceptive peripheral TRPV 1 peripheral sensitization nociceptive central AMPA/kain postsynaptic ate-R transmission nociceptive central + channels postsynaptic transmission nociceptive central mGlu-$ postsynaptic transmission nociceptive central Nav l .3 postsynaptic transmission nociceptive central N 1 postsynaptic transmission nociceptive central NMDA-R postsynaptic transmission nociceptive peripheral Adenosine- presynaptic R transmission nociceptive peripheral mGluR presynaptic transmission nociceptive peripheral VGCC presynaptic transmission nociceptive central ERK signal transduction nociceptive central JNK signal
transduction nociceptive central p38 signal transduction nociceptive central PKA signal
transduction nociceptive central PKC signal isoforms transduction nociceptive peripheral ASIC transduction nociceptive peripheral B 1 transduction nociceptive peripheral BK2 transduction nociceptive peripheral DRASIC transduction nociceptive peripheral MDEG transduction nociceptive peripheral P2X3 transduction nociceptive peripheral TREK- 1 transduction nociceptive peripheral TRPM8 transduction nociceptive peripheral TRPV 1 transduction nociceptive peripheral TRPV2 transduction nociceptive peripheral TRPV3 transduction neuropathic
pain
Inflammatory histamine
pain
Inflammatory ATP
pain
Inflammatory bradykinin
pain
Inflammatory CB2
pain
Inflammatory Endothelins
pain
Inflammatory H+ pain
Inflammatory Interleukins
pain
Inflammatory NGF
pain
Inflammatory prostaglandi
pain ns
Inflammatory serotonin
pain
Inflammatory TNFalpha
pain
VII I. TARGETS: DISORDERS ASSOCIATED WITH DISEASE CAUSING ORGANISMS
[0816] Cas9 molecules, typically eiCas9 molecules or eaCas9 molecules, and gRNA molecules, e.g., an eiCas9 molecule/gRNA molecule complex, e.g., an eaCas9 moIecule/gRNA molecule complex, can be used to treat or control diseases associated with disease causing organisms, e.g., to treat infectious diseases. In an embodiment, the infectious disease is treated by editing (e.g., correcting) one or more target genes, e.g., of the organism or of the subject. In other
embodiments, the infectious disease is treated by delivering one or more payloads (e.g., as described herein) to the cell of a disease causing organism or to an infected cell of the subject, e.g., to a target gene. In some embodiments, the target gene is in the infectious pathogen.
Exemplary infectious pathogens include, e.g., viruses, bacteria, fungi, protozoa, or mutlicellular parasites.
[0817] In other embodiments, the target gene is in the host cell. For example, modulation of a target gene in the host cell can result in resistance to the infectious pathogen. Host genes involved in any stage of the life cycle of the infectious pathogen (e.g., entry, replication, latency) can be modulated. In an embodiment, the target gene encodes a cellular receptor or co-receptor for the infectious pathogen. In an embodiment, the infectious pathogen is a virus, e.g., a virus described herein, e.g., HIV. In an embodiment, the target gene encodes a co-receptor for HIV, e.g., CCR5 or CXCR4. [0818] Exemplary infectious diseases that can be treated by the molecules and methods described herein, include, e.g., AIDS, Hepatitis A, Hepatitis B, Hepatitis C, Herpes simplex, HPV infection, or Influenza.
[0819] Exemplary targets are provided in Table VID- 1. The disease and causative organism are provided.
Table VIIl-1
DISK/VSR SOURCE OF DISEASE
Acinetobacler infcclions Acinetobacler baumannii
Actinomyces israelii. Actinomyces gerencseriae and
Actinomycosis Propionibacteri m propionicus
African sleeping sickness (African
trypanosomiasis) Trypanosoma brucei
AIDS (Acquired immunodeficiency
syndrome) HIV (Human immunodeficiency virus)
Amebiasis Entamoeba histolytica
Anaplasmosis Anaplasma genus
Anthrax Bacillus anthracis
Arcanobacterium haemolyticum infection Arcanobacterium haemolyticum
Argentine hemorrhagic fever Junin virus
Ascariasis Ascaris lumbricoides
Aspergillosis Aspergillus genus
Astrovirus infection Astroviridae family
Babesiosis Babesia genus
Bacillus cereus infection Bacillus cereus
Bacterial pneumonia multiple bacteria
Bacterial vaginosis ( BV) multiple bacteria
Bacleroides infection Bacteroides genus
Balantidiasis Balantidium coli
Bavlisasearis infection Baylisascaris genus
B virus infection BK virus
Black picdra Piedraia hortae
Blaslocystis hominis infection Blastocystis hominis
Blaslomvcosis Blastomyces dermalilidis
Bolivian hemorrhagic fever M achupo virus
Borrelia infection Borrelia genus Clostridium botulinum; Note: Botulism is not an infection by Clostridium botulinum but caused by the intake of
Botulism (and Infant botulism) botulinum toxin.
Brazilian hemorrhagic fever Sabia
Brucellosis Brucella genus
Bubonic plague the bacterial family Enterobacteriaceae
usually Burkholderia cepacia and other Burkholderia
Burkholderia infection species
Buruli ulcer Mycobacterium ulcerans
Calicivirus infection (Norovirus and
Sapovinis) Caliciviridae family
Campylobaclcriosis Campylobacter genus
Candidiasis (Moniliasis; Thrush) usually Candida albicans and other Candida species
Cat-scratch disease Bartonella henselae
Cellulitis usually Group A Streptococcus and Staphylococcus
Chagas Disease (American trypanosomiasis) Trypanosoma cruzi
Chancroid Haemophilus ducreyi
Chickenpox Varicella zoster virus (VZV)
Chlamydia Chlamydia trachomatis
Chlamydophila pneumoniae infection
(Taiwan acute respiratory agent or TWA ) Chlamydophila pneumoniae
Cholera Vibrio cholerae
Chromoblastomvcosis usually Fonsccaca pedrosoi
Clonorchiasis Clonorchis sinensis
Clostridium difficile infection Clostridium difficile
Coccidioidomycosis Coccidioides immitis and Coccidioidcs posadasii
Colorado tick fever (CTF) Colorado tick fever virus (CTFV)
Common cold (Acute viral rhinopharyngitis;
Acute coryza) usually rhinoviruses and coronaviruses.
Creutzfeldt-Jakob disease (CJD) PRNP
Crimean-Congo hemorrhagic fever (CCHF) Crimean-Congo hemorrhagic fever virus
Cryptococcosis Cryptococcus neoformans
Cryptosporidiosis Cryptosporidium genus
Cutaneous larva migrans (CLM) usually Ancylostoma braziliense; multiple other parasites
Cyclosporiasis Cyclospora cayetanensis
Cysticercosis Taenia solium
Figure imgf000229_0001
Haemophilus influenzae infection Haemophilus influenzae Enteroviruses, mainly Coxsackie Λ virus and Enterovirus
Hand, loot and mouth disease (HFMD) 71 (EV7 I )
Hantavirus Pulmonary Syndrome (HPS) Sin Nombre virus
Helicobacter pylori infection Helicobacter pylori
Hemolytic-uremic syndrome (HUS) Escherichia coli 0157:H7, Ol 1 1 and O104:H4
Hemorrhagic fever with renal syndrome
(HFRS) Bunyaviridae family
Hepatitis A Hepatitis A Virus
Hepatitis B Hepatitis B Virus
Hepatitis C Hepatitis C Virus
Hepatitis D Hepatitis D Virus
Hepatitis E Hepatitis E Vims
Herpes simplex Herpes simplex virus 1 and 2 (HSV-1 and HSV-2)
Histoplasmosis Histoplasma capsulatum
Hookworm infection Ancylosloma duodenale and Necator americanus
Human bocavinis infection Human bocavirus (HBoV)
Human ewingii ehrlichiosis Ehrlichia ewingii
Human granulocytic anaplasmosis (HGA) Anaplasma phagocylophilum
Human metapneumovirus infection Human metapneumovirus (hMPV)
Human monocytic ehrlichiosis Ehrlichia chaffeensis
Human papillomavirus (I IPV) infection Human papillomavirus (HPV)
Human parainfluenza virus infection Human parainfluenza viruses (HPIV)
Hymenolepiasis Hymenolepis nana and Hymenolepis diminula
Epstein-Barr Virus Infectious
Mononucleosis (Mono) Epstein-Barr Virus (EBV)
Influenza (flu) Orthomyxoviridae family
Isosporiasis Isospora belli
Kawasaki disease unknown; evidence supports that it is infectious
Keratitis multiple
Kingella kingae infection Kingella kingae
Kuru PRNP
Lassa fever Lassa virus
Legionellosis (Legionnaires' disease) Legionella pneumophila
Figure imgf000231_0001
Pelvic inflammatory disease (PID) multiple
Pertussis (Whooping cough) Bordetella pertussis
Plague Yersinia pestis
Pneumococcal infection Streptococcus pneumoniae
Pneumocystis pneumonia (PCP) Pneumocystis jirovecii
Pneumonia multiple
Poliomyelitis Poliovirus
Prevotella infection Prevotella genus
Primary amoebic meningoencephalitis
(PAM) usually Naegleria fovvleri
Progressive multifocal leukoencephalopathy JC virus
Psittacosis Chlamvdophila psittaci
Q fever Coxiella burnetii
Rabies Rabies virus
Rat-bite fever Slreptobacillus moniliformis and Spirillum minus
Respiratory syncytial virus infection Respiratory syncytial virus (RSV)
Rhinosporidiosis Rhinosporidium seeberi
Rhinovinis infection Rhinovirus
Rickettsial infection Rickettsia genus
Rickettsialpox Rickettsia akari
Rift Valley fever (RVF) Rift Valley fever virus
Rocky Mountain spotted fever (RMSF) Rickettsia rickettsii
Rotavirus infection Rotavirus
Rubella Rubella virus
Salmonellosis Salmonella genus
SARS (Severe Acute Respiratory
Syndrome) SARS coronavirus
Scabies Sarcoptes scabiei
Schistosomiasis Schistosoma genus
Sepsis multiple
Shigellosis (Bacillary dysentery) Shigella genus
Shingles (Herpes zoster) Varicella zoster virus (VZV)
Smallpox (Variola) Variola major or Variola minor
Sporotrichosis Sporothrix schenckii
Staphylococcal food poisoning Staphylococcus genus
Staphylococcal infection Staphylococcus genus
Strongyloidiasis Strongyloides stercoralis
Figure imgf000233_0001
Yersiniosis Yersinia enterocolilica Yellow lever Yellow fever virus
Mucorales order (Mucormycosis) and Entomophthorales
Zygomycosis order (Entomophthoramycosis)
AIDS/HIV
HIV genomic structural elements
[0820] Long terminal repeat (LTR) refers to the DNA sequence flanking the genome of integrated proviruses. It contains important regulatory regions, especially those for transcription initiation and polyadenylation.
[0821] Target sequence (TAR) for viral transactivation, the binding site for Tat protein and for cellular proteins; consists of approximately the first 45 nucleotides of the viral mRNAs in HIV- 1 (or the first 100 nucleotides in HIV-2 and SIV.) TAR RNA forms a hairpin stem-loop structure with a side bulge; the bulge is necessary for Tat binding and function.
[0822] Rev responsive element (RPE) refers to an RNA element encoded within the env region of HIV- 1 . It consists of approximately 200 nucleotides (positions 7327 to 7530 from the start of transcription in HIV- 1, spanning the border of gp l20 and gp41). The RRE is necessary for Rev function; it contains a high affinity site for Rev; in all, approximately seven binding sites for Rev exist within the RRE RNA. Other lentiviruses (HIV-2, SIV, visna, CAEV) have similar RRE elements in similar locations within env, while HTLVs have an analogous RNA element (RXRE) serving the same purpose within their LTR; RRE is the binding site for Rev protein, while RXRE is the binding site for Rex protein. RRE (and RXRE) form complex secondary structures, necessary for specific protein binding.
[0823] Psi elements (PE) are a set of 4 stem-loop structures preceding and overlapping the Gag start codon which are the sites recognized by the cysteine histidine box, a conserved motif with the canonical sequence CysX2CysX4HisX4Cys (SEQ ID NO: 41), present in the Gag p7 MC protein. The Psi Elements are present in unspliced genomic transcripts but absent from spliced viral mRNAs.
[0824] SLIP, an ΓΤΤΤΤ slippery site, followed by a stem-loop structure, is responsible for regulating the - 1 ribosomal frameshift out of the Gag reading frame into the Pol reading frame. [0825] Cis-acting repressive sequences (CRS) are postulated to inhibit structural protein expression in the absence of Rev. One such site was mapped within the pol region of HIV- 1. The exact function has not been defined; splice sites have been postulated to act as CRS sequences.
[0826] Inhibitory/Instability RNA sequences (INS) are found within the structural genes of HIV- 1 and of other complex retroviruses. Multiple INS elements exist within the genome and can act independently; one of the best characterized elements spans nucleotides 414 to 631 in the gag region of HIV- 1. The INS elements have been defined by functional assays as elements that inhibit expression posttranscriptionally. Mutation of the RNA elements was shown to lead to INS inactivation and up regulation of gene expression.
Genes and gene products
Essential for Replication
[0827] The genomic region (GAG) encoding the capsid proteins (group specific antigens). The precursor is the p55 myristylated protein, which is processed to p 17 (MAtrix), p24 (CApsid), p7 (NucleoCapsid), and p6 proteins, by the viral protease. Gag associates with the plasma membrane where the virus assembly takes place. The 55 kDa Gag precursor is called assemblin to indicate its role in viral assembly.
[0828] The genomic region, POL, encoding the viral enzymes protease, reverse transcriptase, RNAse, and integrase. These enzymes are produced as a Gag-Pol precursor polyprotein, which is processed by the viral protease; the Gag-Pol precursor is produced by ribosome frameshifting near the end of gag.
[0829] Viral glycoproteins (e.g., ENV) produced as a precursor (gpl60) which is processed to give a noncovalent complex of the external glycoprotein gp l20 and the transmembrane glycoprotein gp41 . The mature gp l20-gp41 proteins are bound by non-covalent interactions and are associated as a trimer on the cell surface. A substantial amount of gpl20 can be found released in the medium. gp l 20 contains the binding site for the CD4 receptor, and the seven transmembrane do- main chemokine receptors that serve as co-receptors for HIV- 1 .
[0830] The transactivator (TAT) of HIV gene expression is one of two essential viral regulatory factors (Tat and Rev) for HIV gene expression. Two forms are known, Tat- 1 exon (minor form) of 72 amino acids and Tat-2 exon (major form) of 86 amino acids. Low levels of both proteins are found in persistently infected cells. Tat has been localized primarily in the nucleolus/nucleus by immunofluorescence. It acts by binding to the TAR RNA element and activating transcription initiation and elongation from the LTR promoter, preventing the LTR AATAAA
polyadenylation signal from causing premature termination of transcription and polyadenylatio'n. It is the first eukaryotic transcription factor known to interact with RNA rather than DNA and may have similarities with prokaryotic anti-termination factors. Extracellular Tat can be found and can be taken up by cells in culture.
[0831] The second necessary regulatory factor for HIV expression is REV. A 19 kDa phosphoprotein, localized primarily in the nucleolus/nucleus, Rev acts by binding to RRE and promoting the nuclear export, stabilization and utilization of the un- spliced viral mRNAs containing RRE. Rev is considered the most functionally conserved regulatory protein of lentiviruses. Rev cycles rapidly between the nucleus and the cytoplasm.
Others
[0832] Viral infectivity factor (VIF) is a basic protein of typically 23 kDa. Promotes the infectivity but not the production of viral particles. In the absence of Vif the produced viral particles are defective, while the cell-to-cell transmission of virus is not affected significantly. Found in almost all lentiviruses, Vif is a cytoplasmic protein, existing in both a soluble cytosolic form and a membrane-associated form. The latter form of Vif is a peripheral membrane protein that is tightly associated with the cytoplasmic side of cellular membranes. In 2003, it was discovered that Vif prevents the action of the cellular APOBEC-3G protein which deaminates DNA:RNA heteroduplexes in the cytoplasm.
[0833] Viral Protein R (VPR) is a 96-amino acid ( 14 kDa) protein, which is incoiporated into the virion. It interacts with the p6 Gag part of the Pr55 Gag precursor. Vpr detected in the cell is localized to the nucleus. Proposed functions for Vpr include the targeting the nuclear import of preintegration complexes, cell growth arrest, transactivation of cellular genes, and induction of cellular differentiation. In HIV-2, SIV-SMM, SIV- RCM, SIV-MND-2 and SIV-DRL the Vpx gene is apparently the result of a Vpr gene duplication event, possibly by recombination. [0834] Viral Protein U (VPU)) is unique to HIV- 1 , SIVcpz (the closest SIV relative of HIV- 1 ), SIV-GSN, SIV-MUS, SIV- MON and SIV-DEN. There is no similar gene in HIV-2, SIV-SMM or other SIVs. Vpu is a 16 kDa (81 -amino acid) type I integral membrane protein with at least two different biological functions: (a) degradation of CD4 in the endoplasmic reticulum, and (b) enhancement of virion release from the plasma membrane of HIV- 1 -infected cells. Env and Vpu are expressed from a bicistronic mRNA. Vpu probably possesses an N-terminal hydrophobic membrane anchor and a hydrophilic moiety. It is phosphorylated by casein kinase II at positions Ser52 and Ser56. Vpu is involved in Env maturation and is not found in the virion. Vpu has been found to increase susceptibility of' HIV- 1 infected cells to Fas killing.
[0835] NEF is amultifunctional 27-kDa myristylated protein produced by an ORF located at the 3 0 end of the primate lentiviruses. Other forms of Nef are known, including nonmyristylated variants. Nef is predominantly cytoplasmic and associated with the plasma membrane via the myristyl residue linked to the conserved second amino acid (Gly). Nef has also been identified in the nucleus and found associated with the cytoskeleton in some experiments. One of the first HIV proteins to be produced in infected cells, it is the most immunogenic of the accessory proteins. The nef genes of HIV and SIV are dispensable in vitro, but are essential for efficient viral spread and disease progression in vivo. Nef is necessary for the maintenance of high virus loads and for the development of AIDS in macaques, and viruses with defective Nef have been detected in some HIV- 1 infected long term survivors. Nef downregulates CD4, the primary viral receptor, and MHC class I molecules, and these functions map to different parts of the protein. Nef interacts with components of host cell signal transduction and clathrin-dependent protein sorting pathways. It increases viral infectivity. Nef contains PxxP motifs that bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of HIV but not for the downregulation of CD4.
[0836] VPX is a virion protein of 12 kDa found in HIV-2, SIV-SMM, SIV-RCM, SIV-MND-2 and SIV-DRL and not in HIV- 1 or other SIVs. This accessory gene is a homolog of HIV- 1 vpr, and viruses with Vpx carry both vpr and vpx. Vpx function in relation to Vpr is not fully elucidated; both are incorporated into virions at levels comparable to Gag proteins through interactions with Gag p6. Vpx is necessary for efficient replication of SIV-SMM in PBMCs. Progression to AIDS and death in SIV-infected animals can occur in the absence of Vpr or Vpx. Double mutant virus lacking both vpr and vpx was attenuated, whereas the single mutants were not, suggesting a redundancy in the function of Vpr and Vpx related to virus pathogenicity.
Hepatitis A Viral Target Sequences
5' untranslated region contains IRES - internal ribosome entry site P I Region of genome - capsid proteins
VP 1
VP2
VP3
VP4
P2 Region of genome
2A
2B
2C
P3 Region of genome
3A
3B
3C - viral protease
3D - RNA polymerase
Hepatitis B Viral Target Sequences
[0837] Precursor Polypeptide encoding all HCV protein is produced and then spliced into functional proteins. The following are the proteins (coding regions) encoded:
C - core protein - coding region consists of a Pre-C and Core coding region
X - function unclear but suspected to play a role in activation of viral transcription process
P - RNA polymerase
S - surface antigen - coding region consists of a Pre-S l , Pre-S2 and Surface antigen coding regions Hepatitis C Viral Target Sequences
[0838] Precursor Polypeptide encoding all HCV protein is produced and then spliced into functional proteins. The following are the proteins (coding regions) encoded:
RES - non-coding internal ribosome entry site (5' to polyprotein encoding sequence)
3' non-coding sequences -
C region - encodes p22 a nucleocapsid protein
E l region - encodes gp35 envelope glycoprotein - important in cell entry E2 region - encodes gp70 envelope glycoprotein - important in cell entry NS 1 - encodes p7 - not necessary for replication but critical in viral morphogenesis
NS2 - encodes p23 a transmembrane protein with protease activity
NS3 - encodes p70 having both serine protease and RNA helicase activities
NS4A - encodes p8 co-factor
NS4B - encodes p27 cofactor - important in recruitment of other viral proteins NS5A - encodes p56/58 an interferon resistance protein - important in viral replication
NS5B - encodes RNA polymerase
Herpes Simplex Virus Target Sequence
Figure imgf000239_0001
Figure imgf000240_0001
Figure imgf000241_0001
UL35 VP26 Γ681 Capsid protein LRP2 LRP2 Latency-related protein
Figure imgf000242_0001
transcript
HPV Target Sequences
Figure imgf000242_0002
LCR Viral long control region (location of early promoters)
Keratinocyte/auxiliary
enhancer
Po7 Promoter Early (E) gene promoter for subtype HPV 16
Pio5 Promoter Early (E) gene promoter for subtype HPV 18
P(,7o Promoter Late (L) gene promoter for HPV 16
P742 Promoter Late (L) gene promoter for HPV31
Influenza A Target Sequences
[0839] Influenza A is the most common flu virus that infects humans. The influenza A virion is made up of 8 different single stranded RNA segments which encodes 1 1- 14 proteins. These segments can vary in sequence, with most variation occun'ing in the hemagglutinin (H or HA) surface protein and neuraminidase (NA or N). The eight RNA segments (and the proteins they encode) are:
HA - encodes hemagglutinin (about'500 molecules of hemagglutinin are needed to make one virion).
NA - encodes neuraminidase (about 100 molecules of neuraminidase are needed to make one virion).
NP encodes nucleoprotein.
M encodes two matrix proteins (the M l and the M2) by using different reading frames from the same RNA segment (about 3000 matrix protein molecules are needed to make one virion). M42 is produced by alternative splicing, and can partially replace an M2.
NS encodes two distinct non-structural proteins (NS 1 and NEP) by using different reading frames from the same RNA segment.
PA encodes an RNA polymerase; an alternate form is sometimes made through a ribosomal skip, with + 1 frameshift, reading through to the next stop codon. PB 1 encodes an RNA polymerase, plus two other transcripts read from alternate start sites, named PB 1 -N40 and PB 1 -F2 protein (induces apoptosis) by using different reading frames from the same RNA segment.
PB2 encodes an RNA polymerase.
M. tuberculosis Target Sequences
[0840] The methods and composition described herein can be used to target M. tuberculosis and treat a subject suffering from an infection with M. tuberculosis.
Other
[0841] In some embodiments, the target gene is associated with multiple drug resistance (MDR), e.g., in bacterial infection. Infectious pathogens can use a number of mechanisms in attaining multi-drug resistance, e.g., no longer relying on a glycoprotein cell wall, enzymatic deactivation of antibiotics, decreased cell wall permeability to antibiotics, altered target sites of antibiotic, efflux pumps to remove antibiotics, increased mutation rate as a stress response, or a
combination thereof.
IX. Targets: Gene Editing/Correction
[0842] Candidate Cas9 molecules, candidate gRNA molecules, and/or candidate Cas9 molecule/gRNA molecule complexes, can be used to modulate genes (e.g., mutated genes) responsible for diseases. In some embodiments, the gene is modulated by editing or correcting a target gene, e.g., as described herein. In other embodiments, the human gene is modulated by delivery of one or more regulators/effectors (e.g., as described herein) inside cells to the target gene. For example, the genes described herein can be modulated, in vitro, ex vivo, or in vivo.
Table IX-1 . Selected Diseases in which a gene can be therapeutically targeted.
o Kinases (cancer)
o Energy metabolism (cancer) o CFTR (cystic fibrosis)
o Color blindness
o Hemochromatosis
o Hemophilia
o Phenylketonuria
o Polycystic kidney disease
o Sickle-cell disease
o Tay-Sachs disease
o Siderius X-linked mental retardation syndrome
o Lysosomal storage disorders, e.g., Alpha-galactosidase A deficiency o Anderson-Fabry disease
o Angiokeratoma Corporis Diffusum
o CADASIL syndrome
o Carboxylase Deficiency, Multiple, Late-Onset
o Cerebelloretinal Angiomatosis, familial
o Cerebral arteriopathy with subcortical infarcts and leukoencephalopathy o Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy
o Cerebroside Lipidosis syndrome
o Choreoathetosis self-mutilation hyperuricemia syndrome
o Classic Galactosemia
o Crohn's disease, fibrostenosing
o Phenylalanine Hydroxylase Deficiency disease,
o Fabry disease
o Hereditary coproporphyria
o Incontinentia pigmenti
o Microcephaly
o Polycystic kidney disease
o Rett's
o Alpha- 1 antitrypsin deficiency
o Wilson's Disease
o Tyrosinemia
o Frameshift related diseases
o Cystic fibrosis
o Triplet repeat diseases (also referred herein as trinucleotide repeat diseases)
[0843] Trinucleotide repeat diseases (also known as triplet repeat disease, trinucleotide repeat expansion disorders, triplet repeat expansion disorders, or codon reiteration disorders) are a set of genetic disorders caused by trinucleotide repeat expansion, e.g., a type of mutation where trinucleotide repeats in certain genes exceed the normal and/or stable threshold. The mutation can be a subset of unstable microsatellite repeats that occur in multiple or all genomic sequences. The mutation can increase the repeat count (e.g., result in extra or expanded repeats) and result in a defective gene, e.g., producing an abnormal protein. Trinucleotide repeats can be classified as insertion mutations or as a separate class of mutations. Candidate Cas9 molecules, candidate gRNA molecules, and/or candidate Cas9 molecule/gRNA molecule complexes, can be used to modulate one or more genes (e.g., mutated genes) associated with a trinucleotide repeat disease, e.g., by reducing the number of (e.g., removing) the extra or expanded repeats, such that the normal or wild-type gene product (e.g., protein) can be produced.
[0844] Exemplary trinucleotide repeat diseases and target genes involved in trinucleotide repeat diseases are shown in Table IX-I A.
Table IX-I A. Exemplary trinucleotide repeat diseases and target genes involved in trinucleotide repeat diseases
Figure imgf000246_0001
[0845] Exemplary target genes include those genes involved in various diseases or conditions, e.g., cancer (e.g., kinases), energy metabolism, cystic fibrosis (e.g., CFTR), color blindness, hemochromatosis, hemophilia, phenylketonuria, polycystic kidney disease, Sickle-cell disease, Tay-Sachs disease, Siderius X-linked mental retardation syndrome, Lysosomal storage disorders (e.g., Alpha-galactosidase A deficiency), Anderson-Fabry disease, Angiokeratoma Corporis Diffusum, CADASIL syndrome, Carboxylase Deficiency, Multiple, Late-Onset, Cerebelloretinal Angiomatosis, familial, Cerebral aiteriopafhy with subcortical infarcts and leukoencephalopathy, Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, Cerebroside Lipidosis syndrome, Choreoathetosis self-mutilation hyperuricemia syndrome, Classic Galactosemia, Crohn's disease, fibrostenosing, Phenylalanine Hydroxylase Deficiency disease, Fabry disease, Hereditary coproporphyria, Incontinentia pigmenti ,. Microcephaly, Polycystic kidney disease, Rett's, Alpha- 1 antitrypsin deficiency, Wilson's Disease,
Tyrosinemia, Frameshift related diseases, and Triplet repeat diseases.
[0846] Exemplary target genes and diseases are described in Tabic IX-2. The left hand column indentifies the disease and the right hand column identifies a gene for manipulation. (Table IX- 2 is provided in Anne IX-2).
[0847] Additional exemplary target genes include genes associated with diseases including, e.g., Crigler-Najjer syndrome, Glycogen storage disease type IV (GSD type IV), Familial
hemophagocytic lymphohistiocytosis (FHL-Perforin deficiency), Ornithine transcarbamylase deficiency (OTC deficiency) or other Urea Cycle Disorders, Primary Hyperoxaluria, Leber congenital amaurosis (LCA), Batten disease, Chronic Granulomatous Disease, Wiskott-Aldrich syndrome, Usher Syndrome, and hemoglobinoapthies.
[0848] Crigler-Najjer syndrome. Crigler-Najjer syndrome is a severe condition characterized by high levels of bilirubin in the blood (hyperbilirubinemia). Bilirubin is produced when red blood cells are broken down. This substance is removed from the body only after it undergoes a chemical reaction in the liver, which converts the toxic form of bilirubin (unconjugated bilirubin) to a nontoxic form (conjugated bilirubin). People with Crigler-Najjar syndrome have a buildup of unconjugated bilirubin in their blood (unconjugated hyperbilirubinemia). Crigler-Najjar syndrome is divided into two types. Type 1 (CN 1 ) is very severe and Type 2 (CN2) is less severe.
[0849] Mutations in the UGT l A 1 gene can cause Crigler-Najjar syndrome. This gene provides instructions for making the bilirubin uridine diphosphate glucuronosyl transferase (bilirubin- UGT) enzyme, which is found primarily in liver cells and is necessary for the removal of bilirubin from the body. The bilirubin-UGT enzyme is involved in glucuronidation, in which the enzyme transfers glucuronic acid to unconjugated bilirubin, converting it to conjugated bilirubin. Glucuronidation makes bilirubin dissolvable in water so that it can be removed from the body.
[0850] Mutations in the UGT1 A l gene that cause Crigler-Najjar syndrome result in reduced or absent function of the bilirubin-UGT enzyme. People with C l have no enzyme function, while people with CN2 can have less than 20 percent of normal function. The loss of bilirubin-UGT function decreases glucuronidation of unconjugated bilirubin. This toxic substance then builds up in the body, causing unconjugated hyperbilirubinemia and jaundice.
[0851] Glycogen storage disease type IV. Glycogen storage disease type IV (also known as GSD type IV, Glycogenosis type IV, Glycogen Branching Enzyme Deficiency (GBED), polyglucosan body disease, or Amylopectinosis) is an inherited disorder caused by the buildup of a complex sugar called glycogen in the body's cells. The accumulated glycogen is structurally abnormal and impairs the function of certain organs and tissues, especially the liver and muscles.
[0852] Mutations in the GBE 1 gene cause GSD IV. The GBE 1 gene provides instructions for making the glycogen branching enzyme. This enzyme is involved in the production of glycogen, which is a major source of stored energy in the body. GBE1 gene mutations that cause GSD IV lead to a shortage (deficiency) of the glycogen branching enzyme. As a result, glycogen is not formed properly. Abnormal glycogen molecules called polyglucosan bodies accumulate in cells, leading to damage and cell death. Polyglucosan bodies accumulate in cells throughout the body, but liver cells and muscle cells are most severely affected in GSD IV. Glycogen accumulation in the liver leads to hepatomegaly and interferes with liver functioning. The inability of muscle cells to break down glycogen for energy leads to muscle weakness and wasting.
[0853] Generally, the severity of the disorder is linked to the amount of functional glycogen branching enzyme that is produced. Individuals with the fatal perinatal neuromuscular type tend to produce less than 5 percent of usable enzyme, while those with the childhood neuromuscular type may have around 20 percent of enzyme function. The other types of GSD IV are usually associated with between 5 and 20 percent of working enzyme. These estimates, however, vary among the different types.
[0854] Familial hemophagocytic lymphohistiocytosis. Familial hemophagocytic
lymphohistiocytosis (FHL) is a disorder in which the immune system produces too many activated immune cells (lymphocytes), e.g., T cells, natural killer cells, B cells, and macrophages (histiocytes). Excessive amounts of cytokines are also produced. This overactivation of the immune system causes fever and damages the liver and spleen, resulting in enlargement of these organs.
[0855] Familial hemophagocytic lymphohistiocytosis also destroys blood-producing cells in the bone marrow, a process called hemophagocytosis. The brain may also be affected in familial hemophagocytic lymphohistiocytosis. In addition to neurological problems, familial hemophagocytic lymphohistiocytosis can cause abnormalities of the heart, kidneys, and other organs and tissues. Affected individuals also have an increased risk of developing cancers of blood-forming cells (leukemia and lymphoma).
[0856] Familial hemophagocytic lymphohistiocytosis may be caused by mutations in any of several genes. These genes provide instructions for making proteins that help destroy or deactivate lymphocytes that are no longer needed. By controlling the number of activated lymphocytes, these genes help regulate immune system function.
[0857] Approximately 40 to 60 percent of cases of familial hemophagocytic lymphohistiocytosis are caused by mutations in the PRF1 or UNC I 3D genes. Smaller numbers of cases are caused by mutations in other known genes such as STX 1 1 or STXBP2. The gene mutations that cause familial hemophagocytic lymphohistiocytosis can impair the body's ability to regulate the immune system. These changes result in the exaggerated immune response characteristic of this condition.
[0858] Ornithine transcarbamylase deficiency. Ornithine transcarbamylase deficiency (OTC) is an inherited disorder that causes ammonia to accumulate in the blood.
[0859] Mutations in the OTC gene cause ornithine transcarbamylase deficiency.
[0860] Ornithine transcarbamylase deficiency belongs to a class of genetic diseases called urea cycle disorders. The urea cycle is a sequence of reactions that occurs in liver cells. It processes excess nitrogen, generated when protein is used by the body, to make a compound called urea that is excreted by the kidneys. [0861] In ornithine transcarbamylase deficiency, the enzyme that starts a specific reaction within the urea cycle is damaged or missing. The urea cycle cannot proceed normally, and nitrogen accumulates in the bloodstream in the form of ammonia.
[0862] Ammonia is especially damaging to the nervous system, so ornithine transcarbamylase . deficiency causes neurological problems as well as eventual damage to the liver.
[0863] Other urea cycle disorders and associate genes include, e.g., N-Acetylglutamate synthase deficiency (NAGS), Carbamoyl phosphate synthetase I deficiency (CPS 1), "AS deficiency" or citrullinemia (ASS), "AL deficiency" or argininosuccinic aciduria (ASL), and "Arginase deficiency" or argininemia (ARG).
[0864] Primary hyperoxaluria. Primary hyperoxaluria, e.g., primary hyperoxaluria type 1 (PH 1 ), is a rare, autosomal recessive inherited genetic condition in which an error in the glyoxylate metabolism pathway in the liver leads to an overproduction of oxalate, which crystalizes in soft tissues including the kidney, bone marrow, and eyes. The disease manifests as progressive deterioration of the kidneys, and treatment is a complicated double transplant of kidney (the damaged organ) and liver (the diseased organ).
[0865] Primary hyperoxaluria is caused by the deficiency of an enzyme that normally prevents the buildup of oxalate. There are two types of primary hyperoxaluria, distinguished by the enzyme that is deficient. People with type 1 primary hyperoxaluria have a shortage of a liver enzyme called alanine-glyoxylate aminotransferase (AGXT). Type 2 primary hyperoxaluria is characterized by a shortage of an enzyme called glyoxylate reductase/hydroxypyruvate reductase (GRHPR).
[0866] Mutations in the AGXT and GRHPR genes cause primary hyperoxaluria. The breakdown and processing of certain sugars and amino acids produces a glyoxylate. Normally, glyoxylate is converted to the amino acid glycine or to glycolate through the action of two enzymes, alanine-glyoxylate aminotransferase and glyoxylate reductase hydroxypyruvate reductase, respectively. Mutations in the AGXT or GRHPR gene cause a shortage of these enzymes, which prevents the conversion of glyoxylate to glycine or glycolate. As levels of glyoxylate build up, it is converted to oxalate. Oxalate combines with calcium to form calcium oxalate deposits, which can damage the kidneys and other organs. [0867] In ah embodiment, the genetic defect in AGXT is corrected, e.g., by homologous recombination, using the Cas9 molecule and gRNA molecule described herein. For example, the functional enzyme encoded by the corrected AGXT gene can be redirected to its proper subcellular organelle. Though >50 mutations have been identified in the gene, the most common (40% in Caucasians) is a missense G 170R mutation. This mutation causes the AGT enzyme to be localized to the mitochondria rather than to the peroxisome, where it must reside to perform its function. Other common mutations include, e.g., I244T (Canary Islands), Fl 521, G41 R, G630A (Italy), and G588A (Italy).
[0868] In an embodiment, one or more genes encoding enzymes upstream in the glyoxylate metabolism pathway are targeted, using the Cas9 molecule and gRNA molecule described herein. Exemplary targets include, e.g., glycolate oxidase (gene HAO l , OMIM ID 605023). Glycolate oxidase converts glycolate into glyoxylate, the substrate for AGT. Glycolate oxidase is only expressed in the liver and, because of its peroxisomal localization, makes it a suitable target in this metabolic pathway. In an embodiment, a double-strand break in the HAO l gene is introduced and upon repair by NHEJ a frame-shift results in a truncated protein. In an embodiment, a transcriptional repressor (e.g., a transcriptional repressor described herein) is delivered as a payload to the HAOl gene to reduce the expression of HAOl .
[0869] lAiber congenital amaurosis. Leber congenital amaurosis (LCA) is an eye disorder that primarily affects the retina. People with this disorder typically have severe visual impairment beginning in infancy. The visual impairment tends to be stable, although it may worsen very slowly over time. At least 13 types of Leber congenital amaurosis have been described. The types are distinguished by their genetic cause, patterns of vision loss, and related eye
abnormalities.
[0870] Leber congenital amaurosis can result from mutations in at least 14 genes, all of which are necessary for normal vision. These genes play a variety of roles in the development and function of the retina. For example, some of the genes associated with this disorder are necessary for the normal development of photoreceptors. Other genes are involved in phototransduction. Still other genes play a role in the function of cilia, which are necessary for the perception of several types of sensory input, including vision. [0871] Mutations in any of the genes associated with Leber congenital amaurosis (e.g., A1PL1 , CEP290, CRB 1 , CRX, GUCY2D, IMPDH 1 , LCA5, LRAT, RD3, RDH 12, RPE65, RPGRIP1 , SPATA7, TULP 1 ) can disrupt the development and function of the retina, resulting in early vision loss. Mutations in the CEP290, CRB 1 , GUCY2D, and RPE65 genes are the most common causes of the disorder, while mutations in the other genes generally account for a smaller percentage of cases.
[0872] Batten disease. Batten disease or juvenile Batten disease is an inherited disorder that primarily affects the nervous system. After a few years of normal development, children with this condition develop progressive vision loss, intellectual and motor disability, and seizures.
[0873] Juvenile Batten disease is one of a group of disorders known as neuronal ceroid lipofuscinoses (NCLs). These disorders all affect the nervous system and typically cause progressive problems with vision, movement, and thinking ability. Some people refer to the entire group of NCLs as Batten disease, while others limit that designation to the juvenile form of the disorder. The different types of NCLs are distinguished by the age at which signs and symptoms first appear.
[0874] Most cases of juvenile Batten disease are caused by mutations in the CLN3 gene. These mutations can disrupt the function of cellular structures called lysosomes. Lysosome malfunction leads to a buildup of lipopigments within these cell structures. These accumulations occur in cells throughout the body, but neurons in the brain seem to be particularly vulnerable to the damage caused by lipopigments. The progressive death of cells, especially in the brain, leads to vision loss, seizures, and intellectual decline in people with juvenile Batten disease.
[0875] A small percentage of cases of juvenile Batten disease are caused by mutations in other genes (e.g., ATP 13A2, CLN5, PPT1 , TPP1). Many of these genes are involved in lysosomal function, and when mutated, can cause this or other forms of NCL.
[0876] Chronic granulomatous disease. Chronic granulomatous disease is a disorder that causes the immune system to malfunction, resulting in a form of immunodeficiency. Individuals with chronic granulomatous disease have recurrent bacterial and fungal infections. People with this condition often have areas of inflammation (granulomas) in various tissues that can be damaging to those tissues. he features of chronic granulomatous disease usually first appear in childhood, although some individuals do not show symptoms until later in life. [0877] Mutations in the CYBA, CYBB, NCF1, NCF2, or NCF4 gene can cause chronic granulomatous disease. There are five types of this condition that are distinguished by the gene that is involved. The proteins produced from the affected genes are subunits of NADPH oxidase, which plays an important role in the immune system. Specifically, NADPH oxidase is primarily active in phagocytes. Within phagocytes, NADPH oxidase is involved in the production of superoxide, which plays a role in . killing foreign invaders and preventing them from reproducing in the body and causing illness. NADPH oxidase also regulates the activity of neutrophils, which play a role in adjusting the inflammatory response to optimize healing and reduce injury to the body.
[0878] Mutations in the CYBA, CYBB, NCF1 , NCF2, and NCF4 genes result in the production of proteins with little or no function or the production of no protein at all. Without any one of its subunit proteins, NADPH oxidase cannot assemble or function properly. As a result, phagocytes are unable to kill foreign invaders and neutrophil activity is not regulated. A lack of NADPH oxidase leaves affected individuals vulnerable to many types of infection and excessive inflammation.
[0879] Wiskott-Aldrich syndrome. Wiskott-Aldrich syndrome is characterized by abnormal immune system function (immune deficiency) and a reduced ability to form blood clots. This condition primarily affects males. Individuals with Wiskott-Aldrich syndrome have
microthrombocytopenia, which is a decrease in the number and size of blood cells involved in clotting (platelets), which can lead to easy bruising or episodes of prolonged bleeding following minor trauma. Wiskott-Aldrich syndrome causes many types of white blood cells to be abnormal or nonfunctional, leading to an increased risk of several immune and inflammatory disorders. Many people with this condition develop eczema, an inflammatory skin disorder characterized by abnormal patches of red, irritated skin. Affected individuals also have an increased susceptibility to infection. People with Wiskott-Aldrich syndrome are at greater risk of developing autoimmune disorders. The chance of developing some types of cancer, such as cancer of the immune system cells (lymphoma), is also greater in people with Wiskott-Aldrich syndrome.
[0880] Mutations in the WAS gene cause Wiskott-Aldrich syndrome. The WAS gene provides instructions for making WASP protein, which is found in all blood cells. WASP is involved in relaying signals from the surface of blood cells to the actin cytoskeleton. WASP signaling activates the cell when it is needed and triggers its movement and attachment to other cells and tissues (adhesion). In white blood cells, this signaling allows the actin cytoskeleton to establish the interaction between cells and the foreign invaders that they target (immune synapse).
[0881] WAS gene mutations that cause Wiskott-Aldrich syndrome lead to a lack of any functional WASP. Loss of WASP signaling disrupts the function of the actin cytoskeleton in developing blood cells. White blood cells that lack WASP have a decreased ability to respond to their environment and form immune synapses. As a result, white blood cells are less able to respond to foreign invaders, causing many of the immune problems related to Wiskott-Aldrich syndrome. Similarly, a lack of functional WASP in platelets impairs their development, leading to reduced size and early cell death.
[0882] Usher syndrome. Usher syndrome is a condition characterized by hearing loss or deafness and progressive vision loss. The loss of vision is caused by retinitis pigmentosa (RP), which affects the layer of light-sensitive tissue at the back of the eye (the retina). Vision loss occurs as the light-sensing cells of the retina gradually deteriorate.
[0883] Three major types of Usher syndrome, designated as types I (subtypes IA through IG), II (subtypes IIA, IIB, and IIC), and III, have been identified. These types are distinguished by their severity and the age when signs and symptoms appear.
[0884] Mutations in the CDH23, CLRN 1 , GPR98, MY07A, PCDH 15, USH 1C, USH 1G, and USH2A genes can cause Usher syndrome. The genes related to Usher syndrome provide instructions for making proteins that play important roles in normal hearing, balance, and vision. They function in the development and maintenance of hair cells, which are sensory cells in the inner ear that help transmit sound and motion signals to the brain. In the retina, these genes are also involved in determining the structure and function of light-sensing cells called rods and cones. In some cases, the exact role of these genes in hearing and vision is unknown. Most of the mutations responsible for Usher syndrome lead to a loss of hair cells in the inner ear and a gradual loss of rods and cones in the retina. Degeneration of these sensory cells causes hearing loss, balance problems, and vision loss characteristic of this condition.
[0885] Usher syndrome type" I can result from mutations in the CDH23, MY07A, PCDH 15, USH 1C, or USH 1 G gene. Usher syndrome type II can be caused by mutations in, e.g., USH2A or GPR98 (also called VLGR 1 ) gene. Usher syndrome type III can be caused by mutations in e.g., CLRN 1 .
[0886] Hemoglobinopathies. Hemoglobinopathies are a group of genetic defects that result in abnormal structure of one of the globin chains of the hemoglobin molecule. Exemplary hemoglobinopathies include, e.g., sickle cell disease, alpha thalassemia, and beta thalassemia.
[0887] In an embodiment, a genetic defect in alpha globulin or beta globulin is corrected, e.g., by homologous recombination, using the Cas9 molecule and gRNA molecule described herein.
[0888] In an embodiment, a hemoglobinopathies-associated gene is targeted, using the Cas9 molecule and gRNA molecule described herein. Exemplary targets include, e.g., genes associated with control of the gamma-globin genes. In an embodiment, the target is BCL1 1 A.
[0889] Fetal hemoglobin (also hemoglobin F or HbF or α2γ2) is a tetramer of two adult alpha- globin polypeptides and two fetal beta-like gamma-globin polypeptides. HbF is the main oxygen transport protein in the human fetus during the last seven months of development in the uterus and in the newborn until roughly 6 months old. Functionally, fetal hemoglobin differs most from adult hemoglobin in that it is able to bind oxygen with greater affinity than the adult form, giving the developing fetus better access to oxygen from the mother's bloodstream.
[0890] In newborns, fetal hemoglobin is nearly completely replaced by adult hemoglobin by approximately 6 months postnatally. In adults, fetal hemoglobin production can be reactivated pharmacologically, which is useful in the treatment of diseases such as hemoglobinopathies. For example, in certain patients with hemoglobinopathies, higher levels of gamma-globin expression can partially compensate for defective or impaired beta-globin gene production, which can ameliorate the clinical severity in these diseases. Increased HbF levels or F-cell (HbF containing erythrocyte) numbers can ameliorate the disease severity of hemoglobinopathies, e.g., beta- thalassemia major and sickle cell anemia.
[0891] Increased HbF levels or F-cell can be associated reduced BCL1 1 A expression in cells. The BCL 1 1 A gene encodes a multi-zinc finger transcription factor. In an embodiment, the expression of BCL1 1 A is modulated, e.g., down-regulated. In an embodiment, the BCL1 1 A gene is edited. In an embodiment, the cell is a hemopoietic stem cell or progenitor cell. Sickle cell diseases
[0892] Sickle cell disease is a group of disorders that affects hemoglobin. People with this disorder have atypical hemoglobin molecules (hemoglobin S), which can distort red blood cells into a sickle, or crescent, shape. Characteristic features of this disorder include a low number of red blood cells (anemia), repeated infections, and periodic episodes of pain.
[0893] Mutations in the HBB gene cause sickle cell disease. T e HBB gene provides instructions for making beta-globin. Various versions of beta-globin result from different mutations in the HBB gene. One particular HBB gene mutation produces an abnormal version of beta-globin known as hemoglobin S (HbS). Other mutations in the HBB gene lead to additional abnormal versions of beta-globin such as hemoglobin C (HbC) and hemoglobin E (HbE). HBB gene mutations can also result in an unusually low level of beta-globin, i.e., beta thalassemia.
[0894] In people with sickle cell disease, at least one of the beta-globin subunits in hemoglobin is replaced with hemoglobin S. In sickle cell anemia, which is a common form of sickle cell disease, hemoglobin S replaces both beta-globin subunits in hemoglobin. In other types of sickle cell disease, just one beta-globin subunit in hemoglobin is replaced with hemoglobin S. The other beta-globin subunit is replaced with a different abnormal variant, such as hemoglobin C. For example, people with sickle-hemoglobin C (HbSC) disease have hemoglobin molecules with hemoglobin S and hemoglobin C instead of beta-globin. If mutations that produce hemoglobin S and beta thalassemia occur together, individuals have hemoglobin S-beta thalassemia
(HbSBetaThal) disease.
Alpha thalassemia
[0895] Alpha thalassemia is a blood disorder that reduces the production of hemoglobin. In people with the characteristic features of alpha thalassemia, a reduction in the amount of hemoglobin prevents enough oxygen from reaching the body's tissues. Affected individuals also have a shortage of red blood cells (anemia), which can cause pale skin, weakness, fatigue, and more serious complications.
[0896] Two types of alpha thalassemia can cause health problems. The more severe type is hemoglobin Bart hydrops fetalis syndrome or Hb Bart syndrome. The milder form is HbH disease. Hb Bart syndrome is characterized, e.g., by hydrops fetalis, a condition in which excess fluid builds up in the body before birth. HbH disease can cause, e.g., mild to moderate anemia, hepatosplenomegaly, and yellowing of the eyes and skin (jaundice).
[0897] Alpha thalassemia typically results from deletions involving the HBA 1 and HBA2 genes. Both of these genes provide instructions for making alpha-globin, which is a subunit of hemoglobin. The different types of alpha thalassemia result from the loss of some or all of these . alleles.
[0898] Hb Bart syndrome can result from the loss of all four alpha-globin alleles. HbH disease can be caused by a loss of three of the four alpha-globin alleles. In these two conditions, a shortage of alpha-globin prevents cells from making normal hemoglobin. Instead, cells produce abnormal forms of hemoglobin, i.e., hemoglobin Bart (Hb Bart) or hemoglobin H (HbH), which cannot effectively carry oxygen to the body's tissues. The substitution of Hb Bart or HbH for normal hemoglobin can cause anemia and the other serious health problems associated with alpha thalassemia.
[0899] Two additional variants of alpha thalassemia are related to a reduced amount of alpha- globin. A loss of two of the four alpha-globin alleles can result in alpha thalassemia trait.
People with alpha thalassemia trait may have unusually small, pale red blood cells and mild anemia. A loss of one alpha-globin allele can be found in alpha thalassemia silent carriers.
Beta thalassemia
[0900] Beta thalassemia is a blood disorder that reduces the production of hemoglobin. In people with beta thalassemia, low levels of hemoglobin lead to a lack of oxygen in many parts of the body. Affected individuals also have a shortage of red blood cells (anemia), which can cause pale skin, weakness, fatigue, and more serious complications. People with beta thalassemia are at an increased risk of developing abnormal blood clots.
[0901] Beta thalassemia is classified into two types depending on the severity of symptoms: thalassemia major (also known as Cooley's anemia) and thalassemia intermedia. Of the two types, thalassemia major is more severe.
[0902] Mutations in the HBB gene cause beta thalassemia. The HBB gene provides instructions for making beta-globin. Some mutations in the HBB gene prevent the production of any beta- globin. The absence of beta-globin is referred to as beta-zero (B°) thalassemia. Other HBB gene mutations allow some beta-globin to be produced but in reduced amounts, i.e., beta-plus (B+) thalassemia. People with both types have been diagnosed with thalassemia major and thalassemia intermedia.
[0903] In an embodiment, a Cas9 molecule/gRNA molecule complex targeting a first gene is used to treat a disorder characterized by second gene, e.g., a mutation in a second gene. By way of example, targeting of the first gene, e.g., by editing or payload delivery, can compensate for, or inhibit further damage from, the affect of a second gene, e.g., a mutant second gene. In an embodiment the allele(s) of the first gene carried by the subject is not causative of the disorder.
Table IX-3. Selected Disorders and Targets for Compensatory Targeting
Figure imgf000258_0001
Comment Miiti-genetic origin. Factor H aHUS due to fH deficiency.
deficiency is a risk factor. C5 antibody has been shown
Controlling the complement to vastly improve prognosis.
cascade, through fi l Can approach disease directly
upregulation or C5 through increasing fH levels
downregulation, may have a or controlling complement
beneficial effect. through C5 downregulation.
Indication Devices: Graft orthopedics- Parkinson's Allergic Epilepsy Barrett's stent, hcaling/wou articular Disease rhinitis esophagus, pacemaker, nd cartilage Stomach hernia mesh- healing/prev repair, ulcer, gastritis local delivery ention of arthritis
to prevent fibrosis
restenosis/
fibrosis
Target mTORC2, VEGF IL-1 1 SNCA, H I H I receptors H2 receptor others LRRK2, Receptors CNS pylorus,
EIF4GI nasal esophagus mucosa
Upregulate down- up-regiilate tip-regulate up-regulate down- up-regulate down-regulate
/ regulate or fix regulate
Downrcgul mutations
ate
Level of everolimus VEGF local animal model H I -antianimal H2-specific evidence: administratio of cartilage histamines, models antihistamines.
Market n aids in repair e.g. Zyrtec e g- proxy or tracheal omeprazole. animal transplant etc.
model animal
models Comment Embodiments Useful, e.g.. In
include, e.g.. in the embodiments. embodiments, local delivery promoting the subject the subject is to tissue via wound sufferes from treated for device or healing arthritis or is late-stage injection to (burns, etc); in need of barren's. prevent Embodiments healing after
fibrosis, include, e.g., injury. In
restenosis local delivery embodiments,
of growth chondrocytes
factors are targeted
post-injury to
promote
healing.
[0904] In an embodiment, Cas9 molecules, gRNA molecules, and/or Cas9 molecule/gRNA
molecule complexes can be used to activate genes that regulate growth factors, such as up
regulation of Epo to drive RBC production.
[0905] In an embodiment, Cas9 molecules, gRNA molecules, and/or Cas9 molecule/gRNA
molecule complexes can be used to target, e.g., result in repression of, knockout of, or alteration of promoter for key transcription factors, such as BCLl 1 A and KLFl for up-regulating of fetal hemoglobin, e.g., for cure for sickle cell anemia and thalassemia.
[0906] Candidate Cas9 molecules, candidate gRNA molecules, and/or candidate Cas9
molecule/gRNA molecule complexes, as described herein, can be used to edit/correct a target
gene or to deliver a regulator/effector inside cells, e.g., as described herein, at various subcellular locations. In some embodiments, the location is in the nucleus. In some embodiments, the
location is in a sub-nuclear domain, e.g., the chromosome territories, nucleolus, nuclear speckles,
Cajal bodies, Gems (gemini of Cajal bodies), or promyelocytic leukemia (PML) nuclear bodies.
In other embodiments, the location is in the mitochondrion.
[0907] Candidate Cas9 molecules, candidate gRNA molecules, and/or candidate Cas9
molecule/gRNA molecule complexes, as described herein, can be used to edit/correct a target
gene or to deliver a regulator/effector inside cells, as described herein, at various time points
[0908] For example, the editing/correction or delivery can occur at different phases of cell cycle, e.g., GO phase, Interphase (e.g., G l phase, S phase, G2 phase), or M phase. As another example, the editing/correction or delivery can occur at different stages of disease progression, e.g., at latent stage or active stage of a disorder (e.g., viral infection), or at any stage or subclassification of a disorder (e.g., cancer).
[0909] Methods of the invention allow for the treatment of a disorder characterized by unwanted cell proliferation, e.g., cancer. In an embodiment, cancer cells are manipulated to make them more susceptible to treatment or to endogenous immune surveillance. In an embodiment a cancer cell is modulated to make it more susceptible to a therapeutic. In an embodiment, a cancer cell is manipulated so as to increase the expression of a gene that increases the ability of the immune system to recognize or kill the cancer cell. E.g., a Cas9 molecule/gRNA molecule complex can be used to deliver a payload, or edit a target nucleic acid so as to increase the expression of an antigen, e.g., in the case where the cancer cell has downregulated expression of the antigen. In an embodiment, a payload, e.g., a payload comprising a transcription factor or other activator of expression is delivered to the cancer cell. In an embodiment, an increase in expression is effected by cleavage of the target nucleic acid, e.g., cleavage and correction or alteration of the target nucleic acid by a template nucleic acid. In an embodiment, a payload that overrides epigenetic silencing, e.g., a modulator of methylation, is delivered.
[0910] In an embodiment, the treatment further comprises administering a second anti-cancer , therapy, e.g., immunotherapy, e.g., an antibody that binds the upregulated antigen.
[0911] In an embodiment, methods described herein, e.g., targeting of a genomic signature, e.g., a somatic translocation, can be used to target the Cas9 molecule/gRNA molecule to a cancer cell.
[0912] In another aspect, the invention features a method of immunizing a subject against an antigen. The method comprises using a method described herein to promote the expression of the antigen from a cell, e.g., a blood cell, such that the antigen promotes an immune response. In an embodiment, the cell is manipulated ex vivo and then returned or introduced into the subject.
X. Modified Nucleosides, Nucleotides, and Nucleic Acids
[0913] Modified nucleosides and modified nucleotides can be present in nucleic acids, e.g., particularly gRN A, but also other forms of RNA, e.g., mRNA, RNAi, or siRN A. As described herein "nucleoside" is defined as a compound containing a five-carbon sugar molecule (a pentose or ribose) or derivative thereof, and an organic base, purine or pyrimidine, or a derivative thereof. As described herein, "nucleotide" is defined as a nucleoside further comprising a phosphate group.
[0914] Modified nucleosides and nucleotides can include one or more of:
(i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage;
(ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar;
(iii) wholesale replacement of the phosphate moiety with "dephospho" linkers;
(iv) modification or replacement of a naturally occurring nucleobase;
(v) replacement or modification of the ribose-phosphate backbone;
(vi) modification of the 3' end or 5' end of the oligonucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety; and
(vii) modification of the sugar.
[0915] The modifications listed above can be combined to provide modified nucleosides and nucleotides that can have two, three, four, or more modifications. For example, a modified nucleoside or nucleotide can have a modified sugar and a modified nucleobase. In an
embodiment, every base of a gRNA is modified, e.g., all bases have a modified phosphate group, e.g., all are phosphorothioate groups. In an embodiment, all, or substantially all, of the phosphate groups of a unimolecular or modular gRNA molecule are replaced with
phosphorothioate groups.
[0916] In an embodiment, modified nucleotides, e.g., nucleotides having modifications as described herein, can be incorporated into a nucleic acid, e.g., a "modified nucleic acid." In some embodiments, the modified nucleic acids comprise one, two, three or more modified nucleotides. In some embodiments, at least 5% (e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%) of the positions in a modified nucleic acid are a modified nucleotides. [0917] Unmodified nucleic acids can be prone to degradation by, e.g., cellular nucleases. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the modified nucleic acids described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward nucleases.
[0918] In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term "innate immune response" includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. In some embodiments, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can disrupt binding of a major groove interacting partner with the nucleic acid. In some embodiments, the modified
nucleosides, modified nucleotides, and modified nucleic acids described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo, and also disrupt binding of a major groove interacting partner with the nucleic acid.
Definitions of Chemical Groups
[0919] As used herein, "alkyl" is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g. , n-propyl and isopropyl), butyl (e.g. , n-butyl, isobutyl, t-butyl), pentyl (e.g. , n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.
[0920] As used herein, "aryl" refers to monocyclic or polycyclic (e.g. , having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl,
phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms.
[0921 ] As used herein, "alkenyl" refers to an aliphatic group containing at least one double bond. [0922] As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing 2- 12 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propargyl, and 3-hexynyl.
[0923] As used herein, "arylalkyl" or "aralkyl" refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2-phenylethyl, 3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.
[0924] As used herein, "cycloalkyl" refers to a cyclic, bicyclic, tricyclic, or polycyclic non- aromatic hydrocarbon groups having 3 to 12 carbons. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl.
[0925] As used herein, "heterocyclyl" refers to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl.
[0926] As used herein, "heteroaryl" refers to a monovalent radical of a heteroaromatic ring system. Examples of heteroaryl moieties include, but are not limited to, imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolyl, and pteridinyl.
Phosphate Backbone Modifications
The Phosphate Group
[0927] In some embodiments, the phosphate group of a modified nucleotide can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified nucleotide, e.g., modified nucleotide present in a modified nucleic acid, can include the wholesale replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with
unsymmetrical charge distribution. [0928] Examples of modified phosphate groups include, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR2 (wherein R can be, e.g., hydrogen, alkyl, or aryl), or OR (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral; that is to say that a phosphorous atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorous atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp).
[0929] Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non-bridging oxygens can also include the replacement of the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl).
[0930] The phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.
Replacement of the Phosphate Group
[0931] The phosphate group can be replaced by non-phosphorus containing connectors. In some embodiments, the charge phosphate group can be replaced by a neutral moiety.
[0932] Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo,
methylenedimethylhydrazo and methyleneoxymethylimino.
Replacement of the Ribophosphate Backbone
[0933] Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.
Sugar Modifications
[0934] The modified nucleosides and modified nucleotides can include one or more
modifications to the sugar group. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'-alkoxide ion. The 2'-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom.
[0935] Examples of "oxy"-2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein "R" can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar);
polyethyleneglycols (PEG), 0(CH2CH20)nCH2CH2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the "oxy"-2' hydroxyl group modification can include "locked" nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g., by a Ci-6 alkylene or Cj-6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, 0(CH2)n-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the "oxy"-2' hydroxyl group modification can include the methoxyethyl group (MOE),
(OCH2CH2OCH3, e.g., a PEG derivative).
[0936] "Deoxy" modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially ds RNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CH2- amino (wherein amino can be, e.g., as described herein), -NHC(0)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl;
thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein.
[0937] The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The nucleotide "monomer" can have an alpha linkage at the Γ position on the sugar, e.g., alpha-nucleosides. The modified nucleic acids can also include "abasic" sugars, which lack a nucleobase at C- . These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L- nucleosides.
[0938] Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary modified nucleosides and modified nucleotides can include, without limitation, replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). In some embodiments, the modified nucleotides can include multicyclic forms (e.g., tricyclo; and "unlocked" forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replaced with a-L-threofuranosyl-(3'-→2')).
Modifications on the Nucleobase
[0939] The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified nucleosides and modified nucleotides that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base.
Uracil
[0940] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include without limitation pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio- uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy- u,ridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3- methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5- oxyacetic acid methyl ester (mcmo^U), 5-carboxymethyl-uridine (cmsU), 1 -carboxymethyl- pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5- methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5- methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5- carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm \s2U), 5-propynyl-uridine, 1 -propynyl-pseudouridine, 5-taurinomethyl-uridine (xcm5U), 1 -taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Trn5s2U), l-taurinomethyl-4- thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1- methyl-pseudouridine (ιτι'ψ). 5-methyl-2-thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (m' s \|/), 4-thio- l-methyl-pseudouridine, 3-methyl-pseudouridine (m'V), 2-thio- l -methyl- pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydroundine (D), dihydropseudoundine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D),
2- thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio- uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N l -methyl-pseudouridine,
3- (3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3- carboxypropy pseudouridine
Figure imgf000269_0001
5-(isopentenylaminomethyl)uridine (inm5U), 5- (isopentenylaminomethy])-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-0-methyl-uridine (Urn), 5,2'-0-dimethyl-uridine (m5Um), 2'-0-methyl-pseudouridine (ψπι), 2-thio-2'-0-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-0-methyl-uridine (mcm 5Um), 5-carbamoylmethyl-2'-0- methyl-uridine (ncm 5Um), 5-carboxymethylaminomethyl-2'-0-methyl-uridine (cmnm 5Um), 3,2'-0-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-0-methyl-uridine (inm 5Um), l -thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2- carbomethoxyvinyl) uridine, 5-[3-( l-E-propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine.
Cytosine
[0941] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include without limitation 5-aza- cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (act), 5- formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo- cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza- pseudoisocytidine, 1-methyl- l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1 -methyl-pseudoisocytidine, lysidine (k2C), a-thio-cytidine, 2'-0-methyl-cytidine (Cm), 5,2'-0-dimethyl-cytidine (m5Cm), N4-acetyl-2'-0- methyl-cytidine (ac4Cm), N4,2'-0-dimethyl-cytidine (m4Cm), 5-formyl-2'-0-methyl-cytidine (f 5Cm), N4,N4,2'-0-trimethyl-cytidine (m4 2Cm), 1 -thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine. Adenine
[0942] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include without limitation 2-amino- purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloi -purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza- 8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m'A), 2-methyl-adenine (m A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6- isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis- hydroxyisopentenyl)adenos'ine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6- methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl- adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m6 2A), N6-hydroxynorvalylcarbamoyl- adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6- acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio- adenosine, 2'-0-methyl-adenosine (Am), N6,2'-0-dimethyl-adenosine (m5Am), N6-Methyl-2'- deoxyadenosine, N6,N6,2'-0-trimethyl-adenosine (m6 2Am), l ,2'-0-dimethyl-adenosine (m' Am), 2'-0-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8- azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-( 19-amino- pentaoxanonadecyl)-adenosine.
Guanine
[0943] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include without limitation inosine (I), 1 - methyl-inosine (m 'l), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyo"sine (imG- 14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undemriodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q),
epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7- deaza-guanosine (preQ0), 7-aminomethyI-7-deaza-guanosine (preQi), archaeosine (G+), 7-deaza- 8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1 -methyl-guanosine (m'G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m2 2G), N2,7-dimethyl-guanosine (m2,7G), N2, N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1 -meth thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2- dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-0-methyl-guanosine (Gm), N2-methyl-2'-0- methyl-guanosine (m¾m), N2,N2-dimethyl-2'-0-methyl-guanosine (m2 2Gm), l -methyl-2'-0- methyl-guanosine (m'Gm), N2,7-dimethyl-2'-0-methyl-guanosine (m2,7Gm), 2'-0-methyl- inosine (Im), l ,2'-0-dimethyl-inosine (m'lm), 06-phenyl-2'-deoxyinosine, 2'-0-ribosylguanosine (phosphate) (Gr(p)), 1 -thio-guanosine, 06-methy]-guanosine, 06-Methyl-2'-deoxyguanosine, 2'- F-ara-guanosine, and 2'-F-guanosine.
Modified gRNAs
[0944] In some embodiments, the modified nucleic acids can be modified gRNAs. In some embodiments, gRNAs can be modified at the 3' end. In this embodiment, the gRNAs can be modified at the 3' terminal U ribose. For example, the two terminal hydroxyl groups of the U ribose can be oxidized to aldehyde groups and a concomitant opening of the ribose ring to afford a modified nucleoside as sown below:
Figure imgf000271_0001
wherein "U" can be an unmodified or modified uridine.
[0945] In another embodiment, the 3' terminal U can be modified with a 2' 3' cyclic phosphate as shown below:
Figure imgf000271_0002
wherein "U" can be an unmodified or modified uridine. [0946] In some embodiments, the gRNA molecules may contain 3' nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In this embodiment, e.g., uridines can be replaced with modified uridines, e.g., 5-(2-amino)propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein. In some embodiments, deaza nucleotides, e.g., 7- deaza-adenosine, can be incoiporated into the gRNA. In some embodiments, O- and N-alkylated nucleotides, e.g., N6-methyl andenosine, can be incorporated into the gRNA. In some embodiments, sugar-modified ribonucleotides can be incorporated, e.g., wherein the 2' OH- group is replaced by a group selected from H, -OR, -R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo, -SH, -SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN). In some embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate group. In some embodiments, the nucleotides in the overhang region of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2'-sugar modified, such as, 2-F 2'-0-methyl, thymidine (T), 2'-0- methoxyethyl-5-methyluridine (Teo), 2'-0-methoxyethyladenosine (Aeo ), 2'-0-methoxyethyl- 5-methylcytidine (m5Ceo ), and any combinations thereof.
[0947] In an embodiment, a one or more or all of the nucleotides in single stranded overhang of an RNA molecule, e.g., a gRNA molecule, are deoxynucleotides.
XI. Linkers
[0948] In some embodiments, the payload can be linked to the Cas9 molecules or the gRNA, e.g., by a covalent linker. This linker may be cleavable or non-cleavable. In some embodiments, a cleavable linker may be used to release the payload after transport to the desired target.
[0949] Linkers can comprise a direct bond or an atom such as, e.g., an oxygen (O) or sulfur (S), a unit such as -NR- wherein R is hydrogen or alkyl, -C(0)-, -C(0)0-, -C(0)NH-, SO, S02, - SO?NH- or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or . unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, heteroarylalkyl. In some embodiments, one or more methylenes in the chain of atoms can be replaced with one or more of O, S, S(O), S02, -S02NH-, -NR-, -C(O)-, -C(0)0-, -C(0)NH-, a cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, and substituted or unsubstituted heterocyclic.
Non-cleavable linkages
[0950] In some embodiments, the payload is attached to the Cas9 molecule or gRNA through a linker that is itself is stable under physiological conditions, such as an alkylene chain, and does not result in release of the payload from the Cas9 molecule and/or gRNA for at least 2, 3, 4, 5, 10, 15, 24 or 48 hours or for at least 1, 2, 3, 4, 5,or 10 days when administered to a subject. In some embodiments, the payload and the Cas9 molecule and/or gRNA comprise residues of a functional groups through which reaction and linkage of the payload to the Cas9 molecule or gRNA was achieved. In some embodiments, the functional groups, which may be the same or different, terminal or internal, of the payload or Cas9molecule and/or gRNA comprise an amino, acid, imidazole, hydroxyl, thio, acyl halide, -HC=CH-, C≡C group, or derivative thereof. In some embodiments, the linker comprises a hydrocarbylene group wherein one or more methylene groups is optionally replaced by a group Y (provided that none of the Y groups are adjacent to each other), wherein each Y, independently for each occurrence, is selected from, substituted or unsubstituted aryl, heteroaryl, cycloalkyl, heterocycloalkyl, or -0-, -C(=X)- (wherein X is NR,, O or S), -NR , -NR,C(0)-, -C(0)NRi-, -S(0)„-, -NRiS(0)n-,- S(0)nNR, -, - NR|C(0)-NR|-; and R| , independently for each occurrence, represents H or a lower alkyl and wherein n is 0, 1 , or 2.
[0951] In some embodiments, the linker comprises an alkylene moiety or a heteroalkylene moiety (e.g., an alkylene glycol moiety such as ethylene glycol). In some embodiments, a linker comprises a poly-L-glutamic acid, polylactic acid, poly(ethyleneimine), an oligosaccharide, an amino acid (e.g., glycine), an amino acid chain, or any other suitable linkage. The linker groups can be biologically inactive, such as a PEG, polyglycolic acid, or polylactic acid chain. In certain embodiments, the linker group represents a derivatized or non-derivatized amino acid (e.g., glycine). Cleavable Linkages
[0952] A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In one embodiment, the cleavable linking group is cleaved at least 10 times or more, or at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
[0953] Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
[0954] A cleavable linkage group, such as a disulfide bond (-S-S-) can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7. 1 -7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH. A linker can include a cleavable linking group that is cleavable by a particular enzyme.
[0955] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. The candidate cleavable linking group can also be tested for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals.
[0956] In some embodiments, the cleavable linkers include redox cleavable linkers, such as a disulfide group (-S-S-) and phosphate cleavable linkers, such as, e.g., -0-P(0)(OR)-0-, -O- P(S)(OR)-0-, -0-P(S)(SR)-0-, -S-P(0)(OR)-0-, -0-P(0)(OR)-S-, -S-P(0)(OR)-S-, -O- P(S)(OR)-S-, -S-P(S)(OR)-0-, -0-P(0)(R)-0-, -0-P(S)(R)-0-, -S-P(0)(R)-0-, -S-P(S)(R)-0-, - S-P(0)(R)-S-, -OP(S)(R)-S-, wherein R is hydrogen or alkyl.
Acid Cleavable Linking Groups
[0957] Acid cleavable linking groups are linking groups that are cleaved under acidic conditions. In some embodiments, acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C(=N)N-, -C(0)0-, or -OC(O)-.
Ester-Based Linking Groups
[0958] Ester-based cleavable linking groups are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula -C(0)0-, or -OC(O)-.
XII. Formulations and Delivery
[0959] Exemplary formulations and methods for delivery of the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component are described herein, e.g., in Table XII- 1 . .
Table XII-1 : DELIVERY SUMMARY
Figure imgf000275_0001
Vector into Non- Integration Molecule
Dividing Expression Delivered Cells
Physical YES Transient NO Nucleic
Acids and Proteins
Viral Retrovirus NO Stable YES RNA
Lentivirus YES Stable YES/NO RNA with
modifications
Adenovirus YES Transient NO DNA
Adeno- YES Stable NO DNA Associated
Virus (AAV)
Vaccinia Virus YES Very NO DNA
Transient
Herpes YES Stable NO DNA
Simplex Virus
Non- Viral Cationic . YES Transient Depends on Nucleic
Liposomes what is Acids delivered Proteins
Polymeric YES Transient Depends on Nucleic Nanoparticles what is Acids delivered Proteins
BIOLOGICAL Attenuated YES Transient NO Nucleic
NON- VIRAL Bacteria Acids
DELIVERY
VEHICLES Engineered YES Transient NO Nucleic
Bacteriophages Acids
Mammalian YES Transient NO Nucleic
Virus-like Acids
Particles
Biological YES Transient NO Nucleic liposomes: Acids Erythrocyte
Ghosts and
Exosomes DELIVERY VEHICLES
[0960] In an embodiment, the delivery vehicle is a physical vehicle. In an embodiment, the vehicle is low density ultrasound. For example, microbubbles containing payload (e.g., made of biocompatible material such protein, surfactant, or biocompatible polymer or lipid shell) can be used and the microbubbles can be destructed by a focused ultrasound bean during microvascular transit. In sembodiments, the vehicle is electroporation. For example, naked nucleic acids or proteins can be delivered by electroporation, e.g., into cell suspensions or tissue environment, such as retina and embryonic tissue. In an embodiment, the vehicle is needle or jet injection. For example, naked nucleic acids or protein can be- injected into, e.g., muscular, liver, skin, brain or heart tissue.
[0961 ] In an embodiment, the delivery vehicle is a viral vector. Types of viruses include, e.g., retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAV), vaccinia viruses, and herpes simplex viruses.
[0962] In an embodiment, the viral vector has the ability of cell type and/or tissue type recognition. For example, the viral vectors can be pseudotyped with different/alternative viral envelope glycoproteins; engineered with cell type-specific receptors (e.g., genetically
modification of viral envelope glycoproteins to incorporate targeting ligands such as peptide ligands, single chain antibodies, growth factors); and/or engineered to have a molecular bridge with dual specificities with one end recognizing viral glycoproteins and the other end
recognizing a moiety of the target cell surface (e.g., ligand-receptor, monoclonal antibodies, avidin-biotin and chemical conjugation).
[0963] In an embodiment, the viral vector achieves cell type specific expression. For example, tissue-specific promoter can be constructed to restrict expression of the transgene (Cas 9 and gRNA) in only the target cells. The specificity of the vectors can also be mediated by microRNA-dependent control of transgene expression. In an embodiment, the viral vector has increased efficiency of fusion of viral vector and target cell membrane. For example, fusion proteins such as fusion-competent hemagglutin (HA) can be incorporated to increase viral uptake into cells. In an embodiment, the viral vector has the ability of nuclear localization. For example, certain viruses that require the breakdown of the cell wall (during cell division) will not infect non-diving cell. Incorporated nuclear localization peptides into the matrix proteins of the virus allow transduction into non-proliferating cells.
[0964] In an embodiment, the delivery vehicle is a non-viral vector. In an embodiment, the non- viral vector is an inorganic nanoparticle (e.g., attached to the payload to the surface of the nanoparticle). Exemplary inorganic nanoparticles include, e.g., magnetic nanoparticles (e.g., Fe Mn02), silica (e.g., can integrate multi-functionality, e.g., conjugate the outer surface of the nanoparticle with a positively charged polymer (e.g., polyethylenimine, polylysine, polyserine) which allows for attachment (e.g., conjugation or entrapment) of payload and internal magnetic component, mesaporous silica nanoparticles with a positive charged polymer loaded with chloi oquine to enhance transfection of the non-viral vector in vitro, high density lipoproteins and gold nanoparticles, gold nanoparticles coated with payload which gets released when nanoparticles are exposed to increased temperature by exposure to near infrared light, gold, iron or silver nanoparticles with surface modified with polylysine or another charge polymer to capture the nucleic acid cargo. In an embodiment, the non-viral vector is an organic nanoparticle (e.g., entrapment of the payload inside the nanoparticle). Exemplary organic nanoparticles include, e.g., SNALP liposomes that contain cationic lipids together with neutral helper lipids which are coated with polyethylene glycol (PEG) and protamine and nucleic acid complex coated with lipid coating.
[0965] Exemplary lipids and polymers for gene transfer are shown below in Tables XII-2 and XII-3.
[0966] Exemplary lipids for gene transfer are shown below in Table XII-2.
Table XII-2: Lipids Used for Gene Transfer
Figure imgf000278_0001
Figure imgf000279_0001
[0967] Exemplary polymers for gene transfer are shown below in Table XII-3.
Table XII-3: Polymers Used for Gene Transfer
Figure imgf000279_0002
Poly(amidoethylenimine) SS-PAEI
Triethylenetetramine TETA
Poly( -aminoester)
Poly(4-hydroxy-L-proline ester) PHP
Poly(allylamine)
Poly(a-[4-aminobuty]]-L-glycolic acid) PAGA
Poly(D,L-lactic-co-glycolic acid) PLGA
Poly(N-ethyl-4-vinylpyridinium bromide)
Poly(phosphazene)s PPZ
Poly(phosphoester)s PPE
Poly(phosphoramidate)s PPA
Poly(N-2-hydroxypropylmethacrylamide) pHPMA
Poly (2-(dimethylamino)ethyl methacrylate) pD AEMA
Poly(2-aminoethyl propylene phosphate) PPE-EA
Chitosan
Galactosylated chitosan
N-Dodacylated chitosan
Hi stone
Collagen
Dextran-spermine D-SPM
[0968] In an embodiment, the vehicle has targeting modifications to increase target cell update of nanoparticles and liposomes, e.g., cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars, and cell penetrating peptides. In an embodiment, the vehicle uses fusogenic and endosome-destabilizing peptides/polymers. In an embodiment, the vehicle undergoes acid-triggered conformational changes (e.g., to accelerate endosomal escape of the cargo). In an embodiment, a stimuli-cleavable polymer is used, e.g., for release in a cellular compartment. For example, disulfide-based cationic polymers that are cleaved in the reducing cellular environment can be used.
[0969] In an embodiment, liposomes are used for delivery, e.g., to blood or bone marrow, e.g., as a way of targeting hematopoietic stem cells (HSCs) and progenitors. For example, long-term treatment can be enabled by direct delivery using liposomes for conditions where obtaining HSCs is difficult (e.g., HSCs are not stable or HSCs are rare). These conditions can include, e.g., sickle cell anemia, Fanconi anemia, and aplastic anemia. In an embodiment, liposomes are used for delivery to localized specific tissues, e.g., to liver or lung, via intravenous delivery or via localized injection to target organ or its blood flow. For example, long-term treatment can be enable to concentrate effect in that specific orgari or tissue type. These conditions can include urea cycle disorders, alpha- 1 -anti-trypsin or cystic fibrosis.
[0970] In an embodiment, the delivery vehicle is a biological non-viral delivery vehicle. In an embodiment, the vehicle is an attenuated bacterium (e.g., naturally or artificially engineered to be invasive but attenuated to prevent pathogenesis and expressing the transgene (e.g., Listeria monocytogenes, certain Salmonella strains, Bifidobacterium longum, and modified Escherichia coli), bacteria having nutritional and tissue-specific tropism to target specific tissues, bacteria having modified surface proteins to alter target tissue specificity). In an embodiment, the vehicle is a genetically modified bateriophase (e.g., engineered phages having large packaging capacity, less immunogenic, containing mammalian plasmid maintenance sequencesn and having incorporated targeting ligands). In an embodiment, the vehicle is a mammalian virus-like particle. For example, modified viral particles can be generated (e.g., by purification of the "empty" particles followed by ex vivo assembly of the virus with the desired cargo). The vehicle can also be engineered to incorporate targeting ligands to alter target tissue specificity. In an embodiment, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (e.g., erythrocyte ghosts, which are red blood cells broken down into spherical structures derived from the target patient (e.g., tissue targeting can be achieved by attachment of various tissue or cell-specific ligands), or secretory exosomes - patient derived membrane-bound nanovescicle (30 - 100 nm) of endocytic origin (e.g., can be produced from various cell types and can therefore be taken up by cells without the need of for targeting ligands).
[0971] In an embodiment, delivery of Cas by nanoparticles in the bone marrow is an in vivo approach to curing blood and immune diseases.
[0972] In an embodiment, the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component described herein is delivered by nucleofection. For example, Nucleofector™ (Lonza Cologne AG) is a transfection technology that can be used for delivery to primary cells and difficult-to-transfect cell lines. It is a non-viral method based on a combination of electrical parameters and cell-type specific solutions. It allows transfected nucleic acids to directly enter the nucleus (e.g., without relying on cell division for the transfer of nucleic acids into the nucleus), providing the ability to transfect non-dividing cells, such as neurons and resting blood cells. In an embodiment, nucleofection is used as an ex vivo delivery method.
[0973] In an embodiment, the components of a Cas system, e.g,, the Cas9 molecule component and the gRNA molecule component described herein is delivered by methods utilizing
endogenous receptor-mediate transporters, e.g., antibody-based molecular Trojan Horses
(ArmaGen). Such methods can allow for non-invasive delivery of therapeutics to locations that are otherwise difficult to reach, e.g., brain (e.g., to cross blood brain barrier (BBB), e.g., via
endogenous receptor-mediated transport processes).
/
[0974] In an embodiment, one or more nucleic acid molecules (e.g., DNA molecules) other than the components of a Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule component described herein, are delivered. In an embodiment, the nucleic acid molecule is
delivered at the same time as one or more of the compoments of the Cas system are delivered. In an embodiment, the nucleic acid molecule is delivered before or after (e.g., less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) one or more of the components of the Cas system are delivered. In an
embodiment, the nucleic acid molecule is delivered by a different means than one or more of the components of the Cas system, e.g., the Cas9 molecule component and/or the gRNA molecule compoment, are delivered. The nucleic acid molecule can be delivered by any of the delivery methods described herein. For example, the nucleic acid molecule can be delivered by a viral vector, e.g., an integration-deficient lentivirus, and the Cas9 molecule component and/or the
gRNA molecule component can be delivered by electroporation, e.g., such that the toxicity
caused by nucleic acids (e.g., DNAs) can be reduced. In an embodiment, the nucleic acid
molecule encodes a therapeutic protein, e.g., a protein described herein. In an embodiment, the nucleic acid molecule encodes an RNA molecule, e.g, an RNA molecule described herein.
XIII. Bi- odal or Differential Delivery of Components
[0975] Separate delivery of the components of a Cas system, e.g., the Cas9 molecule component and the gRNA molecule component, and more particularly, delivery of the components by
differing modes, can enhance performance, e.g., by improving tissue specificity and safety. [0976] In an embodiment, the Cas9 molecule and the gRNA molecule are delivered by different modes, or as sometimes referred to herein as differential modes. Different or differential modes, as used herein, refer modes of delivery, that confer different pharmacodynamic or
pharmacokinetic properties on the subject component molecule, e.g., a Cas9 molecule, gRNA molecule, template nucleic acid, or payload. E.g., the modes of delivery can result in different tissue distribution, different half-life, or different temporal distribution, e.g., in a selected compartment, tissue, or organ.
[0977] Some modes of delivery, e.g., delivery by a nucleic acid vector that persists in a cell, or in progeny of a cell, e.g., by autonomous replication or insertion into cellular nucleic acid, result in more persistent expression of and presence of a component. Examples include viral, e.g., adeno associated virus or lentivirus, delivery.
[0978] By way of example, the components, e.g., a Cas9 molecule and a gRNA molecule, can be delivered by modes that differ in terms of resulting half life or persistent of the delivered component the body, or in a particular compartment, tissue or organ. In an embodiment, a gRNA molecule can be delivered by such modes. The Cas9 molecule component can be delivered by a mode which results in less persistence or less exposure of its to the body or a particular compartment or tissue or organ.
[0979] More generally, in an embodiment, a first mode of delivery is used to deliver a first component and a second mode of delivery is used to deliver a second component. The first mode of delivery confers a first pharmacodynamic or pharmacokinetic property. The first pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ. The second mode of delivery confers a second pharmacodynamic or pharmacokinetic property. The second pharmacodynamic property can be, e.g., distribution, persistence, or exposure, of the component, or of a nucleic acid that encodes the component, in the body, a compartment, tissue or organ.
[0980] In an embodiment, the first pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure, is more limited than the second pharmacodynamic or pharmacokinetic property. [0981 ] In an embodiment, the first mode of delivery is selected to optimize, e.g., minimize, a pharmacodynamic or pharmacokinetic property, e.g., distribution, persistence or exposure.
[0982] In an embodiment, the second mode of delivery is selected to optimize, e.g., maximize, a pharmacodynamic or pharmcokinetic property, e.g., distribution, persistence or exposure.
[0983] In an embodiment, the first mode of delivery comprises the use of a relatively persistent element, e.g., a nucleic acid, e.g., a plasmid or viral vector, e.g., an AAV or lentivirus. As such vectors are relatively persistent product transcribed from them would be relatively persistent.
[0984] In an embodiment, the second mode of delivery comprises a relatively transient element, e.g., an RNA or protein.
[0985] In an embodiment, the first component comprises gRNA, and the delivery mode is relatively persistent, e.g., the gRNA is transcribed from a plasmid or viral vector, e.g., an AAV or lentivirus. Transcription of these genes would be of little physiological consequence because the genes do not encode for a protein product, and the gR As are incapable of acting in isolation. The second component, a Cas9 molecule, is delivered in a transient manner, for example as mRNA or as protein, ensuring that the full Cas9 molecule/gRNA molecule complex is only present and active for a short period of time.
[0986] Furthermore, the components can be delivered in different molecular form or with different delivery vectors that complement one another to enhance safety and tissue specificity.
[0987] Use of differential delivery modes can enhance performance,' safety and efficacy. For example, the likelihood of an eventual off-target modification can be reduced. Delivery of immunogenic components, e.g., Cas9 molecules, by less persistent modes can reduce
immunogenicity, as peptides from the bacterially-derived Cas enzyme are displayed on the surface of the cell by MHC molecules. A two-part deliveiy system can alleviate these drawbacks.
[0988] Differential delivery modes can be used to deliver components to different, but overlapping target regions. The formation active complex is minimized outside the overlap of the target regions. Thus, in an embodiment, a first component, e.g., a gRNA molecule is delivered by a first delivery mode that results in a first spatial, e.g., tissue, distribution. A second component, e.g., a Cas9 molecule is delivered by a second delivery mode that results in a second spatial, e.g., tissue, distribution. In an embodiment, the first mode comprises a first element selected from a liposome, nanoparticle, e.g., polymeric nanoparticle, and a nucleic acid, e.g., viral vector. The second mode comprises a second element selected from the group. In an embodiment, the first mode of delivery comprises a first targeting element, e.g., a cell specific receptor or an antibody, and the second mode of delivery does not include that element. In an embodiment, the second mode of delivery comprises a second targeting element, e.g., a second cell specific receptor or second antibody.
[0989] When the Cas9 molecule is delivered in a virus delivery vector, a liposome, or polymeric nanoparticle, there is the potential for delivery to and therapeutic activity in multiple tissues, when it may be desirable to only target a single tissue. A two-part delivery system can resolve this challenge and enhance tissue specificity. If the gRNA molecule and the Cas9 molecule are packaged in separated delivery vehicles with distinct but overlapping tissue tropism, the fully functional complex is only be formed in the tissue that is targeted by both vectors.
XIV. Targeting of Genomic Signatures
[0990] Cas9 molecules, gRNA molecules, and in particular, Cas9 molecule/gRNA molecule complexes, can be used to target a cell by virtue of sequence specific interaction with a target nucleic acid comprising a selected genomic signature. This provides for targeted destruction of cells having a selected genomic signature. Method and compositions disclosed herein can be used to treat disorders characterized by a selected genomic signature, e.g., a genomic signature present in the germline or a genomic signature that arise as a result of a sporadic or somatic change in the genome, e.g., a germline or acquired mutation in a cancer cell, a viral infection, or other germline or acquired changes to the genome.
[0991 ] While not wishing to be bound by theory, it is believed that complementarity between the targeting domain of a gRNA molecule and the target sequence of a target nucleic acid mediates target sequence-specific interaction of the Cas9 molecule/gRNA molecule complex with the target sequence. This allows targeting of specific sequences or genomic signatures, e.g., rearrangements, e.g., translocations, insertions, deletions, and inversions, and other mutations. A Cas9 molecule/gRNA molecule complex can be used to target specific sequence, e.g., mutations, that are genriline, mitochondrial, or somatic. Depending on the Cas9 molecule/gRNA molecule complex used, specific editing, the delivery of a payload, or both, can be effected. In an embodiment, both cleavage and delivery of a payload is effected.
[0992] In an embodiment, the Cas9 molecule/gRNA molecule complex that promotes cell death upon recognition of its target genomic sequence. In an embodiment, an eaCas9 molecule/gRNA molecule complex cleaves the target nucleic acid. In an embodiment, it does not deliver a payload. While not wishing to be bound by theory is it believed that endogenous cellular elements, e.g., elements of the DNA damage apoptosis signaling cascade promote apoptosis in these embodiments.
[0993] In an embodiment, an eaCas9 molecule/gRNA molecule complex cleaves the target nucleic acid and delivers a payload. The payload can comprises a compound that inhibits growth or cell division, or promotes apoptosis, e.g., an element of the DNA damage apoptosis signaling cascade. In an embodiment, a second Cas9 molecule/gRNA molecule complex is used to deliver a payload comprising a second compound that inhibits growth or cell division, or promotes apoptosis, e.g., an element of the DNA damage apoptosis signaling cascade. The Cas9
molecule/gRNA molecule complex that delivers the second payload can comprise an eiCas9 molecule or an eaCas9 molecule. An additional, e.g., third or fourth, Cas9 molecule/gRNA molecule complex, can be used to deliver additional payload, e.g., an additional compound that inhibits growth or cell division, or promotes apoptosis, e.g., an additional element of the DNA damage apoptosis signaling cascade promote.
[0994] In an embodiment, the Cas9 molecule/gRNA molecule complex delivers a payload comprising a compound that inhibits growth or cell division, or promotes apoptosis, e.g., an element of the DNA damage apoptosis signaling cascade, but does not cleave the target nucleic acid. While not wishing to be bound by theoiy is it believed that endogenous cellular elements, e.g., elements of the DNA damage apoptosis signaling cascade promote apoptosis in these embodiments.
[0995] Exemplary compounds that inhibit growth or cell division, or promote apoptosis, e.g., an element of the DNA damage apoptosis signaling cascade, are described herein, e.g., in Table XIV- 1. Table XlV-1
ATM kinases (double-strand breaks)
ATR kinases (single-strand breaks)
RF-C related protein (RAD 17)
The 9- 1 - 1 Complex: RAD 1 , RAD9, and HUS 1
Checkpoint proteins CH 1 , CHK2
P53
ZIP Kinase (ZIPK)
Fast Death-Domain Associated Protein XX (DAXX)
Promyelocytic leukemia protein (PML)
Apoptosis-inducing factor (AIF)
Caspase-activated DNAse (CAD) (in the absence of its inhibitor ICAD)
[0996] In an embodiment, a Cas9 molecule/gRNA molecule complex targets a sequence that includes or is near the breakpoint of a rearrangement, e.g., a translocation, inversion, insertion, or deletion. In an embodiment, the rearrangement confers unwanted properties, e.g., unwanted proliferation, on the cell. In an embodiment, the cell harboring the rearrangement is a cancer cell. In an embodiment, the rearrangement comprises a kinase gene and results in unwanted, increased, or constitutive expression of the kinase activity. In an embodiment, the rearrangement disrupts the expression of a tumor suppressor.
[0997] In an embodiment, the Cas9 molecule/gRNA molecule complex:
specifically targets, and e.g., cleaves, the genome of a cell comprising a rearrangement, e.g., by targeting a mutation, e.g., a breakpoint or junction of a rearrangement; or
targets, e.g., for cleavage or payload delivery, a nucleotide sequence within 200, 100, 150, 100, 50, 25, 10, or 5 nucleotides of a mutation, e.g., a rearrangement breakpoint.
[0998] The invention includes a method of manipulating a cell comprising a genomic signature, comprising:
administering a Cas9 molecule/gRNA molecule complex that targets said genomic signature, thereby manipulating said cell .
[0999] In an embodiment, manipulating comprises inhibiting the growth or division of, or killing, said cell. [1000] In an embodiment, said cell is a cancer cell or cell having a viral infection.
[1001 ] In an embodiment, the method comprises treating a subject, e.g., a human subject, for a disorder characterized by a cell having said genomic signature, e.g., a cancer or a viral infection.
[1002] In an embodiment, a Cas9 molecule/gRNA molecule complex disrupts a rearrangement, e.g., by introduction of a stop codon from a template nucleic acid, e.g., a stop codon is inserted into a fusion protein, e.g., a fusion protein comprising kinase activity.
[1003] The invention includes a method of treating a cancer having a translocation of a kinase gene to a non-kinase gene, which places the kinase domain under the control of the non-kinase gene control region comprising:
administering a Cas9 molecule/gRNA molecule complex that targets the translocation. In an embodiment, the control region, e.g., the promoter, or the coding sequence, of the kinase translocation, is edited to reduce expression.
XV. Combination Therapy
[1004] The Cas9 molecules, gRNA molecules, and in particular, Cas9 molecule/gRNA molecule complexes, can be used in combination with a second therapeutic agent, e.g., a cancer drug. In some embodiments, the second therapeutic agent (e.g., a cancer drug) and the Cas9 molecule, gRNA molecule, and in particular, Cas9 molecule/gRNA molecule complex target different (e.g., non-overlapping) pathways. In other embodiments, the second therapeutic agent (e.g., a cancer drug) and the Cas9 molecule, gRNA molecule, and in pailicular, Cas9 molecule/gRNA molecule complex target a same or overlapping pathway.
[1005] Exemplary combination therapies include, e.g.:
mTOR inhibitors (e.g., Temsirolimus (Torisel®) or Everolimus (Afinitor®)) together with a AKT-specific Cas9/gRNA molecule;
- ' Tyrosine kinase inhibitors such as Imatinib mesylate (Gleevec®); Dasatinib (Sprycel®);
Bosutinib (Bosulif®); Trasruzumab (Herceptin®); Pertuzumab (Perjeta™); Lapatinib (Tykerb®); Gefitinib (Iressa©); Erlotinib (Tarceva®) together with a HDAC-specific Cas9/gRNA molecule; and Any chemotherapeutic agent together with one or more Cas9/gRNAs against multidrug resistance genes such as MDR 1 gene.
XVI. Treatment of genetic disorder, e.g., Duchenne muscular dystrophy (DMD)
[1006] In another aspect, the invention features, a method of altering a cell, e.g., reducing or abolishing the effect of a genetic signature, e.g., a stop codon, e.g., a premature stop codon. The method comprises contacting said cell with:
a Cas9 molecule/gRNA molecule complex that cleaves at or upstream from the genetic signature, e.g., a premature stop codon,
thereby altering the cell.
[1007] While not wishing to be bound by theory it is believed that, in an embodiment, cleavage and subsequent exonuclease activity, and non-homologous end joining results in an altered sequence in which the genetic signature, e.g., a premature stop codon is eliminated, e.g., by being placed in a different frame. In an embodiment, the same series of events restores the proper reading frame to the sequence that follows the signature, e.g., premature stop codon.
[1008] When the method is earned out to correct a frameshift mutation in order to remove a premature stop codon, repair can be earned out at various sites in the DNA. One may direct cleavage at the mutation, thereby correcting the frameshift entirely and returning the protein to its wild-type (or nearly wild-type) sequence. One may also direct cleavage at or near the premature stop codon, so that all (or nearly all) amino acids of the protein C-terminal of the codon where repair was effected are wild-type. In the latter case, the resulting protein may have one or more frameshifted amino acids between the mutation and the repair site; however the protein may still be functional because it is full-length and has wild-type sequence across most of its length.
[1009] A genetic signature is a particular DNA sequence at a particular portion of the genome, that causes a phenotype (such as a genetic disease or a symptom thereof)- For instance, the genetic signature may be a premature stop codon that prevents expression of a protein. In this scenario, the premature stop codon can arise from a mutation that directly creates a stop codon, or from a mutation that causes a frameshift leading to a premature stop codon being formed downstream. A genetic signature may also be a point mutation that alters the identity of an important amino acid in a protein, disrupting the protein's function.
[1010] In an embodiment, the Cas9 molecule/gRNA molecule complex mediates a double stranded break in said target nucleic acid.
[1011 ] In certain embodiments, the genetic signature, e.g., a premature stop codon, results from a point mutation, an insertion, a deletion, or a rearrangement. In some embodiments, a mutation causes a frameshift, resulting in a genetic signature, e.g., a premature stop codon downstream of the mutation..
[1012] In certain embodiments, the premature stop codon is within the target nucleic acid. In other embodiments, the target nucleic acid is upstream of the premature stop codon. The mutation may be upstream of the target nucleic acid, within the target nucleic acid, or downstream of the target nucleic acid.
[1013] In some embodiments the double stranded break is within 500, 200, 100, 50, 30, 20, 10, 5, or 2 nucleotides of the mutation. In some embodiments the double stranded break is within 500, 200, 100, 50, 30, 20, 10, 5, or 2 nucleotides of the genetic signature, e.g., a premature stop codon.
[1014] In certain embodiments, the Cas9 molecule/gRNA molecule complex mediates exonuclease digestion of the target nucleic acid. In certain embodiments, the Cas9
molecule/gRNA molecule complex removes 1 , 2, 3, 4, or 5 nucleotides at the double stranded „ break.
[1015] In some embodiments, the double stranded break is resolved by non-homologous end joining.
[1016] In some embodiments the mutation and/or genetic signature, e.g., premature stop codon is in the dystrophin gene, e.g., in exon 51 , or in the intron preceding or following exon 51. The premature stop codon may also be caused by a mutation in the dystrophin gene at one or more of codons 54, 645, 773, 3335, and 3340. In some embodiments, the premature stop codon in the dystrophin gene results from a deletion of codons 2305 through 2366.
[1017] In some embodiments, contacting the cell with a Cas9 molecule/gRNA molecule complex comprises contacting the cell with a nucleic acid encoding a Cas9 molecule. In certain embodiments, contacting the cell with a Cas9 molecule/gRNA molecule complex comprises transfecting the cell with a nucleic acid, e.g., a plasmid, or using a viral vector such as adeno- associated virus (AAV).
[1018] In certain embodiments, the method results in increased levels of the protein in which the genetic signature, e.g., a premature stop codon, was previously located. For instance, protein levels (e.g., dystrophin levels) may be increased by at least 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% in a cell or in a tissue. In some embodiments, the method results in increased levels of the mRNA in which the premature stop codon was previously located, for instance by preventing the mRNA from undergoing nonsense-mediated mRNA decay.
[1019] In some embodiments, one or more of the target nucleic acid, the genetic signature, e.g., premature stop codon, and the mutation are located in the dystrophin gene (which is mutated in DMD). One or more of the target nucleic acid, the genetic signature, e.g., premature stop codon, and the mutation may also be located in the COL7A 1 gene (mutated in type VH-associated dystrophic epidermolysis bullosa), the FKTN gene (mutated in Fukuyama congenital muscular dystrophy), the dysferlin gene (mutated in limb-girdle muscular dystrophy type 2B), the CFTR gene (mutated in cystic fibrosis), HEXA (mutated in Tay-Sachs disease), the IDS gene (mutated in Hunter syndrome), the FVUI gene (mutated in hemophilia), the IDUA gene (mutated in Hurler syndrome), the PPT 1 gene (mutated in infantile neuronal ceroid lipofuscinosis), a tumor suppressor such as the ATM gene (mutated in cancers like gliomas and B-Cell Chronic
Lymphocytic Leukemia), RP2 (mutated in X-linked retinitis pigmentosa), the CTNS gene (mutated in nephropathic cystinosis), and the AVPR2 gene (mutated in Congenital nephrogenic diabetes insipidus).
[1020] In some embodiments, the method is perfoiTned in cultured cells. In some embodiments, the method further comprises administering the cell to a patient. The cell may be, for example, an induced pluripotent stem cell, a bone marrow derived progenitor, a skeletal muscle progenitor, a CD 133+ cell, a mesoangioblast, or a MyoD-transduced dermal fibroblast.
[1021 ] In some embodiments, the method comprises contacting the cell with a template nucleic acid under conditions that allow for homology-directed repair between the target nucleic acid and the template nucleic acid to correct the mutation or the premature stop codon. [1022] In another aspect, the invention features a method of treating a human subject having a disorder associated with a genetic signature, e.g., premature stop codon, e.g., DMD, comprising providing to the human subject:
1) a Cas9 molecule/gRNA molecule complex that cleaves at or upstream from the premature stop codon or
2) a cell that has been contacted with such complex,
thereby treating the subject.
[1023] In an embodiment,the Cas9 molecule/gRNA molecule complex mediates a double stranded break in said target nucleic acid.
[1024] In certain embodiments, genetic signature, e.g., premature stop codon results from a point mutation, an insertion, a deletion, or a rearrangement. In some embodiments, a mutation causes a frameshift, resulting in a premature stop codon downstream of the mutation.
[1025] In some embodiments the double stranded break is within 500, 200, 100, 50, 30, 20, 10, 5, or 2 nucleotides of the mutation. In some embodiments the double stranded break is within 500, 200, 100, 50, 30, 20, 10, 5, or 2 nucleotides of the premature stop codon.
[1026] In certain embodiments, the genetic signature, e.g., premature stop codon is within the target nucleic acid of the Cas9 molecule/gRNA molecule complex. In other embodiments, the target nucleic acid is upstream of the genetic signature, e.g., premature stop codon. The mutation may be upstream of the target nucleic acid, within the target nucleic acid, or downstream of the target nucleic acid.
[1027] In certain embodiments, the Cas9 molecule/gRNA molecule complex mediates exonuclease digestion of the target nucleic acid. In certain embodiments, the Cas9
molecule/gRNA molecule complex removes 1 , 2, 3, 4, or 5 nucleotides at the double stranded break.
[ 028] In some embodiments the double stranded break is resolved by non-homologous end joining.
[1029] In some embodiments the mutation and/or genetic signature, e.g., premature stop codon is in the dystrophin gene, e.g., in exon 51, or in the intron preceding or following exon 51. The premature stop codon may also be caused by a mutation in the dystrophin gene at one or more of codons 54, 645, 773, 3335, and 3340. In some embodiments, the premature stop codon in the dystrophin gene results from a deletion of codons 2305 through 2366.
[1030] In some embodiments, contacting the cell with a Cas9 molecule/gRNA molecule complex comprises contacting the cell with a nucleic acid encoding a Cas9 molecule. In certain embodiments, contacting the cell with a Cas9 molecule/gRNA molecule complex comprises transfecting the cell with a nucleic acid, e.g., a plasmid, or using a viral vector such as aderio- associated virus (AAV).
[1031] In certain embodiments, the method results in increased levels of the protein in which the genetic signature, e.g., premature stop codon was previously located. For instance, protein levels (e.g., dystrophin levels) may be increased by at least 3%, 4%, 5%, 10%, 15%, 20%, 25%, or 30% in a cell or in a tissue. In some embodiments, the method results in increased levels of the mRNA in which the premature stop codon was previously located, for instance by preventing the mRNA from undergoing nonsense-mediated mRNA decay.
[1032] In some embodiments, one or more of the target nucleic acid, the genetic signature, e.g., premature stop codon, and the mutation are located in the dystrophin gene (which is mutated in DMD). One or more of the target nucleic acid, the genetic signature, e.g., premature stop codon, and the mutation may also be located in the COL7A 1 gene (mutated in type VH-associated dystrophic epidermolysis bullosa), the FKTN gene (mutated in Fukuyama congenital muscular dystrophy), the dysferlin gene (mutated in limb-girdle muscular dystrophy type 2B), the CFTR gene (mutated in cystic fibrosis), HEXA (mutated in Tay-Sachs disease), the IDS gene (mutated in Hunter syndrome), the FVIII gene (mutated in hemophilia), the IDUA gene (mutated in Hurler syndrome), the PPT1 gene (mutated in infantile neuronal ceroid lipofuscinosis), a tumor suppressor such as the ATM gene (mutated in cancers like gliomas and B-Cell Chronic
Lymphocytic Leukemia), RP2 (mutated in X-linked retinitis pigmentosa), the CTNS gene (mutated in nephropathic cystinosis), and the AVPR2 gene (mutated in Congenital nephrogenic diabetes insipidus).
[1033] In some embodiments, the method is performed in cultured cells. In some embodiments, the method further comprises administering the cell to a patient. The cell may be, for example, an induced pluripotent stem cell, a bone marrow derived progenitor, a skeletal muscle progenitor, a CD 133+ cell, a mesoangioblast, or a MyoD-transduced dermal fibroblast.
[1034] In some embodiments the method comprises contacting the cell with a template nucleic acid under conditions that allow for homology-directed repair between the target nucleic acid and the template nucleic acid to correct the mutation or the premature stop codon.
[1035] In some embodiments, the subject has a disorder selected from Duchenne Muscular Dystrophy (DMD), collagen type VH-associated dystrophic epidermolysis bullosa, Fukuyama congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B, cystic fibrosis, lysosomal storage disorders (such as Tay-Sachs disease, Hunter syndrome, and nephropathic cystinosis), hemophilia, Hurler syndrome, infantile neuronal ceroid lipofuscinosis, X-linked retinitis pigmentosa (RP2), cancers (such as gliomas and B-Cell Chronic Lymphocytic
Leukemia), and Congenital nephrogenic diabetes insipidus.
XVII. Treatment of disorders characterized by lack of mature specialized cells, e.g., impaired hearing, with loss of hair cells, supporting cells, or spiral ganglion neurons; or for diabetes, with loss of beta islet cells
[1036] In another aspect, the invention features, a method of altering a cell, e.g., to promote the development of other mature specialized cells, e.g, in regeneration therapy. For example, proliferation genes can be upregulated and/or checkpoint inhibitors can be inhibited, e.g., to drive down one or more differenation pathways.
[1037] In an embodiment, the method includes induction of proliferation and specified lineage maturation.
[1038] In an embodiment, the method comprises, e.g., for restoration or improvement of hearing, contacting said cell with:
a Cas9 molecule/gRNA molecule complex that up-regulates a gene that promotes the development of hair cells, or down-regulates a gene that inhibits the development of hair cells thereby altering the cell.
[1039] In an embodiment, the Cas9 molecule/gRNA molecule delivers a payload that up- regulates a gene that promotes hair cell development. [1040] In an embodiment, the Cas9 molecule/gR A molecule delivers a payload that down- regulates a gene that inhibits hair growth.
[1041] In an embodiment, the Cas9 molecule/gRNA molecule complex edits the genome of a cell to up-regulate a gene that promotes hair growth. In an embodiment, a template nucleic acid is used to effect a Cas9 molecule/gRNA molecule complex alteration to the genome that up- regulates a gene that promotes hair growth.
[1042] In an embodiment, the Cas9 molecule/gRNA molecule complex edits the genome of a cell to down-regulate a gene that inhibits hair growth. In an embodiment, a template nucleic acid is used to effect a Cas9 molecule/gRNA molecule complex alteration to the genome that down- regulates a gene that promotes hair growth.
[1043] In an embodiment, said cell is an iPS cell, a native hair cell progenitor, or a mature hair cell.
[1044] In an embodiment, the Cas9 molecule/gRNA molecule and modifies expression of a gene, e.g., by modifying the structure of the gene (e.g., by editing the genome) or by delivery of a payload that modulates a gene. In an embodiment, the gene is a transcription factor or other regulatory gene.
[1045] In an embodiment, for hair cell or other mature cell regeneration, the method includes one or more or all of the following:
contacting the cell with a Cas9 molecule/gRNA molecule complex that results in up- regulation one or more of the following for cell proliferation: c-Myc, GATA3, Oct4, Sox2, Wnt, TCF3;
contacting the cell with a Cas9 molecule/gRNA molecule complex that results in down- regulation one or more of the following for check point: BCL2, BMP, Hesl , Hes5, Notch, p27, Prox 1 , TGF|3; and
contacting the cell with a Cas9 molecule/gRNA molecule complex that results in turning on a maturation pathway. For hair cells this would include one or more of the following: Atoh 1 (Malh l ). Barhl 1 , Gfi l , Myo7a, p63, PAX2, PAX 8, Pou4f3 arid for neurons would include one or more of the following: N EFH, Neurod l , Neurog l , POU4F1 . [1046] In an embodiment, the method comprises generation of inner ear hair cells, outer ear hair cells, spiral ganglion neurons, and ear supporting cells.
[1047] In an embodiment, one or more growth factors can be modulated, e.g., upregulated, e.g., TPO can be upregulated for production of platelets and GCSF can be upregulated for production of neutrophils.
[1048] In another aspect, the invention provides altered cell described herein, e.g., in this Section XVII.
[1049] In another aspect, the invention features a method of treating impaired hearing. The method comprises administering to said subject, an altered cell described herein, e.g., in this section XVII. In an embodiment, the cell is autologous. In an embodiment, the cell is allogeneic. In an embodiment, the cell is xenogeneic.
[1050] In another aspect, the invention features a method of treating subject, e.g., for impaired hearing. The method comprises administering to said subject:
a Cas9 molecule/gRNA molecule complex that up-regulates a gene that promotes the growth of hair, or down-regulates a gene that inhibits the growth of hair thereby altering the cell.
[1051] In an embodiment, the Cas9 molecule/gRNA molecule delivers a payload that up- regulates a gene that promotes hair growth.
[1052] In an embodiment, the Cas9 molecule/gRNA molecule delivers a payload that down- regulates a gene that inhibits hair growth.
[1053] In an embodiment, the Cas9 molecule/gRNA molecule complex edits the genome of a cell to up-regulate a gene that promotes hair growth. In an embodiment, a template nucleic acid is used to effect a Cas9 molecule/gRNA molecule complex alteration to the genome that up- regulates a gene that promotes hair growth.
[1054] In an embodiment, the Cas9 molecule/gRNA molecule complex edits the genome of a cell to down-regulate a gene that inhibits hair growth. In an embodiment, a template nucleic acid is used to effect a Cas9 molecule/gRNA molecule complex alteration to the genome that down- regulates a gene that promotes hair growth. [1055] In an embodiment, the Cas9 molecule/gRNA molecule and modifies expression of a gene, e.g., by modifying the structure of the gene (e.g., by editing the genome) or by delivery of a payload that modulates a gene. In an embodiment, the gene is a. transcription factor or other regulatory gene.
[1056] In an embodiment, the method includes one or more or all of the following:
administering a Cas9 molecule/gRNA molecule complex that results in up-regulation one or more of the following: c-Myc, GATA3, Oct4, Sox2, Wnt, TCF3;
administering a Cas9 molecule/gRNA molecule complex that results in turning on a maturation pathway. For hair cells this would include one or more of the following: A toll 1 (Math l), Barhl l , Gfi l, Myo7a, p63, PAX2, PAX8. Pou4f3 and for neurons would include one or more of the following: N EFH, Neurod l , Neurog l , POU4F1 .
Annexes are included as part of the application.
INCORPORATION BY REFERENCE
[1057] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
EQUIVALENTS
[1058] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. While this invention has been disclosed with reference to. specific aspects, it is apparent that other aspects and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such aspects and equivalent variations.
Figure imgf000299_0001
BHLHE23
BHLHE40
j BHLHE41
\ C13orf38- j SOHLH2
; Obesity and metabolic syndrome, j CLOCK
1 gliomas, Alzheimer's, infertility
I FERD3L
j FIGLA i premature ovarian failure type 6 i HANDl
: HAND2
j HELT
HES1
j HES2
! HES3
; HES4
i HES5
\ HES6
\ HES7
i HEY1
: osteopenia and chondrocyte
; HEY2
hypertrophy in bone and glioblastoma
! HEYL
: Multiple cancers (regulating
; HIF1A
: angiogenesis) and ischemic disease i ID1 \ Pancreatic carcinoma
: ID2
: ID3
: ID4
: KIAA2018
: LOC388553
\ LYL1
i MAX ! Hereditary pheochromocytoma i MESP1
j Spondylocostal dysostosis, autosomal
\ MESP2
I recessive 2
\ MITF
! MLX
: MLXIP
: MLXIPL
j MNT
i MSC
i MSGN1
: MXD1 ; Acute leukemia, melanoma
; MXD3
i MXD4
; MXI1 Hematopoietic tumors, leukemias and
MYC
lymphomas, including Burkitt lymphoma
MYCL1
MYCN Neuroblastoma
MYF5
MYF6 Myopathy, centronuclear
! Squamous cell carcinomas and
\ MYOD1
: malignant melanomas i MYOG
i NEUROD1 I Type 2 diabetes
i NEUROD2
I NEUROD4
i NEUROD6
j NEUROG1
j NEUROG2
NEUROG3 I Diarrhea 4, malabsorptive, congenital j NHLH1
; NHLH2
: NPAS1
i NPAS2
NPAS3 : Schizophrenia and mental retardation
\ NPAS4
: OLIG1 ; NSCLC and mutiple sclerosis
I Alzheimer's disease, Down syndrome,
\ OLIG2
: pilocytic astrocytoma
: OLIG3
PTF1A
; SCXA
SCXB
: SIM1
j SOHLH1
j SOHLH2
; SREBF1
: SREBF2
I TALI \ ALL
; TAL2
i TCF12
j TCF15
j TCF21
\ TCF23
i TCF3
I TCF4
j TCFL5
TFAP4
i TFE3 j Renal cell carcinoma, papillary, 1
; TFEB
TFEC
TWIST1 I Saethre-Chotzen syndrome. j TWIST2
j USF1 I Familial combined hyperlipidemia i USF2
I C/EBP Regulate cell differentiation, ! Cutaneous squamous cell carcinoma
\ ATF3
growth, survival, and ; and other cancers inflammation i ATF7 i Breast cancer
: BATF3
i CEBPA i AML
; CEBPB
; CEBPD
; CEBPE
i CEBPG : AML, non-bronchogenic carcinoma
! CREB5
! XBP1
\ T-ALL (T-cell acute lymphoblastic
; leukemia),[25] CADASIL (Cerebral
: Autosomal-Dominant Arteriopathy with
\ CBFB ; Sub-cortical Infarcts and
; Leukoencephalopathy), MS (Multiple \. Sclerosis), Tetralogy of Fallot, Alagille 1 syndrome
\ CG-1 Calcium signaling by direct I CAMPTAl, CAMPTA2: Cardia
; CAMTA1
binding of calmodulin. : hypertrophy, congenital ataxia
i CAMTA2
: EBF1 I EBF1 throught 4: Hodgkin Lymphoma
\ glioblastoma multiforme and gastric
! EBF2
; carcinoma
i EBF3
EBF4
COU P Involved in widely diverse i NR2F1
physiological functions, j NR2F2
including control of
embryonic development,
cell differentiation and NR2F6
homeostasi j Colon cancer
! CP2 ; , Progressive autosomal dominant
: GRHL1
; hearing loss
: GRHL2
: GRHL3
j Hepatocellular carcinoma, Alzheimer's
[ TFCP2
: disease
: TFCP2L1
UBP1
: CSD Cold shock domain proteins I CARHSPl
protect the cells when
j CSDA : Hepatocellular carcinoma, gastric cancer temperatures go below
\ CSDC2
optimum growth
i CSDEl
temperatures
: LIN28A : LIN28B
j YBX1
: YBX2 i Male factor infertility
i CSL Universal transcriptional : CSL
effector of Notch signaling.
Notch signaling is
dysregulated in many i Prostate cancer cancers, and faulty notch
signaling is implicated in
many diseases
\ CTF/NFI Functions both in viral DNA
! NFIA, NFIB, NFIC, NFIX: Lymphedema- replication and in the
NFIA 1 distichiasis, developmental verbal regulation of gene
; dyspraxia, autoimmune diseases expression
Breast and head and neck adenoid
; NFIB
; cystic carcinoma
; NFIC
\ NFIX
CUT i cuxi : AML, major depressive disorder
CUX2
; ONECUT1
; O ECUT2
i ONECUT3
: SATB1 : Prostate cancer
i 1 Isolated cleft palate and mental SATB2
retardation
; DM Believed to function as j Defective testicular development and XY
\ DMRT1
transcription factors on i feminization
downstream sex- DMRT2 i Gonadal dysgenesis and XY sex reversal determination genes i DM T3
j DMRTAl
\ DMRTA2
\ DMRTB1
! DM TC2
: E2F involved in the cell cycle E2F1
regulation and synthesis of : E2F2 : Involvement in most cancers
DNA. Some are activators
\ E2F3
and others are suppressors
: E2F4
i E2F5
I E2F6
i E2F7
! E2F8
i TFDP1
; TFDP2
\ TFDP3
i Ecdystd involved in widely diverse ; NR1H2 ; Preeclampsia
receptor physiological functions,
including control of ! NR1H3
embryonic development, ; Atherosclerosis
Figure imgf000304_0001
: Anterior segment mesenchymal
\ FOXE3
! dysgenesis
; Alveolar capillary dysplasia with
\ FOXF1
i misalignment of pulmonary veins i FOXF2
I FOXG1 : Rett syndrome, congenital variant i FOXH1
FOXI1 i Enlarged vestibular aqueduct
\ FOXI2
: FOXJ1
; FOXJ2
; FOXJ3
i FOXK1
I FOXK2
! FOXL1
i Blepharophimosis-ptosis-epicanthus
\ FOXL2
i inversus syndrome
: FOXM1 ; Cancer
! T-cell immunodeficiency, congenital
\ FOXN1
i alopecia, and nail dystrophy
; FOXN2
! FO 3
\ FOXN4
\ FOXOl ; Autoimmune diseases
FOX03
FOX04
! FOX06
1 FOXP1
I FOXP2 : Developmental verbal dystraxia
; IPEX (immunodysregulation
I polyendocrinopathy enteropathy X- i FOXP3
; linked syndrome) and autoimmune i diseases
\ FOXP4
i FOXQl
FOXR1
FOXR2
FOXS1
KIAA0415
GCM Regulators of gliogensis GCMl
severe congenital isolated
GCM2
hypoparathyroidism.
GCR Control of embryonic
development, cell
NR3C1
differentiation and
homeostasis Generalized glucocorticoid resistance
GTF2I Involvement in the GTF2I Williams-Beuren Syndrome
morphological and neuro- GTF2IRD1 Williams-Beuren Syndrome developmental anomalies
GTF2IRD2 Williams-Beuren Syndrome j GTF2IRD2B : Williams-Beuren Syndrome
H G Involved in the regulation of BBX
patterns of anatomical CIC
development
HBP1
HMG20A
HMG20B
HMGB1
HMGB2
HMGB3
HMGXB3
HMGXB4
LEF1
PBRM1
PINX1 i Parkinson diseased, early onset
P S1
SMARCE1
: SOX1 ; Hepatocellular carcinoma
\ Waardenburg-Shah and Waardenburg-
\ SOX10
: Hirschsprung disease
: SOX11 : Epithlian ovarian cancer
SOX12
i SOX13
: SOX 14
i SOX15
i SOX17
Hypotrichosis-lymphedem telangiectasia syndrome
SOX2 Microphthalmia, syndromic 3
SOX21
Mental retardation, X-linked, with
SOX3
isolated growth hormone deficiency
SOX30
SOX4
I SOX5
: sox6
\ SOX7
\ SOX8
: sox9 i Acampomelic campomelic dysplasia SRY ; Swyer syndorme and XX male syndrome
: SSRP1
\ TCF7
\ TCF7L1
\ TCF7L2
! TFAM
i TOX
i TOX2
TOX3
: TOX4 UBTF
UBTFL1
UBTFL2
WHSC1 Wolf-Hirschhorn syndrome
HMGI/ HMGY Non-histone nuclear : Cancer, fibrosis and autoimmune proteins which constitute H GA1
diseases
important components
chromatin structure. HMGA2 Liposarcoma
Homeobox Over 200 different genes
involved in development, ADNP
differentiation and tumor
ALX1 Axenfeld-Rieger syndrome suppression. Following
branchiootorenal syndrome homeobox genes have a ALX3
coloboma
genetic disease association; ALX4
combined pituitary hormone deficiency ALX4, ARX, H ESX1, HOXA13,
ARGFX congenital central hypoventilation LMX1B, MSX1, MSX2, OTX2,
syndrome
PAX2, PAX3, PAX6, PAX8, ARX
congenital fibrosis of the extraocular PHOX2A, PHOX2B, PITX2, BARHL1
muscles
POU3F4, PROP1, SHOX, SIX1,
BARHL2 congenital hypothyroidism
SIX3, SIX5, TGIF1, and ZEB2.
BARX1 craniofaciai-deafness-hand syndrome enlarged parietal foramina
BARX2
hand-foot-genital syndrome
BSX Langer mesomelic dysplasia
CDX1 Leri-Weill dyschondrosteosis
microphthalmia
CDX2
Mowat-Wilson syndrome
CDX4 nail-patella syndrome
DBX1 neuroblastoma
nonsyndromic deafness
DBX2
nonsyndromic holoprosencephaly
DLX1 Partington syndrome
DLX2 Potocki-Shaffer syndrome
renal coloboma syndrome
DLX3
septo-optic dysplasia
DLX4 Turner syndrome
DLX5 Waardenburg syndrome
Wilms tumor, aniridia, genitourinary DLXG
anomalies, and mental retardation D BX1 syndrome
DPRX Wolf-Hirschhorn syndrome
X-linked infantile spasm syndrome DRGX
X-linked lissencephaly with abnormal
DUX4L2 genitalia
DUX4L3
DUX4L5
DUX4L6
DUXA EMX1
EMX2 EM
EN2
ESX1
EVX1
EVX2
GBX1
GBX2
GSC
GSC2
GSX1
GSX2
HDX
Growth hormone deficiency with
HESX1
pituitary anomalies
HHEX
HLX
HMBOX1
HMX1
HMX2
HMX3
HNF1A MODY
HNF1B MODY
HOMEZ
HOPX
HOXA1
HOXA10
HOXA11
HOXA13
Microtia, hearing impairment, and cleft
HOXA2
palate
HOXA3
HOXA4
HOXA5
HOXA6
HOXA7
HOXA9
HOXB1
HOXB13
HOXB2
HOXB3 HOXB4
HOXB5 ' :
HOXB6
HOXB7
HOXB8
HOXB9
HOXC10
HOXC11
HOXC12
HOXC13
HOXC4
HOXC5
HOXC6
HOXC8
HOXC9
HOXD1
HOXD10 i Vertical talus, congenital
HOXD11
HOXD12
HOXD13 Brachydactyly type D
HOXD3
HOXD4
HOXD8
HOXD9
IRX1
IRX2
IRX3
IRX4
IRX5
IRX6
ISL1
ISL2
ISX
LASS2
LASS 3
LASS4
LASS5
LASS6
LBX1 LBX2
LEUTX LHXl
LHX2
LHX3 ! Pituitary disease
LHX4
LHX5
LHX6
LHX8
LHX9
LMX1A
LMX1B
MEIS1
MEIS2
MEIS3
ME0X1
MEOX2
MIXL1
MNX1
i MSX1 ; Witkop syndrome
\ MSX2 I Craniosynostosis, type 2
; NANOG
NKXl-1
i NKX2-1
i NKX2-2 j Chorea, hereditary benign
NKX2-3
NKX2-4
NKX2-5
NKX2-6 : Persistent truncus arteriosus
NKX2-8
NKX3-1
NKX3-2
NKX6-1
NKX6-2
NKX6-3
NOBOX
NOTO
OTP
PBXl : PBX2
PBX3
PBX4
\ PDX1
; Fibrosis of extraocular muscles, j PHOX2A
i congenital, 2
: PHOX2B
j PITX1 i Clubfoot, congenital
; PITX2 i Iridogoniodysgenesis, type 2
Anterior segment mesenchymal j PITX3
i dysgenesis
; PKNOX1
\ PKNOX2
\ PROP1
\ PRRXl
PRRX2
i RAX
\ RAX2
j RHOXF1
\ RHOXF2
\ RHOXF2B
I SEBOX
1 SHOX
j SHOX2
i SIX1 i Brachiootic syndrome 3
\ SIX2
; SIX3 j Holoprosencephaly-2
i SIX4
\ SIX5 : Branchiootorenal syndrome 2
i Microphthalmia, isolated, with cataract SIX6
\ 2
j TGIFl
i TGIF2
I TGIF2LX
; TGIF2LY
TLXl
TLX 2
TLX 3
UNCX
VAX1
VAX2 i VENTX
i vsxi
\ VSX2
: ZEB1
i ZFHX3
I ZFHX4 ; Ptosis, congenital
ZHX1
: ZHX2
ZHX3
HSF ; Activators of heat shock I utiple myeloma and other malignant
\ HSF1
ϊ proteins ■i cancers
: HSF4 I Congenital and age related catarats
; HSFX1
; HSFX2
\ HSFY1
\ HSFY2
LCOR Involved in development i LCOR
and metabolism regulation
: LCORL
IRF :: Important in the regulation ; IRF1 ! AML
\ of interferons in response to
i IRF2
I infection by virus and in the L Pancreatic cancer
regulation of interferon- i IRF3 ; Increased susceptibility to hepatitis virus ; inducible genes : diffuse large B-cell and follicular
\ IRF4
; lymphomas.
IRF5 T Melanoma
; Woude syndrome, popliteal pterygium IRF6 : syndrome and non-syndromic orofacial
; cleft type 6
i IRF7 ! Systemic lupus erythematosus.
IRF8 i Dendritic-cell immunodeficiency i IRF9
MBD genomic imprinting, BAZ2A
transposon and
: BAZ2B
chromosome X inactivation,
: MBD1 : Pancreatic cancer
differentiation, and cancer
: MBD2
i MBD3
: MBD4 i Rheumatoid arthritis
; MECP2 j Rett Syndrome
i SETDB1
! SETDB2
MH1 ! Inflammation, cancer, and i SMAD 1 through 7 and SMAD 9:
i metabolic disorders S AD1 . i Colorectal, ovarian, NSCLC and prostate :
! cancer, lupus
S AD2
SMAD3 SMAD4
SMAD5
SMAD6
SMA07
SMAD9
; MYB Regulator of proliferation, i CCDC79 i ALL
differentiation and cell fate/ ; CDC5L
25 different genes, yet the ; D TF1 i NSCLC
most representative is MYB
i DNAJCl i Ovarian cancer
i DNAJC2
: MIER3
; MYB i Breast and prostate cancer
; MYBL1
\ MYBL2
I MYSMl
\ NCORl
i NCOR2
i RCOR1 i Advanced osteoarthritic chondrocytes. i RCOR2
: RCOR3
i SMARCC1
i SMARCC2 \ Gastric and colorectal cancer
: SNAPC4
j TADA2A
: TADA2B
: TERF1 ! NSCLC
! TERF1P2
\ TERF2
; TTF1
: ZZZ3
NDT80/PhoG : Hematopoiesis and
\ Cllorfa
development of bone tissue
NF-YA \ NFYA
NF-YB/C ; NFYB
! NFYC
Nrfl i NRF1
Nuclear orphan Involved in widely diverse i NR4A1 : Gastric cancer
! receptor physiological functions,
including control of \ NR4A2 ; Rheumatoid arthritis
embryonic development,
cell differentiation and ! NR4A3 i Extraskeletal myxoid chondrosarcomas homeostasis.
Estrogen Control of embryonic
receptor development, cell i Estrogen resistance, familial breast
ESR1
differentiation and ! cancer, migraine, myocardial infarction homeostasis Other nuclear Hepatocellular carcinoma and X-linked receptor NR0B1 congenital adrenal hypoplasia and hypogonadotropic hypogonadism
Fibrolamellar carcinoma and
NR0B2
hepatocellular carcinoma.
P53 Control development and TP53 Large variety of cancers
differentiation and play a
role in tumor suppression : Ectodermal dysplasia, and cleft
lip/palate syndrome 3 (EEC3); split- hand/foot malformation 4 (SHFM4);
TP63 ankyloblepharon-ectodermal defects- cleft lip/palate; ADULT syndrome (acro- dermato-ungual-lacrimal-tooth); limb- mammary syndrome; Rap-Hodgkin syndrome (RHS)
TP73 myelodysplastic syndrome.
PAX critical role in the formation Cervical cancer, vertebral
PAX1
of tissues and organs during malformations, Klippel-Feil syndrome embryonic development
Optic nerve colobomas and renal
PAX2
hypoplasia
Waardenburg syndrome, craniofacial-
PAX3 deafness-hand syndrome, and alveola rhabdomyosarcoma
MODY, early onset-age type 2 diabetes,
PAX4
melanoma
PAX5 Lymphomas
ocular disorders such as aniridia and
PAX6
Peter's anomaly
PAX7 Alveolar rhabdomyosarcoma
Thyroid dysgenesis, thyroid follicular
PAX8 carcinomas and atypical follicular thyroid adenomas
PAX9 Oligodontia
PC4 Replication, DNA repair and
SUB1
transcription
POU Wide variety of functions, all POU Genes: Aautoimmune arthritis, of which are related to the asthma, septic shock, lung fibrosis,
POU1F1
function of the glomerulonephritis, atherosclerosis, and neuroendocrine system and AIDS
the development of an POU2F1
organism
POU2F2
POU2F3
Pituitary hormone deficiency,
POU3F1
combined, 1
POU3F2
POU3F3
POU3F4 Deafness, X-linked 2
POU4F1 POU4F2
POU4F3 Deafness, autosomal dominant 15
POU5F1
POU5F1B
POU6F1
\ POU6F2
: PPAR receptor involved in cell proliferation, ; PPAR A
cell differentiation and in \ CLL, bladder cancer
immune and inflammation PPARD : Colorectal cancer responses.
j PPARG ; Thyroid cancer
Progesterone Plays a central role in
: receptor reproductive events
associated with the \ PGR
establishment and
maintenance of pregnancy i Breast and endometrial cancer
Proxl Essential for the ; PROXi : Multiple cancers
development and
maintenance of lymphatic \ PROX2
vasculature
Retinoic acid Control of embryonic ! ESRRA
receptor development, cell NR2C1
differentiation and
i NR2C2
homeostasis
I NR2E1
i NR5A1
; NR5A2
j NR6A1
RFX Regulate development and RFX1 : Bardet-Biedl syndrome
function of cilia : RFX2
RFX3
: RFX4
: RFX5 ! Bare lymphocyte syndrome, type II
: RFX6
i RFX 7
i RFX8
RH D ; 10 different genes. One of the most
; important is NFKB: Autoimmune
: NFAT5 ! arthritis, asthma, septic shock, lung
: fibrosis, glomerulonephritis, atherosclerosis, and AIDS
NFATC1
NFATC2
NFATC3
NFATC4
i Cancer, inflammatory and autoimmune NFKB1 j diseases, septic shock, viral infection,
I and improper immune development Inflammatory events associated with
j NFKB2 ! autoimmune arthritis, asthma, septic
I shock, lung fibrosis, glomerulonephritis, j atherosclerosis, and AIDS
i REL
j RELA
j RELB
RO receptor Control of embryonic \ NR1D2
development, cell j RORA : Autism
differentiation and i RORB j Bipolar disorder homeostasis
i RORG i Inflammatory auto immune
Runt Essential for osteoblastic j RUNX1 i Different leukemia
differentiation and skeletal
morphogenesis \ RUNX3 ; Ranal cell carcinoma, psoriatic arthritis
SAND j Autoimmune polyendocrinopathy with
\ AIRE ; candidiasis and ectodermal dystrophy
\ (APECED)
i DEAF1 \ Colorectaladenocarcinomas
\ GMEB1
\ GMEB2
i SP100 i Brain tumors
! SP110 i Tubercolosis, tubercolosis
I SP140
\ SP140L
SRF Involved incell proliferation ; LOC729991- and differentiation \ EF2B
MEF2A : Coronary artery disease
j MEF2B
! Parkinson disease and myotonic i EF2D
i dystrophy
I Gastric cancer and hepatocellular
\ SRF
: carcinoma
STAT Regulates many aspects of
; STATs: Angiogenesis, enhanced tumor growth, survival and STAT1
; survival and immunosuppression. differentiation in cells.
\ STAT2
i STAT3 I Hyper-lgE recurrent infection syndrome i STAT4
i STAT5A
I Growth hormone insensitivity with
STAT5B
i immunodeficiency
! STAT6
T-box Involved in limb and heart
: T-Box genes involved in: Alzheimer's, development
i Parkinson's, ulnar-mammary syndrome
\ EOMES ; chronic and congenital heart defects like Holt-Oram syndrome and obstructive coronary diseases
MGA i T : Oral squamous cell carcinoma.
i TBR1
\ TBX1 ; Conotruncal anomaly face syndrome
TBX10
j TBX15 \ Cousin syndrome
i TBX18
I TBX19
i TBX2
TBX20
; TBX21 : Cousin syndrome
: TBX22
! TBX3
i TBX4 : Small patella syndrome
\ TBX5
j TBX6
TEAD Essential for cardiac, i TEAD1 ; Sveinsson's chorioretinal atrophy skeletal, and smooth i TEAD2
development
\ TEAD3
i TEAD4
TF bZIP Important regulators of ; Angiomatoid fibrous histiocytoma and
! ATFl
development such as : clear cell sarcoma proliferation, differentiation \ ATF2
and transformation \ ATF4
; ATF5
\ ATF6
I ATF6B
: BACHl
: BACH2
i BATF
! BATF2
I i Histiocytoma, angiomatoid fibrous, CREB1
: somatic
! CREB3 : Cerical cancer
j CREB3L1
i CREB3L2
: CREB3L3
: CREB3L4
: CREBL2
\ CREM i
! DBP
; DDIT3
! FOS : Multiple cancers
; FOSB : Multiple cancers
! FOSLl I Multiple cancers
i FOSL2 ; Multiple cancers
i HLF
i JDP2
JUN Breast and lung cancer
Figure imgf000318_0001
\ HNF4G j MODY, obesity
! ARG
; RXRA Acute promyelocyte leukemia
ESRRG Breast cancer
i RXRB : type II vitamin D-resistant rickets
TSC22 i Gliobastoma, slaivary gland and
1 prostate cancer i Inflammation, RA
; Tub ; Associated with neuronal
; differentiation and TUB
Obesity and sensorineural degradation ■ development
i TULP1 i Retinitis pigmentosa
TULP2
TULP3
TULP4
! ZBTB Regulate lymphoid \ BCL6 i Diffuse large-cell lymphoma (DLCL) development and function j BCL6B : Gastric cancer
i GZF1
i HICl
\ HIC2
\ MYNN
; PATZ1
i ZBTB1
i ZBTB10
\ ZBTB11
ZBTB12
; Skeletal defects, genital hypoplasia, and
: ZBTB16
! mental retardation
ZBTB17
; ZBTB2
! ZBTB20 i Hepatocellular carcinoma
: ZBTB24
j ZBTB25
; ZBTB26
j ZBTB3
i ZBTB32
; ZBTB33
ZBTB34
I ZBTB37
: ZBTB38
I ZBTB4
; ZBTB40
\ ZBTB41
ZBTB42
i ZBTB43
i ZBTB44
I ZBTB45 ZBTB46
ZBTB47
ZBTB48
ZBTB49
ZBTB6
ZBTB7A
ZBTB7B
ZBTB7C
ZBTB8A
ZFP161
ZNF131
ZNF238
ZNF295
zf-BED I Cllorf95 ; Chondroid lipoma
\ ZBEDl
i ZBED2
: ZBED3
: ZBED4
zf-C2H2 Large family of transcription ; X-linked spinal, bulbar muscular atrophy factors group together : BCL11A
I and prostate cancer
because of the presence of
\ BCL11B ; B-Cell malignancies
zinc fingers in their
: BNC2
structure. Invoved with in
i CTCF
regulating a multitude of
functions \ CTCFL
\ E4F1
\ EGR1 ; Prostate and breast cancer
Congenital hypomyelinating neuropathy and Charot-Marie-Tooth type 1
EGR3
EGR4
FEZF1
FEZF2
FIZ1
GFU
GFI1B
GUI
GLI2 Holoprosencephaly-9
GLI3 Polydactyly, postaxial, types Al and B
GLI4
GLIS1
GLIS2 Nephronophthisis 7
Diabetes mellitus, neonatal, with
GLIS3
congenital hypothyroidism
GTF3A
HINFP
HIVEP1
HIVEP2
HIVEP3 HK 1
IKZF1
IKZF2
IKZF3
IKZF4
IKZF5
INSM1
INSM2
KLF1
KLF10
KLF11
KLF12
KLF13
KLF14
KLF15
KLF16
KLF17
KLF2
KLF3
KLF4
KLF5
KLF6
KLF7
KLF8
KLF9
LOC100131539
LOC100132396
LOC100287841
MAZ
MECOM
MTF1
MZF1
OSR1
OSR2
OVOL1
OVOL2
OVOL3
PEG3
PLAG1 i Salivary gland pleomorphic adenomas
PLAGL1
PLAGL2
PRD 1
PRDM10
PRDM12
PRDM13
PRDM14
PRDM15
PRDM16 PRDM2
j PRDM4
: PRDM5
! PRD 6
PR DM8
i PRDM9
RBAK
: REPIN1
\ REST ; Glioblastoma
\ RREBl
j SALL1
i SALL2
: SALL3
i SALL4
j SCRT1
i SCRT2
: SNAI1 : Various cancers
i SNAI2
: SNAI3
i SP1 j Various cancers
i SP2
; SP3
j SP4
j SP5
i SPG
SP7
j SP8
; S 9
TRERFl
\ TSHZ1
j TSHZ3
i VEZF1
! wiz
; wTi ; Nephrotic syndrome and Wilms tumor YYl
YY2
: ZEB2
i ZFAT
i ZFP1
j ZFP112
: ZFP14
j ZFP2
: ZFP28
! ZFP3
\ ZFP30
! ZFP37
ZFP41
ZFP42 i ZFP57 ! Diabetes mellitus, transient neonatal, 1 i ZFP62
j ZFP64
j ZFP82
\ ZFP90
ZFP91
: ZFPM1
\ ZFPM2 i Diaphragmatic hernia 3
i ZFX
; ZFY
: ZlCl j Isolated Dandy-Walker malformation
\ ZIC2 I Alobar holoprosencephaly
! Isolated congenitally uncorrected
\ ZIC3
i transposition of the great arteries
: ZIC4 ; Isolated Dandy-Walker malformation
\ ZIC5
: ziKi
: ZI 2
ZIM3
i ZKSCAN1
! ZKSCAN2
I Z SCAN3
: ZKSCAN4
; ZKSCAN5
; ZNF10
ZNF100
ZNF101
i ZNF107
i ZNF114
i ZNF117
i ZNF12
ZNF121
j ZNF124
ZNF132
ZNF133
i ZNF134
ZNF135
! ZNF136
: ZNF138
: ZNF14
! ZNF140
: ZNF141 i Postaxial Polydactyly type A, bilateral
! ZNF142
ZNF143
ί ZNF146
ZNF148
j ZNF154
! ZNF155 ZNF157
ZNF16
ZNF160
ZNF165
ZNF167
ZNF169
ZNF17
ZNF174
ZNF175
ZNF177
ZNF18
ZNF180
ZNF181
ZNF182
ZNF184
ZNF189
ZNF19
ZNF192
ZNF193
ZNF195
ZNF197
ZNF2
ZNF20
ZNF200
ZNF202
ZNF205
ZNF208
ZNF211
ZNF212
ZNF213
ZNF214
ZNF215
ZNF217
ZNF219
ZNF22
ZNF221
ZNF222
ZNF223
ZNF224
ZNF225
ZNF226
ZNF227
ZNF229
ZNF23
ZNF230
ZNF232
ZNF233
ZNF234 ZNF235
ZNF236
ZNF239
ZNF24
ZNF248
ZNF25
ZNF250
ZNF251
ZNF253
ZNF254
ZNF256
ZNF257
ZNF26
ZNF263
ZNF264
ZNF266
ZNF267
ZNF268
ZNF273
ZNF274
ZNF275
ZNF276
ZNF28
ZNF280D
ZNF281
ZNF282
ZNF283
ZNF284
ZNF285
ZNF286A
ZNF287
ZNF292
ZNF296
ZNF3
ZNF30
ZNF300
ZNF302
ZNF304
ZNF311
ZNF317
ZNF319
ZNF32
ZNF320
ZNF322A
ZNF322B
ZNF323 ZNF329
ZNF331
ZNF333
ZNF334
ZNF335
ZNF337
ZNF33A
ZNF33B
ZNF34
ZNF341
ZNF343
ZNF345
ZNF347
ZNF35
ZNF350
ZNF354A
ZNF354B
i ZNF354C
i ZNF358
i ZNF362
i ZNF366
ZNF367
; ZNF37A
ZNF382
j ZNF383
i ZNF384
ZNF391
ZNF394
: ZNF396
! ZNF397
I ZNF398
; ZNF407
i ZNF408 ;. Familial exudative vitreoretinopathy
! X-linked nonsyndromic intellectual
; ZNF41
i deficit
j ZNF410
j ZNF415
\ ZNF416
ZNF417
i ZNF418
i ZNF419
: ZNF420
; Joubert syndrome with oculorenal
ZNF423
: defect
ZNF425
ZNF426
ZNF429
ZNF43 ZNF430
ZNF431
ZNF432
ZNF433
ZNF434
ZNF436
ZNF439
ZNF44
ZNF440
ZNF441
ZNF442
ZNF443
ZNF444
ZNF445
ZNF446
ZNF449
ZNF45
ZNF451
ZNF454
ZNF4G0
ZNF461
ZNF467
ZNF468
ZNF469 i Brittle cornea syndrome
ZNF470
ZNF471
ZNF473
ZNF479
ZNF48
ZNF480
ZNF483
ZNF484
ZNF485
ZNF486
ZNF490
ZNF491
ZNF492
ZNF493
ZNF496
ZNF497
ZNF498
ZNF500
ZNF501
ZNF502
ZNF506
ZNF507
ZNF510
ZNF512
Figure imgf000328_0001
\ ZNF574 : ZNF575 \ ZNF576 \ ZNF577 : ZNF579 ; ZNF580 \ ZNF581 \ ZNF582 ZNF583 \ ZNF584 ! ZNF585A
ZNF585B : ZNF586 \ ZNF587
ZNF589 \ ZNF594 j ZNF596 ZNF597 ; ZNF599 : ZNF600 : ZNF606 \ ZNF607 : ZNF610 i ZNF611 i ZNF613 \ ZNF614 : ZNF615 ; ZNF616 \ ZNF619 \ ZNF620 ; ZNF621 \ ZNF623 : ZNF624 \ ZNF625 : ZNF626 i ZNF627 ! ZNF628 j ZNFS29 i ZNF630
ZNF639 \ ZNF641 \ ZNF642 i ZNF643 ■ ZNF644 ZNF646 ; ZNF648 i ZNF649 I ZNF652 ZNF653
ZNF655
ZNF658
ZNF660
ZNF662
ZNF66
ZNF665
ZNF667
ZNF668
ZNF669
ZNF670
ZNF671
ZNF672
X-linked nonsyndromic intellectual ZNF674
deficit
ZNF675
ZNF676
ZNF677
ZNF678
ZNF679
ZNF680
ZNF681
ZNF682
ZNF683
ZNF684
ZNF687
ZNF688
ZNF689
ZNF69
ZNF691
ZNF692
ZNF696
ZNF697
ZNF699
ZNF7
ZNF70
ZNF700
ZNF701
ZNF705A
ZNF705D
ZNF705G
ZNF707
ZNF708
ZNF709
ZNF71
ZNF710
X-linked nonsyndromic intellectual
ZNF711
deficit ZNF713
ZNF714
ZNF716
ZNF717
ZNF732
ZNF736
ZNF737
ZNF74
ZNF740
ZNF746
ZNF749
ZNF75A
ZNF75D
ZNF76
ZNF763
ZNF764
ZNF765
ZNF766
ZNF768
ZNF77
ZNF770
ZNF771
ZNF772
ZNF773
ZNF774
ZNF775
ZNF776
ZNF777
ZNF778
ZNF780A
ZNF780B
X-linked nonsyndromic intellectual
ZNF781
deficit
ZNF800
ZNF805
ZNF808
ZNF81
ZNF813
ZNF814
ZNF816
ZNF821
ZNF823
ZNF827
ZNF83
ZNF831
ZNF836
ZNF837
ZNF84
ZNF841
ZNF844
ZNF845
ZNF846
ZNF85
ZNF852
ZNF860
ZNF865
ZNF878
ZNF879
ZNF880
ZNF90
ZNF91
ZNF92
ZNF93
ZNF98
ZNF99
ZSCAN1
ZSCAN10
ZSCAN12
ZSCAN16
ZSCAN18
ZSCAN2
ZSCAN20
ZSCA 21
ZSCAN22
ZSCAN23
ZSCAN29
ZSCAN30
ZSCAN4
ZSCAN5A
ZSCAN5B
ZSCAN5C ZXDA
ZXDB
ZXDC
zf-C2HC L3MBTL1
L3MBTL4
YST2
MYT1
MYT1L
ST18
zf-GATA GATA1 Congenital heart defects and cancer
GATA2
Hypoparathyroidism, sensorineural
GATA3
deafness, and renal dysplasia
GATA4
GATA5
GATA6
GATAD1
GATAD2B
MTA1
MTA2
MTA3
ERE
TRPS1
ZGLP1
zf-LITAF-like C16orf5
LITAF
zf-MIZ PIAS1
PIAS2
PIAS3
PIAS4
RNF138
ZMIZ1
ZMI 2
zf-NF-Xl NFX1
NFXL1
ESR Control of embryonic ESR
development, cell Estrogen resistance, familial breast differentiation and cancer, migraine, myocardial infarction homeostasis
Table IX-2 (Annex IX-2) .
Disease Name tt of Genes
per Disease Gene Name
Duane-radial ray syndrome 1 SALL4
Achalasia-Addisonianism-Alacrimia syndrome 1 AAAS
Charcot-Marie-Tpoth disease, axonal, type 2N 1 AARS
Saccharopinuria 1 AASS
GABA-transaminase deficiency 1 ABAT
H DL deficiency, type 2 1 ABCA1
Ichthyosis, harlequin 1 ABCA12
Ichthyosis, lamellar 2 1 ABCA12
Surfactant metabolism dysfunction, pulmonary, 3 1 ABCA3
Fundus flavimaculatus 1 ABCA4 >
Macular degeneration, age-related, 2 1 ABCA4
Retinal dystrophy, early-onset severe 1 ABCA4
Cholestasis, benign recurrent intrahepatic, 2 1 ABCB11
Cholestasis, progressive familial intrahepatic 2 1 ABCB11
Cholestasis, familial intrahepatic, of pregnancy 1 ABCB4
Cholestasis, progressive familial intrahepatic 3 1 ABCB4
Gallbladder disease 1 1 ABCB4
Anemia, sideroblastic, with ataxia 1 ABCB7
Pseudoxanthoma elasticum, forme fruste 1 ABCC6
Diabetes mellitus, noninsulin-dependent 1 ABCC8
Diabetes mellitus, permanent neonatal 1 ABCC8
Diabetes mellitus, transient neonatal 2 1 ABCC8
Hyperinsulinemic hypoglycemia, familial, 1 1 ABCC8
Hypoglycemia of infancy, leucine-sensitive 1 ABCC8
Cardiomyopathy, dilated, 10 1 ABCC9
Adrenoleukodystrophy 1 ABCD1
Gallbladder disease 4 1 ABCG8
Chanarin-Dorfman syndrome 1 ABHD5
ACAD9 deficiency 1 ACAD9
Acyl-CoA dehydrogenase, long chain, deficiency of 1 ACADL
Acyl-CoA dehydrogenase, medium chain, deficiency of 1 ACADM
Acyl-CoA dehydrogenase, short chain, deficiency of 1 ACADS
VLCAD deficiency 1 ACADVL
Osteochondritis dissecans, short stature, and early-onset osteoarthritis 1 ACAN
Alpha-methylacetoacetic aciduria 1 ACAT1
1
Table IX-21781338.1 Lysosomal acid phosphatase deficiency 1 ACP2
Mast syndrome 2 ACP33
Mental retardation, X-linked nonspecific, 63 1 ACSL4
Myopathy, actin, congenital, with excess of thin myofilaments 1 ACTA1
Myopathy, congenital, with fiber-type disproportion 1 1 ACTAl
Myopathy, nemaline, 3 1 ACTAl
Aortic aneurysm, familial thoracic 6 1 ACTA2
Dystonia, juvenile-onset 1 ACTB
Cardiomyopathy, dilated, 1R 1 ACTC1
Cardiomyopathy, familial hypertrophic, 11 1 ACTC1
Deafness, autosomal dominant 20/26 1 ACTG1
Cardiomyopathy, dilated, 1AA 1 ACT 2
Glomerulosclerosis, focal segmental, 1 1 ACTN4
Hereditary hemorrhagic telangiectasia-2 1 ACVRL1
Aminoacylase 1 deficiency 1 ACY1
Alzheimer disease-10 6 AD10
Alzheimer disease-5 6 AD5
Alzheimer disease 6 6 AD6
Alzheimer disease 8 6 AD8
Adenosine deaminase deficiency, partial 1 ADA
Thrombotic thrombocytopenic purpura, familial 1 ADAMTS13
' Adiponectin deficiency 1 ADIPOQ
Leukemia, acute myeloid 14 AF10
Mental retardation, X-linked, FRAXE type 1 AFF2
Spinocerebellar ataxia 28 1 AFG3L2
Lipodystrophy, congenital generalized, type 1 1 AGPAT2
Myasthenia, limb-girdle, familial 2 AGRN
Hypertension, essential 3 AGTR1
Hyperoxaluria, primary, type 1 1 AGXT
Immunodeficiency with hyper-lgM, type 2 1 AICDA
Combined oxidative phosphorylation deficiency 6 1 AIFM1
Pituitary adenoma, ACTH-secreting 1 AIP
Pituitary adenoma, growth hormone-secreting 1 AIP
Pituitary adenoma, prolactin-secreting 1 AIP
Autoimmune polyendocrinopathy syndrome , type 1, with or without
reversible metaphyseal dysplasia 1 AIRE
Long QT syndrome-11 1 AKAP9
Bile acid synthesis defect, congenital, 2 1 AKR1D1
Breast cancer, somatic 4 AKT1
Colorectal cancer, somatic 6 AKT1
Ovarian cancer, somatic 2 AKT1
2
Table IX-21781338.1 Diabetes mellitus type II 1 AKT2
Porphyria, acute hepatic 1 ALAD
Anemia, sideroblastic, X-linked 1 ALAS2
Protoporphyria, erythropoietic, X-linked dominant 1 ALAS2
Epilepsy, pyridoxine-dependent 1 ALDH7A1
Fructose intolerance 1 ALDOB
Congenital disorder of glycosylation, type Ik 1 ALG1
Congenital disorder of glycosylation, type Ig ' 1 ALG12
Congenital disorder of glycosylation, type li 1 ALG2
Congenital disorder of glycosylation, type Id 1 ALG3
Congenital disorder of glycosylation, type lc 1 ALG6
Congenital disorder of glycosylation, type Ih 1 ALG8
Congenital disorder of glycosylation, type II 1 ALG9
Ichthyosiform erythroderma, congenital, nonbullous, 1 2 ALOX12B
Ichthyosiform erythroderma, congenital, nonbullous, 1 2 ALOXE3
Hypophosphatasia, adult 1 ALPL
Hypophosphatasia, childhood 1 ALPL
Hypophosphatasia, infantile 1 ALPL
Amyotrophic lateral sclerosis, juvenile 1 ALS2
Primary lateral sclerosis, juvenile 1 ALS2
Spastic paralysis, infantile onset ascending 1 ALS2
Frontorhiny 1 ALX3
Bile acid synthesis defect, congenital, 4 1 AMAC
Amelogenesis imperfecta, hypoplastic/hypomaturation type 1 AMELX
Leukemia, acute myelogenous 3 AMLCR2
Megaloblastic anemia-1, Norwegian type 1 AMN
Glycine encephalopathy 3 AMT
Spherocytosis, type 1 1 ANK1
Cardiac arrhythmia, ankyrin-B-related 1 ANK2
Long QT syndrome-4 1 ANK2
Chondrocalcinosis 2 1 ANKH
Gnathodiaphyseal dysplasia 1 AN05
Muscular dystrophy, limb-girdle, type 2L 1 AN05
Fibromatosis, juvenile hyaline 1 ANTXR2
Hyalinosis, infantile systemic 1 ANTXR2
Mental retardation, X-linked 59 1 AP1S2
Cerebral palsy, spastic quadriplegic, 3 1 AP4M1
Adenomatous polyposis coli 1 APC
Colorectal cancer, somatic 6 APC
Gastric cancer, somatic 8 APC
Amyloidosis, 3 or more types 1 APOA1
3
Table IX-21781338.1 Hypoalphalipoproteinemia 1 APOA1
Hypercholesterolemia, familial, modification of 1 APOA2
Hyperchylomicronemia, late-onset 1 APOA5
Hypercholesterolemia, due to ligand-defective apo B 1 APOB
Hyperlipoproteinemia, type lb 1 APOC2
Alzheimer disease-2 1 APOE
Alzheimer disease 1, familial 6 APP
Cerebral amyloid angiopathy, Dutch, Italian, Iowa, Flemish, Arctic
variants 1 APP
Ataxia, early-onset, with oculomotor apraxia and hypoalbuminemia 1 APTX
Coenzyme Q10 deficiency 6 APTX
Diabetes insipidus, nephrogenic 1 AQP2
Androgen insensitivity 1 AR
Androgen insensitivity, partial, with or without breast cancer 1 AR
Hypospadias 1, X-linked 1 AR
Spinal and bulbar muscular atrophy of Kennedy 1 AR
Leukemia, juvenile myelomonocytic 3 ARHGAP26
Slowed nerve conduction velocity, AD 1 ARHGEF10
Leukemia, acute myeloid 14 ARHGEF12
Mental retardation, X-linked nonspecific, type 46 1 ARHGEF6
Maroteaux-Lamy syndrome, several forms 1 ARSB
Chondrodysplasia punctata, X-linked recessive 1 ARSE
Epilepsy, myoclonic, with mental retardation and spasticity 1 ARX
Epileptic encephalopathy, early infantile, 1 1 ARX
Hydranencephaly with abnormal genitalia 1 ARX
Lissencephaly, X-linked 2 1 ARX
Mental retardation, X-linked 36/43/54 1 ARX
Proud syndrome 1 ARX
• Farber lipogranulomatosis 1 ASA HI
Central hypoventilation syndrome, congenital 5 ASCL1
Skin/hair/eye pigmentation 9, dark/light hair 1 ASIP
Canavan disease 1 ASPA
Microcephaly, primary autosomal recessive, 5, with or without
simplified gyral pattern 1 ASPM
Alveolar soft part sarcoma 1 ASPSCR1
Citrullinemia , . 1 ASS1
Antithrombin III deficiency 1 AT3
Ataxia, cerebellar, Cayman type 1 ATCAY
Parkinson disease 9 1 ATP13A2
Migraine, familial basilar 1 ATP1A2
Migraine, familial hemiplegic, 2 1 ATP1A2
4
Table IX-21781338.1 Dystonia-12 1 AT 1 A3
Hailey-Hailey disease 1 ATP2C1
Mental retardation, X-linked, with epilepsy 1 ATP6AP2
Renal tubular acidosis with deafness 1 ATP6B1
Cutis laxa, autosomal recessive, type II 1 ATP6V0A2
Renal tubular acidosis, distal, autosomal recessive 1 ATP6V0A4
Menkes disease 1 ATP7A
Spinal muscular atrophy, distal, X-linked 3 1 ATP7A
Cholestasis, benign recurrent intrahepatic 1 ATP8B1
Cholestasis, progressive familial intrahepatic 1 1 ATP8B1
ATP synthase deficiency, nuclear-encoded 1 ATPAF2
Alpha-thalassemia mental retardation syndrome 1 ATRX
Alpha-thalassemia myelodysplasia syndrome, somatic 1 ATRX
Mental retardation-hypotonic facies syndrome, X-linked 1 ATRX
Machado-Joseph disease 1 ATXN3
Spinocerebellar ataxia 8 1 ATXN8
Spinocerebellar ataxia 8 1 ATX N 80S
Diabetes insipidus, neurohypophyseal 1 AVP
Diabetes insipidus, nephrogenic 1 AVPR2
Atrioventricular canal defect 2 AVSD1
Caudal duplication anomaly 1 AXIN1
Hepatocellular carcinoma, somatic 3 AXIN1
Colorectal cancer . 6 AXIN2
Hypoproteinemia, hypercatabolic 1 B2M
Congenital disorder of glycosylation, type lid 1 B4GALT1
Hypercholanemia, familial 3 BAAT
Myopathy, myofibrillar, BAG3-related 1 BAG3
Maple syrup urine disease, type la 1 BCKDHA
Maple syrup urine disease, type lb 1 BCKDHB
Hypercarotenemia and vitamin A deficiency, autosomal dominant 1 BCMOl
Leukemia, acute lymphocytic 1 BCR
Leukemia, chronic myeloid 1 BCR
Mitochondrial complex III deficiency 1 BCS1L
Central hypoventilation syndrome, congenital 5 BDNF
Spinocerebellar ataxia 31 1 BEAN
Microcornea, rod-cone dystrophy, cataract, and posterior staphyloma 1 BEST1
Vitelliform macular dystrophy, adult-onset 1 BEST1
Vitreoretinochoroidopathy. 1 BEST1
Cataract, cortical, juvenile-onset 1 BFSP1
Cataract, autosomal dominant, multiple types 1 1 BFSP2
Cataract, congenital 1 BFSP2
5
Table IX-21781338.1 Cataract, juvenile-onset 1 BFSP2
Myopathy, centronuclear, autosomal recessive 1 BIN1
Lymphoproliferative syndrome, X-linked, 2 1 BIRC4
Maturity-onset diabetes of the young, type 11 1 BLK
Agammaglobulinemia 4 1 BLNK
Ovarian dysgenesis 2 1 BMP15
Premature ovarian failure 4 1 BMP15
Microphthalmia, syndromic 6 1 BMP4
Orofacial cleft 11 1 BMP4
Polyposis syndrome, hereditary mixed, 2 1 BMPR1A
Polyposis, juvenile intestinal 2 BMPR1A
Chrondrodysplasia, acromesomelic, with genital anomalies 1 BMPR1B
Pulmonary hypertension, familial primary 1 BMPR2
Adenocarcinoma of lung, somatic 3 BRAF
Fanconi anemia, complementation group Dl 1 BRCA2
Pancreatic cancer 2 BRCA2
Prostate cancer 1 BRCA2
Wilms tumor 1 BRCA2
' Breast cancer, early-onset 1 BRIP1
Fanconi anemia, complementation group J 1 BRIP1
Mental retardation, X-linked 93 1 BRWD3
Lipodystrophy, congenital generalized, type 2 1 BSCL2
Neuropathy, distal hereditary motor, type V 1 BSCL2
Silver spastic paraplegia syndrome 1 BSCL2
Bartter syndrome, type 4a 1 BSND
Sensorineural deafness with mild renal dysfunction 1 BSND
Agammaglobulinemia and isolated hormone deficiency 1 BTK
Agammaglobulinemia, type 1, X-iinked 1 BTK
Colorectal cancer 6 BUB1B
Mosaic variegated aneuploidy syndrome 1 BUB1B
Mitochondrial DNA depletion syndrome, hepatocerebral form 1 C10orf2
Progressive external ophthalmoplegia with mitochondrial DNA deletions
3 1 C10orf2
Tn syndrome 1 C1GALT1C1
Angioedema, hereditary, types 1 and II 1 C1NH
Complement component 4, partial deficiency of 1 C1NH
Retinal degeneration, late-onset, autosomal dominant 1 C1QTNF5
Brown-Vialetto-Van Laere syndrome 1 C20orf54
Mitochondrial complex 1 deficiency 1 C20orf7
Homocystinuria, cbID type, variant 1 1 C2orf25
Methylmalonic aciduria, cbID type, variant 2 1 C2orf25
6
Table IX-21781338.1 C5 deficiency 1 C5
C6 deficiency •1 C6
C7 deficiency . 1 C7
Trichothiodystrophy, nonphotosensitive 1 1 C7orfll
Cerebral cavernous malformations-2 1 C7orf22
Osteopetrosis, autosomal recessive 3, with renal tubular acidosis 1 CA2
Cerebellar ataxia and mental retardation with or without quadrupedal
locomotion 3 1 CA8
Coenzyme Q10 deficiency 6 CABC1
Night blindness, congenital stationary, type 2B 1 CABP4
Episodic ataxia, type 2 1 CACNA1A
Brugada syndrome 3 1 CACNA1C
Night blindness, congenital stationary, X-linked, type 2A 1 CACNA1F
Retinal cone dystrophy 4 1 CACNA2D4
Brugada syndrome 4 1 CACNB2
Desbuquois dysplasia 1 CANT1
Muscular dystrophy, limb-girdle, type 2A 1 CAPN3
Candidiasis, familial chronic mucocutaneous, autosomal recessive 1 CARD9
FG syndrome 4 1 CASK
Mental retardation and microcephaly with pontine and cerebellar
hypoplasia 1 CASK
Gastric cancer, somatic .8 CASP10
Non-Hodgkin lymphoma, somatic 1 CASP10
Hepatocellular carcinoma, somatic 3 CASP8
Ventricular tachycardia, catecholaminergic polymorphic, 2 1 CASQ2
Hyperparathyroidism, neonatal 1 CAS
Hypocalcemia, autosomal dominant 1 CAS
Hypocalciuric hypercalcemia, type 1 1 CASR
Male infertility, nonsyndromic, autosomal recessive 1 CATSPER1
Lipodystrophy, congenital generalized, type 3 1 CAV1
Cardiomyopathy, familial hypertrophic 2 CAV3
Creatine phosphokinase, elevated serum 1 CAV3
Long QT syndrome-9 1 CAV3
Muscular dystrophy, limb-girdle, type IC 1 CAV3
Corticosteroid-binding globulin deficiency 1 CBG
Homocystinuria, B6-responsive and nonresponsive types 1 CBS
Thrombosis, hyperhomocysteinemic 1 CBS
46XY gonadal dysgenesis, complete, CBS2-related 1 CBX2
Mental retardation, autosomal recessive 3 1 CC2D1A
COACH syndrome 3 CC2D2A
Deafness, autosomal dominant 44 1 CCDC50
7
Table IX-21781338.1 Thyroid papillary carcinoma 1 CCDC6
Cavernous malformations of CNS and retina 1 CCM1
Cerebral cavernous malformations-1 1 CCM1
Hyperkeratotic cutaneous capillary-venous malformations associated
with cerebral capillary malformations 1 CCM1 von Hippel-Lindau disease, modification of 1 CCND1
Neuropathy, hereditary sensory, with spastic paraplegia 1 CCT5
Nephropathy with pretibial epidermolysis bullosa and deafness 1 CD151
Immunodeficiency, common variable, 3 1 CD19
Immunodeficiency due to defect in CD3-zeta 1 CD247
Glomerulosclerosis, focal segmental, 3 1 CD2AP
Methylmalonic aciduria due to transcobalamin receptor defect 1 CD320
Platelet glycoprotein IV deficiency 1 CD36
Severe combined immunodeficiency, T cell-negative, B-cell/natural
killer-cell positive 3 CD3D
Severe combined immunodeficiency, T cell-negative, B-cell/natural
killer-cell positive 3 CD3E
CD59 deficiency 1 CD59
Immunodeficiency, hypogammaglobulinemia, and reduced B cells 1 CD79B
Immunodeficiency, common variable, 6 1 CD81
CD8 deficiency, familial 1 CD8A
C-like syndrome 1 CD96
Anemia, congenital dyserythropoietic, type 1 1 CDAN1
Gastric cancer, familial diffuse 1 CDH1
Mental retardation, autosomal dominant 3 1 CDH15
Deafness, autosomal recessive 12 1 CDH23
Ectodermal dysplasia, ectrodactyly, and macular dystrophy 1 CDH3
Melanoma 1 CDK4
Microcephaly, primary autosomal recessive, 3 1 CDK5RAP2
Angelman syndrome-like 1 CDKL5
Epileptic encephalopathy, early infantile, 2 1 CDKL5
Beckwith-Wiedemann syndrome 1 CDKN1C
Pancreatic cancer/melanoma syndrome 1 CDKN2A
Leukemia, acute myeloid 14 CEBPA
Specific granule deficiency 1 CEBPE
Maturity-onset diabetes of the young, type VIII 1 CEL
Microcephaly, primary autosomal recessive, 6 1 CEMPJ
Hyperalphalipoproteinemia 1 CETP
Heterotaxy, visceral, 2, autosomal 1 CFC1
Complement factor 1 deficiency 1 CFI
Nemaline myopathy 7 1 CFL2
Myasthenic syndrome, congenital, associated with episodic apnea 1 CHAT
8
Table IX-21781338.1 Hypogonadotropic hypogonadism 6 CHD7
Dementia, familial, nonspecific 1 CHMP2B
Cataract, posterior polar, 3 1 CHMP4B
Multiple pterygium syndrome, lethal type 3 CHRNA1
Myasthenic syndrome, fast-channel congenital 3 CHRNA1
Myasthenic syndrome, slow-channel congenital 4 CHRNA1
Epilepsy, nocturnal frontal lobe, type 4 1 CHRNA2
Epilepsy, nocturnal frontal lobe, 1 1 CHRNA4
Myasthenic syndrome, congenital, associated with acetylcholine
receptor deficiency 4 CHRNB1
• Myasthenic syndrome, slow-channel congenital 4 CHRNB1
Epilepsy, nocturnal frontal lobe, 3 1 CHRNB2
Multiple pterygium syndrome, lethal type 3 CHRND
Myasthenic syndrome, fast-channel congenital 3 CHRND
Myasthenic syndrome, slow-channel congenital 4 CHRND
Myasthenic syndrome, congenital, associated with acetylcholine
receptor deficiency 4 CHRNE
Myasthenic syndrome, fast-channel congenital 3 CHRNE
Myasthenic syndrome, slow-channel congenital 4 CHRNE
Escobar syndrome 1 CHRNG
Multiple pterygium syndrome, lethal type 3 CHRNG
C ediak-Higashi syndrome 1 CHS1
Spondyloepiphyseal dysplasia with congenital joint dislocations 1 CHST3
Cocoon syndrome 1 CHUK
Microphthalmia, isolated 2 1 CHX10
Microphthalmia, isolated, with coloboma 3 1 CHX10
Cirrhosis, North American Indian childhood type 1 CIRH1A
Wolfram syndrome 2 1 CISD2
Myotonia congenita, dominant 1 CLCNl
Myotonia congenita, recessive 1 CLCN1
Hypophosphatemic rickets 1 CLCN5
Nephrolithiasis, type 1 1 CLCN5
Proteinuria, low molecular weight, with hypercalciuric nephrocalcinosis 1 CLCN5
Osteopetrosis, autosomal dominant 2 1 CLCN7
, Osteopetrosis, autosomal recessive 4 1 CLCN7
Bartter syndrome, type 4b, digenic 1 CLCNKA
Bartter syndrome, type 3 1 CLCNKB
Bartter syndrome, type 4, digenic 1 CLCNKB
Ichthyosis, leukocyte vacuoles, alopecia, and sclerosing cholangitis 1 CLDN1
Hypomagnesemia, primary 1 CLDN16
Hypomagnesemia, renal, with ocular involvement 1 CLDN19
9
Table IX-21781338.1 Candidiasis, familial chronic mucocutaneous, autosomal dominant 1 CLEC7A
Ceroid lipofuscinosis, neuronal 3, juvenile 1 CLN3
Ceroid-lipofuscinosis, neuronal-5, variant late infantile 1 CLN5
Ceroid-lipofuscinosis, neuronal-6, variant late infantile 1 CLN6
Ceroid lipofuscinosis, neuronal 8 1 CLN8
Ceroid lipofuscinosis, neuronal, 8, Northern epilepsy variant 1 CLN8
Macular degeneration juvenile 1 CNGB3
Myopathy, congenital, Compton-North 1 CNTN1
Pitt-Hopkins like syndrome 1 1 CNTNAP2
Deafness, autosomal dominant 9 1 COCH
Cerebrocostomandibular-like syndrome 1 COG1
Congenital disorder of glycosylation, type llg 1 COGl
Congenital disorder of glycosylation, type llj 1 COG4
Congenital disorder of glycosylation, type Me 1 COG7
Congenital disorder of glycosylation, type llh . 1 COG8
Deafness, autosomal dominant 13 1 COLHA2
Deafness, autosomal recessive 53 1 COL11A2
Epidermolysis bullosa, junctional, non-Herlitz type 4 COL17A1
Knobloch syndrome, type 1 1 COL18A1
Caffey disease 1 COL1A1
01 type II 1 COL1A1
01 type III 1 COL1A1
01 type IV 1 COL1A1
Epiphyseal dysplasia, multiple, with myopia and deafness 1 COL2A1
Osteoarthritis with mild chondrodysplasia 1 COL2A1
SED congenita 1 COL2A1 ,
SMED, Strudwick type 1 COL2A1
Angiopathy, hereditary, with nephropathy, aneurysms, and muscle
cramps 1 COL4A1
Brain small vessel disease with Axenfeld-Rieger anomaly 1 COL4A1
Brain small vessel disease with hemorrhage 1 COL4A1
Porencephaly 1 COL4A1
Alport syndrome, autosomal recessive 1 COL4A3
Hematuria, benign familial 1 COL4A3
Alport syndrome, autosomal recessive 1 COL4A4
Alport syndrome 1 COL4A5
Leiomyomatosis, diffuse, with Alport syndrome 1 COL4A6
Ullrich congenital muscular dystrophy 3 COL6A1
Ullrich congenital muscular dystrophy 3 COL6A2
Ullrich congenital muscular dystrophy 3 COL6A3
EBD inversa 1 COL7A1
10
Table IX-21781338.1 EBD, Bart type 1 COL7A1
Epidermolysis bullosa dystrophica, AD 1 COL7A1
Epidermolysis bullosa dystrophica, AR 1 COL7A1
Epidermolysis bullosa pruriginosa 1 COL7A1
Epidermolysis bullosa, pretibial 1 COL7A1
Toenail dystrophy, isolated 1 COL7A1
Corneal dystrophy polymorphous posterior, 2 1 COL8A2
Corneal dystrophy, Fuchs endothelial, 1 1 COL8A2
Epiphyseal dysplasia, multiple, 2 1 COL9A2
Epiphyseal dysplasia, multiple, 3 1 COL9A3
Endplate acetylcholinesterase deficiency 1 COLQ
Epiphyseal dysplasia, multiple 1 1 COMP
Coenzyme Q10 deficiency 6 COQ2
Coenzyme Q10 deficiency 6 COQ9
Exocrine pancreatic insufficiency, dyserythropoietic anemia, and
calvarial hyperostosis 1 COX4I2
Cytochrome C oxidase deficiency 1 COX6B1
Cerebellar ataxia 1 CP
Hemosiderosis, systemic, due to aceruloplasminemia 1 CP
Carboxypeptidase N deficiency 1 CPN1
Coproporphyria 1 CPOX
Harderoporphyria 1 CPOX
Carbamoyl phosphate synthetase 1 deficiency 1 CPS1
CPT deficiency, hepatic, type IA 1 CPT1A
CPT deficiency, hepatic, type II 1 CPT2
CPT II deficiency, lethal neonatal 1 CPT2
Myopathy due to CPT II deficiency 1 CPT2
Pigmented paravenous chorioretinal atrophy 1 CRB1
Mental retardation, autosomal recessive 2A 1 CRBN
Histiocytoma, angiomatoid fibrous, somatic 1 CREB1
Atrioventricular septal defect, partial, with heterotaxy syndrome 2 CRELD1
Inflammatory bowel disease 25 1 CRFB4
Myopathy, cardioskeletal, desmin-related, with cataract 1 CRYAB
Cataract, congenital zonular, with sutural opacities 1 CRYBA1
Cataract, lamellar 2 1 CRYBA4
Microphthalmia, isolated, with cataract 4 1 CRYBA4
Cataract, congenital nuclear, autosomal recessive 3 1 CRYBB1
Cataract, cerulean, type 2 1 CRYBB2
Cataract, sutural, with punctate and cerulean opacities 1 CRYBB2
Cataract, congenital nuclear, 2 1 CRYBB3
Cataract, congenital, cerulean type, 3 1 CRYGD
11
Table IX-21781338.1 Cataract, crystalline aculeiform 1 CRYGD
Cataract, nonnuclear polymorphic congenital 1 CRYGD
Pulmonary alveolar proteinosis 1 CSF2RA
Neutrophilia, hereditary 11 CSF3R
Chondrosarcoma, extraskeletal myxoid 3 CSMF
Night blindness, congenital stationary, type 1 1 CSNB1
Cornelia de Lange syndrome 3 1 CSPG6
Cardiomyopathy, dilated, 1M 1 CSRP3
Cardiomyopathy, familial hypertrophic, 12 1 CSRP3
Cerebral amyloid angiopathy 1 CST3
Macular degeneration, age-related, 11 1 CST3
Epilepsy, progressive myoclonic 1 1 CSTB
Hepatocellular carcinoma 2 CTNNB1
Ovarian cancer 1 CTNNB1
Mental retardation in cri-du-chat syndrome 1 CTNND2
Cystinosis, late-onset juvenile or adolescent nephropathic 1 CTNS
Cystinosis, nephropathic 1 CTNS
Cystinosis, ocular nonnephropathic 1 CTNS
Periodontitis, juvenile 1 CTSC
Ceroid lipofuscinosis, neuronal, 10 1 CTSD
Megaloblastic anemia-1, Finnish type 1 CUBN
Mental retardation syndrome, X-linked, Cabezas type 1 CUL4B
Mental retardation-hypotonic facies syndrome, X-linked, 2 1 CUL4B
Methemoglobinemia, type IV 1 CYB5A
Methemoglobinemia, type 1 1 CYB5R3
Methemoglobinemia, type II 1 CYB5R3
Chronic granulomatous disease, autosomal, due to deficiency of CYBA 1 CYBA
Chronic granulomatous disease, X-linked 1 CYBB
Thrombocytopenia 4 1 CYCS
Brooke-Spiegler syndrome 1 CYLD1
Cylindromatosis, familial 1 CYLD1
Trichoepithelioma, multiple familial, 1 1 CYLD1
Lipoid congenital adrenal hyperplasia 1 CYP11A
Adrenal hyperplasia, congenital, due to 11-beta-hydroxylase deficiency 1 CYP11B1
Aldosteronism, glucocorticoid-remediable 1 CYP11B1
Hypoaldosteronism, congenital, due to CMO 1 deficiency 1 CYP11B2
Hypoaldosteronism, congenital, due to CMO II deficiency 1 CYP11B2
17,20-lyase deficiency, isolated 2 CYP17A1
17-alpha-hydroxylase/17,20-lyase deficiency 2 CYP17A1
Glaucoma 3A, primary congenital 1 CYP1B1
Glaucoma, primary open angle, adult-onset 1 CYP1B1
12
Table IX-21781338.1 Glaucoma, primary open angle, juvenile-onset 1 CYP1B1
Vitamin D-dependent rickets, type 1 1 CYP27B1
Coumarin resistance 1 CYP2A6
Clopidogrel, impaired responsiveness to 1 CYP2C
Mephenytoin poor metabolizer 1 CYP2C
Opremazole poor metabolizer 1 CYP2C
Proguanil poor metabolizer 1 CYP2C
Warfarin sensitivity 1 CYP2C9
Rickets due to defect in vitamin D 25-hydroxylation 1 CYP2R1
Ichthyosis, lamellar, 3 1 CYP4F22
Spastic paraplegia-5A 1 CYP7B1
D-2-hydroxyglutaric aciduria 1 D2HGDH
Adrenal hypoplasia, congenital, with hypogonadotropic hypogonadism 1 DAX1
Dosage-sensitive sex reversal 1 DAX1
Maple syrup urine disease, type II 1 DBT
Mirror movements, congenital 1 DCC
Severe combined immunodeficiency, Athabascan type 1 DCLRE1C
Corneal dystrophy, congenital stromal 1 DCN
Neuropathy, distal hereditary motor, type VIIB 1 DCTN1
Spondylometaepiphyseal dysplasia, short limb-hand type 1 DDR2
Cardiomyopathy, dilated, 11 1 DES
Myopathy, desmin-related, cardioskeletal 1 DES
Deafness, autosomal dominant 5 1 DFNA5
Hyperoxaluria, primary, type III 1 DHDPSL
Miller syndrome 1 DHODH
Pseudohyperkalemia, familial 1 DHS
Deafness, autosomal dominant 1 1 DIAPH1
Premature ovarian failure 1 DIAPH2
Mental retardation, FRA12A type 1 DIP2B
Renal cell carcinoma 2 DIRC2
Schizophrenia 1 DISC2
Parkinson disease 7, autosomal recessive early-onset 1 DJ1
Maple syrup urine disease, type III 1 DLD
Esophageal cancer 1 DLEC1
Lung cancer 5 DLEC1
Spondylocostal dysostosis, autosomal recessive, 1 1 DLL3
Amelogenesis imperfecta, hypomaturation-hypoplastic type, with
taurodontism 1 DLX3
Cardiomyopathy, dilated, 3B 1 DMD
Hypophosphatemic rickets, AR 1 DMP1
Myotonic dystrophy 1 DMPK
13
Table IX-21781338.1 Ciliary dyskinesia, primary, 7, with or without situs inversus 1 DNAH11
Ciliary dyskinesia, primary, 3, with or without situs inversus 1 DNAH5
Ciliary dyskinesia, primary, 1, with or without situs inversus 1 DNAI1
Ciliary dyskinesia, primary, 9, with or without situs inversus 1 DNAI2
3-Methylglutaconic aciduria type V 1 DNAJC19
Charcot-Marie-Tooth disease, axonal, type 2M 1 DNM2
Charcot-Marie-Tooth disease, dominant intermediate B 1 DNM2
Myopathy, centronuclear 2 DNM2
Immunodeficiency-centromeric instability-facial anomalies syndrome 1 DNMT3B
Hyper-lgE recurrent infection syndrome, autosomal recessive 1 DOCK8
Myasthenia, limb-girdle, familial 2 DOK7
Congenital disorder of glycosylation, type Ij 1 DPAGT2
Congenital disorder of glycosylation, type le 1 DPMI
Congenital disorder of glycosylation, type lo 1 DPM3
Ventricular fibrillation, paroxysmal familial, 2 1 DPP6
5-fluorouracil toxicity 1 DPYD
Dystonia, myoclonic 1 D D2
Arrhythmogenic right ventricular dysplasia, familial, 11 1 DSC2
Hypotrichosis and recurrent skin vesicles 1 DSC3
Arrhythmogenic right ventricular dysplasia, familial, 10 1 DSG2
Cardiomyopathy, dilated, IBB 1 DSG2
Hypotrichosis, localized, autosomal recessive 1 DSG4
Arrhythmogenic right ventricular dysplasia 8 1 DSP
Dilated cardiomyopathy with woolly hair and keratoderma 1 DSP
Epidermolysis bullosa, lethal acantholytic 1 DSP
Deafness, autosomal dominant 36, with dentinogenesis 1 DSPP
Thryoid dyshormonogenesis 6 1 DUOX2
Thyroid dyshormonogenesis 5 1 DUOXA2
Cornelia de Lange syndrome 2 1 DXS423E
Asphyxiating thoracic dystrophy 3 1 DYNC2H1
Muscular dystrophy, limb-girdle, type 2B 1 DYSF
Myopathy, distal, with anterior tibial onset 1 DYSF
Dystonia-1, torsion 1 DYTl
Chondrodysplasia punctata, X-linked dominant 1 EBP
Ectodermal dysplasia, anhidrotic, X-linked 1 EDI
Tooth agenesis, selective, X-linked 1 1 EDI
Ectodermal dysplasia, hypohidrotic, autosomal dominant 1 EDAR
Ectodermal dysplasia, hypohidrotic, autosomal recessive 1 EDAR
Ectodermal dysplasia, anhidrotic, autosomal dominant 1 EDARADD
Ectodermal dysplasia, anhidrotic, autosomal recessive 1 EDARADD
Central hypoventilation syndrome, congenital 5 EDN3
14
Table IX-21781338.1 Migraine, resistance to 1 EDNRA
ABCD syndrome 1 EDNRB
Doyne honeycomb degeneration of retina 1 EFEMP1
Cutis laxa, autosomal recessive, type 1 1 EFEMP2
Hypomagnesemia 4, renal 1 EGF
Adenocarcinoma of lung, response to tyrosine kinase inhibitor in 1 EGFR
Nonsmall cell lung cancer, response to tyrosine kinase inhibitor in 1 EGFR
Erythrocytosis, familial, 3 1 EGLN1
Neuropathy, congenital hypomyelinating, 1 1 EGR2
Leukoencephalopathy with vanishing white matter 4 EIF2B1
Leukoencephalopathy with vanishing white matter 4 EIF2B2
Leukoencephalopathy with vanishing white matter 4 EIF2B3
Leukoencephaly with vanishing white matter 1 EIF2B4
Leukoencephalopathy with vanishing white matter 4 EIF2B5
Hematopoiesis, cyclic 1 ELANE
Neutropenia, severe congenital, autosomal dominant 1 1 ELANE
Cutis laxa, AD 1 ELN
Macular dystrophy, autosomal dominant, chromosome 6-linked 1 ELOVL4
Emery-Dreifuss muscular dystrophy 1 EMD
Bowen-Conradi syndrome . 1 EMG1
Amelogenesis imperfecta, type IB 1 ENAM
Amelogenesis imperfecta, type IC 1 ENAM
Hereditary hemorrhagic telangiectasia-1 1 ENG
Arterial calcification, generalized, of infancy 1 ENPP1
Hypophosphatemia rickets, autosomal recessive, 2 1 ENPP1
Ossification of posterior longitudinal ligament of spine 1 ENPP1
Colorectal cancer 6 EP300
Erythrocytosis, familial, 4 1 EPAS1
Elliptocytosis-1 1 EPB41
Spherocytosis, hereditary, type 5 1 EPB42
Stomatocytosis 1 1 EPB72
Colorectal cancer, hereditary nonpolyposis, type 1 1 EPCAM
Diarrhea 5, with tufting enteropathy, congenital 1 EPCAM
Cataract, posterior polar, 1 1 EPHA2
Prostate cancer, progression and metastasis of 1 EPHB2
Hypercholanemia, familial 3 EPHX1
Epilepsy, myoclonic, Lafora type 2 EPM2A
Eosinophil peroxidase deficiency 1 EPX
Adenocarcinoma of lung, somatic 3 ERBB2
Gastric cancer, somatic 8 ERBB2
Glioblastoma, somatic 1 ERBB2
15
•Table IX-21781338.1 Cerebrooculofacioskeletal syndrome 4 1 ERCC1
Cerebrooculofacioskeletal syndrome 2 1 ERCC2
Trichothiodystrophy 1 ERCC2
Trichothiodystrophy 1 ERCC3
XFE progeroid syndrome 1 ERCC4
Cerebrooculofacioskeletal syndrome 1 1 ERCC6
Cockayne syndrome type B 1 ERCC6
De Sanctis-Cacchione syndrome 1 ERCC6
Cockayne syndrome type A 1 ERCC8
Deafness, autosomal recessive 36 1 ESPN
Deafness, autosomal recessive 35 1 ESRRB
Glutaricaciduria, type IIA 1 ETFA
Glutaricaciduria, type MB 1 ETFB
Glutaricaciduria, type IIC 1 ETFDH
Leukemia, acute myeloid, somatic 1 ETV6
Neuroepithelioma 1 EWSR1
Exostoses, multiple, type 1 1 EXT1
Exostoses, multiple, type 2 1 EXT2
Branchiootorenal syndrome with cataract 1 EYA1
Cardiomyopathy, dilated, 1J 1 EYA4
Deafness, autosomal dominant 10 1 EYA4
Factor XI deficiency, autosomal dominant 1 Fll
Factor XI deficiency, autosomal recessive 1 Fll
Angioedema, hereditary, type III 1 F12
Factor XII deficiency 1 F12
Factor XIIIA deficiency 1 F13A1
Factor XIIIB deficiency 1 F13B
Factor V deficiency 1 F5
Hemophilia B 1 F9
Thrombophilia, X-linked, due to factor IX defect 1 F9
Leukodystrophy, dysmyelinating, and spastic paraparesis with or
without dystonia 1 FA2H
Fanconi anemia, complementation group B 1 FAAP95
Osteopathia striata with cranial sclerosis 1 FAM123B
Leukodystrophy, hypomyelinating, 5 1 FAM126A
Neuropathy, hereditary sensory and autonomic, type IIB 1 FAM134B
STAR syndrome 1 FAM58A
Amelogenesis imperfecta, type 3 1 FAM83H
Fanconi anemia, complementation group A 1 FANCA
Fanconi anemia, complementation group 1 1 FANCI
Mitochondrial complex IV deficiency 1 FASTKD2
16
Table IX-21781338.1 Cutis laxa, autosomal dominant 1 FBLN5
Cutis laxa, autosomal recessive 1 FBLN5
Macular degeneration, age-related, 3 1 FBLN5
MASS syndrome 2 FBN1
Contractural arachnodactyly, congenital 1 FBN2
Parkinson disease 15, autosomal recessive 1 FBX07
Thrombocytopenic purpura, autoimmune 1 FCGR2C
Polycystic kidney and hepatic disease 1 FCYT
Protoporphyria, erythropoietic, autosomal dominant 1 FECH
Protoporphyria, erythropoietic, autosomal recessive 1 FECH
Afibrinogenemia, congenital 2 FGA
Amyloidosis, hereditary renal 1 FGA
Afibrinogenemia, congenital 2 FGB
Aplasia of lacrimal and salivary glands 1 FGF10
LADD syndrome 2 FGF10
Hypophosphatemia rickets, autosomal dominant 1 FGF23
Tumoral calcinosis, hyperphosphatemic, familial 1 FGF23
Deafness, congenital with inner ear agenesis, microtia, and microdontia 1 FGF3
Hypogonadotropic hypogonadism 6 FGFR1
Jackson-Weiss syndrome 2 FGFR1
Trigonocephaly 1 FGFR1
Beare-Stevenson cutis gyrata syndrome 1 FGFR2
Gastric cancer, somatic 8 FGFR2
. Saethre-Chotzen syndrome 2 FGFR2
Bladder cancer, somatic 3 FGFR3
CATSHL syndrome 1 FGFR3
Cervical cancer, somatic 1 FGFR3
Colorectal cancer, somatic 6 FGFR3
LADD syndrome 2 FGFR3
Nevus, keratinocytic, nonepidermolytic 1 FGFR3
Leiomyomatosis and renal cell cancer 1 FH
Multiple cutaneous and uterine leiomyomata 1 FH
Emery-Dreifuss muscular dystrophy 6 1 FHL1
Myopathy, reducing body, X-linked, childhood-onset 1 FHL1
Myopathy, reducing body, X-linked, severe early-onset 1 FHL1
Myopathy, X-linked, with postural muscle atrophy 1 FHL1
Premature ovarian failure 6 1 FIGLA
Cardiomyopathy, dilated, IX 1 FKTN
Colorectal cancer, somatic 6 FLCN
Pneumothorax, primary spontaneous 1 FLCN
Renal carcinoma, chromophobe, somatic 1 FLCN
17
Table IX-21781338.1 Ichthyosis vulgaris 1 FLG
Thrombocytopenia-2 1 FU14813
Premature ovarian failure 2B 1 FU22792
FG syndrome 2 1 FLNA
Heterotopia, periventricular 1 FLNA
Heterotopia, periventricular, ED variant 1 FLNA
Intestinal pseudoobstruction, neuronal 1 FLNA
Melnick-Needles syndrome 1 FLNA
Larsen syndrome . 1 FLNB
Spondylocarpotarsal synostosis syndrome 1 FLNB
Myopathy, myofibrillar, filamin C-related 1 FLNC
Leukemia, acute myeloid 14 FLT3
Hemangioma, capillary infantile, somatic 2 FLT4
Lymphedema, hereditary 1 1 FLT4
Proliferative vasculopathy and hydraencephaly-hydrocephaly syndrome 1 FLVCR2
Glomerulopathy with fibronectin deposits 2 1 FN1
Neurodegeneration due to cerebral folate transport deficiency 1 FOLR1
Iridogoniodysgenesis, type 1 1 FOXC1
Iris hypoplasia and glaucoma 1 FOXC1
Rieger or Axenfeld anomalies 1 FOXC1
Lymphedema-distichiasis syndrome 1 FOXC2
Lymphedema-distichiasis syndrome with renal disease and diabetes
mellitus 1 FOXC2
Anterior segment mesenchymal dysgenesis 2 FOXE3
Aphakia, congenital primary 1 FOXE3
Alveolar capillary dysplasia with misalignment of pulmonary veins 1 FOXF1
Rett syndrome, congenital variant 1 FOXG1B
Enlarged vestibular aqueduct 2 FOXI1
Premature ovarian failure 3 1 FO L2
T-cell immunodeficiency, congenital alopecia, and nail dystrophy 1 FOXN1
Rhabdomyosarcoma, alveolar 1 FOXOIA
Speech-language disorder-1 1 FOXP2
Immunodysregulation, polyendocrinopathy, and enteropathy, X-linked 1 FOXP3
Bifid nose with or without anorectal and renal anomalies 1 FREM1
Nystagmus 1, congenital, X-linked 2 FRMD7
Follicle-stimulating hormone deficiency, isolated 1 FSHB
Ovarian dysgenesis 1 1 FSHR
Ovarian response to FSH stimulation 1 FSHR
Polycystic ovary syndrome 1 FST
Glutamate formiminotransferase deficiency 1 FTCD
Hyperferritinemia-cataract syndrome 1 FTL
18
Table IX-21781338.1 Neurodegeneration with brain iron accumulation 3 1 FTL
Growth retardation, developmental delay, coarse facies, and early
death 1 FTO
Mental retardation, X-linked-9 1 FTSJ1
Hypomagnesemia-2, renal 1 FXYD2
Exudative vitreoretinopathy 1 FZD4
Retinopathy of prematurity 1 FZD4
Darsun syndrome 1 G6PC3
Neutropenia, severe congenital, autosomal recessive 4 1 G6PC3
Epilepsy, juvenile myoclonic, susceptibility to 1 GABRD
Epilepsy, generalized, with febrile seizures plus, type 3 1 GABRG2
Febrilel, convulsions, familial 1 GABRG2
Myoclonic epilepsy, severe, of infancy 1 GABRG2
Cerebral palsy, spastic, symmetric, autosomal recessive 1 GAD1
Krabbe disease 1 GALC
Galactose epimerase deficiency 1 GALE
Tumoral calcinosis, hyperphosphatemic, familial 1 GALNT3
Galactosemia 1 GALT
GAMT deficiency 1 GAMT
Charcot-Marie-Tooth disease type 2D 1 GARS
Neuropathy, distal hereditary motor, type V 1 GARS
Dyserythropoietic anemia with thrombocytopenia 1 GATA1
Leukemia, megakaryoblastic, of Down syndrome 1 GATA1
Leukemia, megakaryoblastic, with or without Down syndrome 1 GATA1
Macrothrombocytopenia 1 GATA1
Thrombocytopenia with beta-thalassemia, X-linked 1 GATA1
Hypoparathyroidism, sensorineural deafness, and renal dysplasia 1 GATA3
AGAT deficiency 1 GATM
Glutaricaciduria, type 1 1 GCDH
Dystonia, DOPA-responsive, with or without hyperphenylalainemia 1 GCH1
Hyperpehnylalaninemia, BH4-deficient, B 1 GCH1
Diabetes mellitus, gestational 1 GCK
Diabetes mellitus, noninsulin-dependent, late onset 1 GCK
Diabetes mellitus, permanent neonatal 1 GCK
Hyperinsulinemic hypoglycemia, familial, 3 1 GCK
Hemolytic anemia due to gamma-glutamylcysteine synthetase
deficiency 1 GCLC
Hypoparathyroidism, familial isolated 1 GCMB
Adult i phenotype with congenital cataract 1 GCNT2
Adult i phenotype without cataract 1 GCNT2
Congenital disorder of glycosylation, type lib 1 GCS1
19
Table IX-21781338.1 Glycine encephalopathy 1 GCSH
Charcot-Marie-Tooth disease, axonal, type 2K 1 GDAP1
Charcot-Marie-Tooth disease, axonal, with vocal cord paresis 1 GDAP1
Charcot-Marie-Tooth disease, recessive intermediate, A 1 GDAP1
Chondrodysplasia, Grebe type 1 GDF5
Symphalangism, proximal 2 GDF5
Klippel-Feil syndrome, autosomal dominant 1 GDF6
Microphthalmia, isolated 4 1 GDF6
Spondylocostal dystostosis 4, autosomal dominant 1 GDF6
Mental retardation, X-linked nonspecific 1 GDI1
Central hypoventilation syndrome 1 GDNF
Myopathy, mitochondrial progressive, with congenital cataract, hearing
loss, and developmental delay 1 GFER
Neutropenia, nonimmune chronic idiopathic, of adults 1 GFI1
Neutropenia, severe congenital, autosomal dominant 2 1 GFI1
Combined oxidative phosphorylation deficiency 1 1 GFM1
Pseudoxanthoma elasticum-like disorder with multiple coagulation
factor deficiency 1 GGCX
Vitamin K-dependent coagulation defect 1 GGCX
Growth hormone deficiency, isolated, type IA 1 GH1
Growth hormone deficiency, isolated, type IB 1 GH1
Growth hormone deficiency, isolated, type II . 1 GH1
Kowarski syndrome 1 GH1
Short stature, idiopathic 1 GHR
Growth hormone deficiency, isolated, type IB 1 GHRHR
Short stature 1 GHSR
Intrinsic factor deficiency 1 GIF
Parkinson disease 11 1 GIGYF2
Fibromatosis, gingival, 2 ■ 1 GINGF2
Atrioventricular septal defect 2 GJA1
Hallermann-Streiff syndrome 1 GJA1
Hypoplastic left heart syndrome 1 GJA1
Cataract, zonular pulverulent-3 1 GJA3
Atrial fibrillation 1 GJA5
Cataract, zonular pulverulent-1 1 GJA8
Cataract-microcornea syndrome 1 GJA8
Charcot-Marie-Tooth neuropathy, X-linked dominant, 1 1 GJB1
Bart-Pumphrey syndrome 1 GJB2
Deafness, autosomal dominant 3A 1 GJB2
Deafness, autosomal recessive 1A 1 GJB2
Hystrix-like ichthyosis with deafness 1 GJB2
20
Table IX-21781338.1 Keratitis-ichthyosis-deafness syndrome 1 GJB2
Vohwinkel syndrome 1 GJB2
Deafness, autosomal dominant 2B 1 GJB3
Deafness, digenic, GJB2/GJB3 1 GJB3
Deafness, autosomal dominant 3B 1· GJB6
Deafness, autosomal recessive IB 1 GJB6
Deafness, digenic GJB2/GJB6 1 GJB6
Ectodermal dysplasia, hidrotic 1 GJB6
Leukodystrophy, hypomyelinating, 2 . 1 GJC2
Lymphedema, hereditary, IC 1 GJC2
Spastic paraplegia, 44 1 GJC2
GMl-gangliosidosis, type 1 1 GLB1
Morquio syndrome B 1 GLB1
Glaucoma IB, primary open angle, adult onset 1 GLC1B
Glycine encephalopathy 3 GLDC
Arthrogryposis, lethal, with anterior horn cell disease 1 GLE1
Holoprosencephaly-9 1 GLI2
Polydactyly, postaxial, types Al and B 1 GLI3
Polydactyly, preaxial, type IV 1 GLI3
Nephronophthisis 7 1 GLIS2
Diabetes mellitus, neonatal, with congenital hypothyroidism 1 GLIS3
Startle disease/hyperekplexia, autosomal dominant 1 GLRA1
Hyperekplexia, autosomal recessive 1 GLRB
Anemia, sideroblastic, pyridoxine-refractory, autosomal recessive 1 GLRX5
Glutamine deficiency, congenital 1 GLUL
Leukemia, acute myelogenous 3 GMPS
Ventricular tachycardia, idiopathic 1 GNAI2
Osseous heteroplasia, progressive 1 GNAS
Night blindness, congenital stationary, autosomal dominant 3 1 GNAT1
Inclusion body myopathy, autosomal recessive 1 GNE
Nonaka Myopathy 1 GNE
Glycine N-methyltransferase deficiency 1 GNMT
Chondrodysplasia punctata, rhizomelic, type 2 1 G PAT
Hypogonadotropic hypogonadism due to GNRH deficiency 1 GNRH1
Fertile eunuch syndrome 1 GNRHR
Mucopolysaccharidosis type MID 1 GNS
Thyroid carcinoma, papillary 6 GOLGA5
Globozoospermia 2 GOPC
Wilms tumor, somatic 1 GPC3
Omodysplasia 1 1 GPC6
Brugada syndrome 2 1 GPD1L
21
Table IX-21781338.1 Hyperekplexia 2 GPHN
Molybdenum cofactor deficiency, type C 1 GPHN
Hemolytic anemia, nonspherocytic, due to glucose phosphate isomerase
deficiency 1 GPI
Nystagmus 6, congenital, X-linked 2 GPR143
Ocular albinism, type 1, Nettleship-Falls type 1 GPR143
Polymicrogyria, bilateral frontoparietal 1 GPR56
Convulsions, familial febrile, 4 1 GPR98
Deafness, autosomal dominant 28 1 GRHL2
Hyperoxaluria, primary, type II 1 GRHPR
Mental retardation, X-linked 94 1 GRIA3
Mental retardation, autosomal recessive, 6 1 GRIK2
Night blindness, congenital stationary, type IB 1 GRM6
Frontotemporal lobar degeneration with ubiquitin-positive inclusions 1 GRN
Deafness, autosomal recessive 25 1 GRXCR1
Amyloidosis, Finnish type 1 GSN
Hemolytic anemia due to glutathione synthetase deficiency 1 GSS
Trichothiodystrophy, complementation group A 1 GTF2H5
Cone dystrophy-3 1 GUCA1A
Mucopolysaccharidosis VII 1 GUSB
Glycogen storage disease 0, muscle 1 GYS1
Silver-Russell syndrome 1 H19
Wilms tumor 2 1 H19
Cortisone reductase deficiency 1 H6PD
Fatty liver, acute, of pregnancy 1 HADHA
HELLP syndrome, maternal, of pregnancy 1 HADHA
LCHAD deficiency 1 HADHA
Trifunctional protein deficiency 2 HADHA
Trifunctional protein deficiency 2 HADHB
3-hydroxyacyl-coa dehydrogenase deficiency 1 HADHSC
Hyperinsulinemic hypoglycemia, familial, 4 1 HADHSC
Neutropenia, severe congenital, autosomal recessive 3 1 HAX1
Heinz body anemias, alpha- 1 HBA1
Thalassemias, alpha- 2 HBA1
Thalassemia, alpha- 2 HBA2
Heinz body anemias, beta- 1 HBB
Thalassemia-beta, dominant inclusion-body 1 HBB
Thalassemias, beta- 1 HBB
Fetal hemoglobin quantitative trait locus 1 2 • HBG1
Fetal hemoglobin quantitative trait locus 1 2 HBG2
Microphthalmia, syndromic 7 1 HCCS
22
Table IX-21781338.1 Thrombophilia due to heparin cofactor II deficiency 1 HCF2
Brugada syndrome 8 1 HCN4
Growth hormone deficiency with pituitary anomalies 1 HESX1
GM2-gangliosidosis, several forms 1 HEXA
Basal laminar drusen 1 HF1
Complement factor H deficiency 1 HF1
Hemolytic uremic syndrome, atypical, susceptibility to, 1 1 HF1
Membranoproliferative glomerulonephritis with CFH deficiency 1 HF1
Deafness, autosomal recessive 39 1 HGF
3-hydroxyisobutryl-CoA hydrolase deficiency 1 HIBCH
Hemolytic anemia due to hexokinase deficiency 1 HK1
Porphyria, acute intermittent 1 HMBS
Porphyria, acute intermittent, nonerythroid variant 1 HMBS
HMG-CoA synthase-2 deficiency 1 HMGCS2
Diabetes mellitus, insulin-dependent, 20 1 HNF1A
Hepatic adenoma 1 HNF1A
Renal cell carcinoma 2 HNF1A
Diabetes mellitus, noninsulin-dependent 1 HNF1B
Microtia, hearing impairment, and cleft palate 1 HOXA2
Vertical talus, congenital 1 HOXD10
Brachydactyly type D 1 HOXD13
Brachydactyly-syndactyly syndrome 1 HOXD13
Syndactyly, type V 1 HOXD13
Digital clubbing, isolated congenital 1 HPGD
Hypertrophic osteoarthropathy, primary, autosomal recessive 1 HPGD
HPRT-related gout 1 HPRT1
Urofacial syndrome 1 HPSE2
Hypotrichosis, hereditary, Marie Unna type, 1 1 HR
Costello syndrome 1 HRAS
Thrombophilia due to elevated HRG 1 HRG
Thrombophilia due to HRG deficiency 1 HRG
Hyperparathyroidism, familial primary 1 HRPT2
Hyperparathyroidism-jaw tumor syndrome 1 HRPT2
Parathyroid adenoma with cystic changes 1 HRPT2
Parathyroid carcinoma 1 HRPT2
Cortisone reductase deficiency 1 HSD11B1
17-beta-hydroxysteroid dehydrogenase X deficiency 1 HSD17B10
Mental retardation, X-linked 17/31, microduplication 1 HSD17B10
Mental retardation, X-linked syndromic 10 1 HSD17B10
Pseudohermaphroditism, male, with gynecomastia 1 HSD17B3
D-bifunctional protein deficiency 1 HSD17B4
23
Table IX-21781338.1 Cholestasis, progressive familial intrahepatic 4 1 HSD3B7
Cataract, lamellar 1 HSF4
Cataract, Marner type 1 HSF4
Charcot-Marie-Tooth disease, axonal, type 2F 1 HSPB1
Neuropathy, distal hereditary motor, type MB 1 HSPB1
Neuronopathy, distal hereditary motor, type IIC 1 HSPB3
Charcot-Marie-Tooth disease, axonal, type 2L 1 HSPB8
Neuropathy, distal hereditary motor, type IIA 1 HSPB8
Leukodystrophy, hypomyelinating, 4 1 HSPD1
Spastic paraplegia-13 1 HSPD1
Parkinson disease 13 1 HT A2
Mental retardation, X-linked syndromic, Turner type 1 HUWE1
Cleft lip/palate-ectod.ermal dysplasia syndrome 1 HVEC
Orofacial cleft 7 1 HVEC
Mucopolysaccharidosis type IX 1 HYAL1
Hydrolethalus syndrome 1 HYLS1
Ichthyosis, congenital, autosomal recessive 1 ICHYN
Immunodeficiency, common variable, 1 1 ICOS
BCG infection, generalized familial 1 IFNGR1
Mycobacterial infection, atypical, familial disseminated 2 IFNGR1
Cranioectodermal dysplasia 1 IFT122
Asphyxiating thoracic dystrophy 2 1 IFT80
Corpus callosum, agenesis of, with mental retardation, ocular coloboma
and micrognathia 1 IGBP1
Growth retardation with deafness and mental retardation due to IGF1
deficiency 1 IGF1
Insulin-like growth factor 1, resistance to 1 IGF1R
Agammaglobulinemia 1 1 IGHM
Neuronopathy, distal hereditary motor, type VI 1 IGHMBP2
Agammaglobulinemia 2 1 IGLL1
Dysautonomia, familial 1 IKBKAP
Ectodermal dysplasia, hypohidrotic, with immune.deficiency 1 IKBKG
Ectodermal, dysplasia, anhidrotic, lymphedema and immunodeficiency 1 IKBKG
Immunodeficiency, isolated 1 IKBKG
BCG and salmonella infection, disseminated 1 IL12B
Mental retardation, X-linked, 21/34 1 IL1RAPL1
Interleukin 1 receptor antagonist deficiency 1 IL1RN lnterleukin-2 receptor, alpha chain, deficiency of 1 IL2RA
Combined immunodeficiency, X-linked, moderate 1 IL2RG
Severe combined immunodeficiency, X-linked 1 IL2RG
Severe combined immunodeficiency, T-cell negative, B-cell/natural killer
cell-positive type. 1 IL7R
24
Table IX-21781338.1 Glomerulosclerosis, focal segmental, 5 1 INF2
Mental retardation, truncal obesity, retinal dystrophy, and micropenis 1 INPP5E
Diabetes mellitus, insulin-dependent, 2 1 INS
Diabetes mellitus, permanent neonatal 1 INS
Diabetes mellitus, type 1 1 INS
Maturity-onset diabetes of the young, type 10 1 INS
Cryptorchidism, idiopathic 2 INSL3
Diabetes mellitus, insulin-resistant, with acanthosis nigricans 1 INSR .
Hyperinsulinemic hypoglycemia, familial, 5 1 INSR
Leprechaunism 1 INSR
Nephronophthisis 2, infantile 1 INVS
Pancreatic agenesis 1 IPF1
Mental retardation, X-linked 1 1 IQSEC2
Invasive pneumococcal disease, recurrent isolated, 1 1 IRAK4
I AK4 deficiency 1 IRAK4
Gastric cancer, somatic 8 IRF1
Nonsmall cell lung cancer, somatic 2 IRF1
Orofacial cleft 6 1 IRF6
Popliteal pterygium syndrome 1 IRF6
Myopathy with lactic acidosis, hereditary 1 ISCU
Autoimmune disease, syndromic multisystem 1 ITCH
Epidermolysis bullosa, junctional, with pyloric stenosis 1 ITGA6
Muscular dystrophy, congenital, due to ITGA7 deficiency 1 ITGA7
Epidermolysis bullosa of hands and feet 1 ITGB4
Epidermolysis bullosa, junctional, non-Herlitz type 4 ITGB4
Epidermolysis bullosa, junctional, with pyloric atresia 1 ITGB4
Lymphoproliferative syndrome, EBV-associated, autosomal, 1 1 ITK
Dementia, familial British 1 ITM2B
Dementia, familial Danish 1 ITM2B
Spinocerebellar ataxia 15 1 ITPR1
Thyroid dyshormonogenesis 4 1 IYD
Leukemia, acute myelogenous 3 JAK2
Myelofibrosis, idiopathic 1 JAK2
Polycythemia vera 1 JAK2
Thrombocythemia, essential 3 JAK2
Arrhythmogenic right ventricular dysplasia, familial, 12 1 JUP
Cerebral palsy, spastic quadriplegic 1 KANK1
Charcot-Marie-Tooth disease, recessive intermediate, B 1 KARS
Episodic ataxia/myokymia syndrome 1 KCNA1
Atrial fibrillation, familial, 7 1 KCNA5
' Atrial fibrillation, familial, 4 1 KCNE2
25
Table IX-21781338.1 Brugada syndrome 6 1 KCNE3
Short QT syndrome-1 1 KCNH2
Bartter syndrome, type 2 1 KCNJ 1
Diabetes mellitus, permanent neonatal, with neurologic features 1 KCNJ 11
Diabetes mellitus, transient neonatal, 3 1 KCNJ 11
Diabetes, permanent neonatal 1 KCNJ 11
Hyperinsulinemic hypoglycemia, familial, 2 1 KCNJ 11
Long QT syndrome-7 1 KCNJ2
Short QT syndrome-3 1 KCNJ2
Long QT syndrome 13 1 KCNJ5
Birk-Barel mental retardation dysmorphism syndrome 1 KCNK9
Atrial fibrillation, familial, 3 1 KCNQ1
Long QT syndrome-1 1 KCNQ1
Short QT syndrome-2 1 KCNQ1
Epilepsy, benign, neonatal, type 1 1 KCNQ2
Myokymia with neonatal epilepsy 1 KCNQ2
Epilepsy, benign neonatal, type 2 1 KCNQ3
Deafness,. autosomal dominant 2A 1 KCNQ4
Retinal cone dystrophy 3B 1 KCNV2
Epilepsy, progressive myoclonic 3 1 KCTD7
Mental retardation, X-linked, syndromic, JARIDlC-related 1 KDM5C
Hemangioma, capillary infantile, somatic 2 KDR
Cornea plana congenita, recessive 1 KERA
Spastic paraplegia-8 . 1 KIAA0196
Charcot-Marie-Tooth disease type 2A1 1 KIF1B
Pheochromocytoma 5 KIF1B
Fibrosis of extraocular muscles, congenital, 1 1 KIF21A
Fibrosis of extraocular muscles, congenital, 3B 1 KIF21A
Spastic paraplegia 10 1 KIF5A
Kindler syndrome 2 KI ND1
Leukocyte adhesion deficiency, type I II 1 KI ND3
Mental retardation, autosomal dominant 4 1 KIRREL3
Hypogonadotropic hypogonadism 6 KISS1R
Germ cell tumors 1 KIT
Leukemia, acute myeloid 14 KIT
Hyperpigmentation, familial progressive 1 KITLG
Tumoral calcinosis, hyperphosphatemic 1 KL
Blood group-Lutheran inhibitor 1 KLF1
Maturity-onset diabetes of the young, type VII 1 KLF11
Gastric cancer, somatic 8 KLF6
Prostate cancer, somatic 2 KLF6
26
Table IX-21781338.1 Hodgkin lymphoma 1 KLHDC8B
Amelogenesis imperfecta, type MAI 1 1<LK4
Fletcher factor deficiency 1 KLKB1
Bladder cancer 3 K AS
Breast cancer, somatic 4 KRAS
Lung cancer 5 KRAS
Pancreatic carcinoma, somatic 1 KRAS
Stomach cancer 1 KRAS
Epidermolytic hyperkeratosis 2 KRT1
Ichthyosis, cyclic, with epidermolytic hyperkeratosis 2 KRT1
Plamoplantar keratoderma, epidermolytic 1 KRT1
Epidermolytic hyperkeratosis 2 KRT10
Ichthyosis with confetti 1 KRT10
Ichthyosis, cyclic, with epidermolytic hyperkeratosis 2 KRT10
Nevus, epidermal, epidermolytic hyperkeratotic type 1 KRT10
Epidermolysis bullosa simplex, Dowling-Meara type 2 KRT14
Epidermolysis bullosa simplex, Koebner type 2 KRT14
Epidermolysis bullosa simplex, recessive 1 KRT14
Epidermolysis bullosa simplex, Weber-Cockayne type 2 KRT14
Palmoplantar keratoderma, nonepidermolytic 1 KRT16
Palmoplantar keratoderma, nonepidermolytic, focal 1 KRT16
Palmoplantar verrucous nevus, unilateral 1 KRT16
Steatocystoma multiplex 1 KRT17
Ichthyosis bullosa of Siemens 1 KRT2
Epidermolysis bullosa simplex, Dowling-Meara type 2 KRT5
Epidermolysis bullosa simplex, Koebner type 2 KRT5
Epidermolysis bullosa simplex, Weber-Cockayne type 2 KRT5
Ectodermal dysplasia, 'pure' hair-nail type 1 KRT85
Ciliary dyskinesia, primary, 10 1 KTU
Corpus callosum, partial agenesis of 1 L1CAM
CRASH syndrome 1 L1CAM
Hydrocephalus due to aqueductal stenosis 1 L1CAM
Hydrocephalus with congenital idiopathic intestinal pseudoobstruction 1 L1CAM
Hydrocephalus with Hirschsprung disease and cleft palate 1 L1CAM
Muscular dystrophy, congenital merosin-deficient 1 LAMA2
Muscular dystrophy, congenital, due to partial LAMA2 deficiency 1 LAMA2
Epidermolysis bullosa, generalized atrophic benign 1 LAMA3
Epidermolysis bullosa, junctional, Herlitz type 3 LAMA3
Laryngoonychocutaneous syndrome 1 LAMA3
Nephrosis, congenital, with or without ocular abnormalities 1 LAMB2
Epidermolysis bullosa, junctional, Herlitz type 3 LAMB3
27
Table IX-21781338.1 Epidermolysis bullosa, junctional, non-Herlitz type 4 LAMB3
Epidermolysis bullosa, junctional, Herlitz type 3 LAMC2
Epidermolysis bullosa, junctional, non-Herlitz type 4 LAMC2
Arrhythmogenic right ventricular dysplasia 5 1 LAMR1
Ellis-van Creveld syndrome 1 LBN
Pelger-Huet anomaly 1 LBR
Norum disease 2 LCAT
Thalassemia, Hispanic gamma-delta-beta 1 LCRB
Lactase deficiency, congenital 1 LCT
Cardiomyopathy, dilated 1C 1 LDB3
Myopathy, myofibrillar, ZASP-related 1 LDB3
Hypercholesterolemia, familial 1 LDLR
Hypercholesterolemia, familial, autosomal recessive 1 LDLRAP1
Osteopoikilosis 1 LEMD3
Spondylocostal dysostosis, autosomal recessive 3 1 LFNG
Epilepsy, partial, with auditory features 1 LGI1
Cryptorchidism, bilateral 2 LGR8
Leydig cell hypoplasia with pseudohermaphroditism 1 LHCGR
Luteinizing hormone resistance, female 1 LHCGR
Deafness, autosomal recessive 67 1 LHFPL5
Pituitary hormone deficiency, combined, 3 1 LHX3
Pituitary hormone deficiency, combined, 4 1 LHX4
Hypotrichosis, localized, autosomal recessive 2 1 LIPH
Acheiropody 1 LMBR1
Polydactyly, preaxial type II 1 LMBR1
Lipase deficiency, combined 1 LMF1
Cardiomyopathy, dilated, 1A 1 LM A
Emery-Dreifuss muscular dystrophy, AD 1 LM A
Emery-Dreifuss muscular dystrophy, A 1 LMNA
Huchinson-Gilford Progeria 1 LMNA
Lipodystrophy, familial partial 1 LMNA
Muscular dystrophy, limb-girdle, type IB 1 LMNA
Leukodystrophy, adult-onset, autosomal dominant 1 LMNB1
Lipodystrophy, partial, acquired 1 LMNB2
Osteosarcoma 3 LOH18CR1
Vohwinkel syndrome with ichthyosis 1 LOR
Cutis laxa, recessive, type 1 1 LOX
Deafness, autosomal recessive 77 1 LOXHD1
Myoglobinuria, acute recurrent, autosomal recessive 1 LPIN1
Combined hyperlipidemia, familial 1 LPL
Lipoprotein lipase deficiency 1 LPL
28
Table IX-21781338.1 Leukemia, acute myeloid 14 LPP
Retinal dystrophy, early-onset severe 1 LRAT
Exudative vitreoretinopathy 4 1 LRP5
Hyperostosis, endosteal 1 LRP5
Osteopetrosis, AD type 1 1 LRP5
Osteoporosis-pseudoglioma syndrome 1 LRP5 van Buchem disease, type 2 1 LRP5
Ciliary dyskinesia, primary, 13 " 1 LRRC50
Agammaglobulinemia 5 1 LRRC8A
Parkinson disease-8 1 LRRK2
Deafness, autosomal recessive 63 1 LRTOMT
Glaucoma 3,' primary congenital, D 1 LTBP2
Tooth agenesis, selective, 6 1 LTBP3
Amyloidosis, renal 1 LYZ
Esophageal squamous cell carcinoma 2 LZTS1
Prostate cancer, somatic 2 MAD1L1
Juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome 1 MADH4
Polyposis, juvenile intestinal 2 MADH4
Pulmonary hypertension, primary 1 MADH9
Mental retardation, X-linked 95 1 MAGT1
Hypospadias 2, X-linked 1 MAMLD1
Mannosidosis, alpha-, types 1 and II 1 MAN2B1
Manhosidosis, beta 1 MANBA
Brunner syndrome 1 MAOA
Lung cancer, somatic 1 MAP3K8
Immunodeficiency due to defect in MAPBP-interacting protein 1 MAPBPIP
Epileptic encephalopathy, Lennox-Gastaut type 1 MAPK10
Dementia, frontotemporal, with or without parkinsonism 1 MAPT
Pick disease 2 MAPT
Supranuclear palsy,- progressive 1 MAPT
Supranuclear palsy, progressive atypical 1 MAPT
Deafness, autosomal recessive 49 1 MARVELD2
Hypermethioninemia, persistent, autosomal dominant, due to
methionine adenosyltransferase l/lll deficiency 1 MAT1A
Methionine adenosyltransferase deficiency, autosomal recessive 1 MAT1A
Epiphyseal dysplasia, multiple, 5 1 MATN3
Spondyloepimetaphyseal dysplasia 1 MATN3
Myopathy, distal 2 1 MATR3
Mental retardation, autosomal dominant 1 1 MBD5
Glucocorticoid deficiency, due to ACTH unresponsiveness 1 MC2R
Obesity, autosomal dominant 1 MC4R
29
Table IX-21781338.1 3-Methylcrotonyl-CoA carboxylase 1 deficiency 1 MCCC1
3-Methylcrotonyl-CoA carboxylase 2 deficiency 1 MCCC2
Methylmalonyl-CoA epimerase deficiency 1 MCEE
Factor V and factor VIII, combined deficiency of 1 MCFD2
Lactase persistance/nonpersistance 1 MCM6
Microcephaly, autosomal recessive 1 1 MCPH1
Premature chromosome condensation with microcephaly and mental
retardation 1 MCPH1
Microcephaly, primary autosomal recessive, 2 1 MCPH2
Microcephaly, primary autosomal recessive, 4 1 MCPH4
Angelman syndrome 1 MECP2
Encephalopathy, neonatal severe 1 MECP2
Mental retardation, X-linked, Lubs type 1 MECP2
Mental retardation, X-linked, syndromic 13 1 MECP2
Rett syndrome, preserved speech variant 1 MECP2
Lujan-Fryns syndrome 1 MED12
Opitz-Kaveggia syndrome 1 MED12
Transposition of the great arteries, dextro-looped 1 1 MED13L
Chromosome 5ql4.3 deletion syndrome 1 MEF2C
Mental retardation, stereotypic movements, epilepsy, and/or cerebral
malformations 1 MEF2C
Hyperparathyroidism, AD 1 MEN1
Spondylocostal dysostosis, autosomal recessive 2 1 MESP2
Hepatocellular carcinoma, childhood type 1 MET
Renal cell carcinoma, papillary, familial and sporadic 1 MET
Charcot-Marie-Tooth disease type 2A2 1 MFN2
Microphthalmia, isolated 5 1 MFRP
Ceroid lipofuscinosis, neuronal, 7 1 MFSD8
Congenital disorder of glycosylation, type IIA 1 MGAT2
Bare lymphocyte syndrome, type II, complementation group A 1 MHC2TA
Opitz G syndrome, type 1 1 MIDI
Thyroid carcinoma, follicular 2 MINPP1
Cataract, polymorphic and lamellar 1 MIP
Mirror-image Polydactyly 1 MIPOL1
Deafness, autosomal dominant 50 1 MIR96
Leukemia, acute myeloid 14 MLF1
Colorectal cancer, hereditary nonpolyposis, type 2 1 MLH1
Mismatch repair cancer syndrome 1 MLH1
Colorectal cancer, somatic ' 6 MLH3
Endometrial cancer 1 MLH3
Methylmalonic aciduria, vitamin B12-responsive 1 MMAA
30
Table IX-21781338.1 Methylmalonic aciduria, vitamin B12-responsive( due to defect in
synthesis of adenosylcobalamin, cblB complementation type 1 MMAB
COPD, rate of decline of lung function in 1 MMP1
Amelogenesis imperfecta, type IIA2 1 MMP20
Meningioma 1 MN1
Molybdenum cofactor deficiency, type A 1 MOCS1
Molybdenum cofactor deficiency, type B 1 MOCS2
Congenital disorder of glycosylation, type If 1 MPDU1
Carbohydrate-deficient glycoprotein syndrome, type lb 1 MPI
Thrombocythemia, essential 3 MPL
Thrombocytopenia, congenital amegakaryocytic 1 MPL
Mitochondrial DNA depletion syndrome, hepatocerebral form 1 MPV17
Charcot-Marie-Tooth disease type 21 1 MPZ
Charcot-Marie-Tooth disease type 2J 1 MPZ
Charcot-Marie-Tooth disease, dominant intermediate 3 1 MPZ
Neuropathy/congenital hypomyelinating 1 MPZ
Paroxysmal nonkinesigenic dyskinesia 1 MR1
Glucocorticoid deficiency 2 1 MRAP
Combined oxidative phosphorylation deficiency 2 1 MRPS16
Combined oxidative phosphorylation deficiency 5 ■1 MRPS22
Immunodeficiency, common variable, 5 1 MS4A1
Colorectal cancer, hereditary nonpolyposis, type 1 1 MSH2
Mismatch repair cancer syndrome 1 MSH2
Endometrial cancer, familial 1 MSH6
Mismatch repair cancer syndrome 1 MSH6
Prostate cancer, hereditary 1 MSR1
Orofacial cleft 5 1 MSX1
Tooth agenesis, selective, 1, with or without orofacial cleft 1 MSX1
Witkop syndrome 1 MSX1
Craniosynostosis, type 2 1 MSX2
Myotubular myopathy, X-linked 1 MTM1
Charcot-Marie-Tooth disease type 4B1 1 MTMR2
Abetalipoproteinemia 1 MTP
Homocystinuria-megaloblastic anemia, cbl E type 1 MTRR
Myasthenic syndrome, congenital, associated with acetylcholine
receptor deficiency 4 MUSK
Adenomas, multiple colorectal 1 MUTYH
Colorectal adenomatous polyposis, autosomal recessive, with
pilomatricomas 1 MUTYH
Gastric cancer, somatic 8 MUTYH
Hyper-lgD syndrome 1 MVK
Cardiomyopathy, familial hypertrophic, 4 1 MYBPC3
31
Table IX-21781338.1 Microcephaly and digital abnormalities with normal intelligence 1 MYCN
Myopathy, centronuclear 2 MYF6
Aortic aneurysm, familial thoracic 4 1 MYH11
Deafness, autosomal dominant 4 1 MYH14
Inclusion body myopathy-3 1 MYH2
Arthrogryposis, distal, type 2A 1 MYH3
Arthrogryposis, distal, type 2B 2 MYH3
Cardiomyopathy, dilated, 1EE 1 MYH6
Cardiomyopathy, familial hypertrophic, 14 1 MYH6
Cardiomyopathy, dilated, IS 1 MYH7
Cardiomyopathy, familial hypertrophic, 1 1 MYH7
Myopathy, myosin storage 1 MYH7
Carney complex variant 1 MYH8
Deafness, autosomal dominant 17 1 MYH9
Epstein syndrome 1 MYH9
Fechtner syndrome 1 MYH9
Macrothrombocytopenia and progressive sensorineural deafness 1 MYH9
May-Hegglin anomaly 1 MYH9
Sebastian syndrome 1 MYH9
Cardiomyopathy, familial hypertrophic, 10 1 MYL2
Cardiomyopathy, familial hypertrophic, 8 1 MYL3
Cardiomyopathy, hypertrophic, midventricular, digenic 1 MYLK2
Deafness, autosomal recessive 3 1 MY015A
Deafness, autosomal dominant 48 1 MYOIA
Deafness, autosomal recessive 30 1 MY03A
Deafness, autosomal dominant 22 1 MY06
Deafness, autosomal recessive 37 1 MY06
Deafness, sensorineural, with hypertrophic cardiomyopathy 1 MY06
Deafness, autosomal dominant 11, neurosensory 1 MY07A
Deafness, autosomal recessive 2, neurosensory 1 MY07A
Glaucoma 1A, primary open angle, juvenile-onset 1 MYOC
Kanzaki disease 1 NAGA
Schindler disease, type 1 1 NAGA
Schindler disease, type III 1 NAGA
N-acetylglutamate synthase deficiency 1 NAGS
Familial cold autoinflammatory syndrome 2 1 NALP12
Hydatidiform mole 1 NALP7
Chronic granulomatous disease due to deficiency of NCF-1 1 NCF1
Chronic granulomatous disease due to deficiency of NCF-2 1 NCF2
Thyroid carcinoma, papillary 6 COA4
Exudative vitreoretinopathy, X-linked 1 NDP
32
Table IX-21781338.1 Mitochondrial complex 1 deficiency 1 NDUFA1
Mitochondrial complex 1 deficiency 1 NDUFA11
Mitochondrial complex 1 deficiency 1 NDUFAF2
Mitochondrial complex 1 deficiency 1 NDUFAF3
Mitochondrial complex 1 deficiency 1 NDUFAF4
Mitochondrial complex 1 deficiency 1 NDUFS1
Mitochondrial complex 1 deficiency 1 NDUFS2
Mitochondrial complex 1 deficiency 1 NDUFS4
Complex 1, mitochondrial respiratory chain, deficiency of 1 NDUFS6
Alexander disease 1 NDUFV1
Mitochondrial complex 1 deficiency 1 NDUFV1
Mitochondrial complex 1 deficiency 1 NDUFV2
Nemaline myopathy 2, autosomal recessive 1 NEB
Charcot-Marie-Tooth disease type 2E 1 NEFL
Hypogonadotropic hypogonadism 6 NELF
Maturity-onset diabetes of the young 6 1 NEU OD1
Diarrhea 4, malabsorptive, congenital 1 NEUROG3
Cardiomyopathy, dilated, ICC 1 NEXN
Leukemia, juvenile myelomonocytic 3 NF1
Neurofibromatosis, familial spinal 1 NF1
Neurofibromatosis-Noonan syndrome 1 NF1
Meningioma, NF2-related, somatic 1 NF2
Schwannomatosis 1 NF2
Ectodermal dysplasia, anhidrotic, with T-cell immunodeficiency 1 NFKBIA
Neuropathy, hereditary sensory and autonomic, type V 1 NGFB
Severe combined immunodeficiency with microcephaly, growth
retardation, and sensitivity to ionizing radiation 1 NHEJ1
Epilepsy, myoclonic, Lafora type 2 NHLRC1
Epilepsy, progressive myoclonic 2B 1 NHLRC1
Cataract, congenital, X-linked 1 NHS
Spastic paraplegia-6 1 NIPA1
Chorea, hereditary benign 3 NKX2-1
Choreoathetosis, hypothyroidism, and neonatal respiratory distress 1 NKX2-1
Persistent truncus arteriosus 1 NKX2-6
Atrial septal defect with atrioventricular conduction defects 1 NKX2E
Hypothyroidism, congenital nongoitrous, 5 1 NKX2E
Mental retardation, X-linked 1 NLGN4
Cold-induced autoinflammatory syndrome, familial 1 NLRP3
Neuroblastoma 7 NME1
Premature ovarian failure 5 1 NOBOX
Heterotaxy, visceral, 5 1 NODAL
33
Table IX-21781338.1 Symphalangism, proximal 2 NOG
Synostoses syndrome, multiple, 1 1 NOG
Alzheimer disease, late-onset, susceptibility to 6 NOS3
Aortic valve disease 1 NOTCH1
Cerebral arteriopathy with subcortical infarcts and
leukoencephalopathy 1 NOTCH3
Nephronophthisis 1, juvenile 1 NPHP1
Nephronophthisis 3 1 NPHP3
Nephronophthisis 4 1 NPHP4
Nephrotic syndrome, type 1 1 NPHS1
Leukemia, acute myeloid 14 NPM1
Atrial fibrillation, familial, 6 1 NPPA
Obesity, mild, early-onset 1 NR0B2
Enhanced S-cone syndrome 1 NR2E3
Hypertension, early-onset, autosomal dominant, with exacerbation in
pregnancy 1 NR3C2
Premature ovarian failure 7 1 NR5A1
Anemia, hypochromic microcytic 1 ■ NRAMP2
Colorectal cancer 6 NRAS
Thyroid carcinoma, follicular 2 NRAS
Leukemia, acute myeloid 14 NSD1
Anemia, hemolytic, due to UMPH1 deficiency 1 NT5C3
Glaucoma 1, open angle, 10 1 NTF4
Insensitivity to pain, congenital, with anhidrosis 1 NTRK1
Medullary thyroid carcinoma, familial 1 NTRK1
Leukemia, acute myeloid 14 NUP214
Striatonigral degeneration, infantile 1 NUP62
Three M syndrome 2 1 OBSL1
Albinism, brown oculocutaneous 2 OCA2
Albinism, oculocutaneous, type II 2 OCA2
Lowe syndrome 1 OCRL
Alpha-ketoglutarate dehydrogenase deficiency 1 OGDH
Renal cell carcinoma, clear cell, somatic 1 OGG1
Optic atrophy 1 1 OPA1
Optic atrophy and deafness 1 OPA1
3-Methylglutaconic aciduria type III 1 OP A3
Optic atrophy and cataract 1 OP A3
Mental retardation, X-linked, with cerebellar hypoplasia and distinctive
facial appearance 1 OPHN1
Blue-cone monochromacy 1 OPN1LW
Colorblindness, deutan 1 OPN1MW
Colorblindness, tritan 1 OPN1SW
34
Table IX-21781338.1 Amytrophic lateral sclerosis 12 1 OPTN
Glaucoma 1, open angle, E 1 OPTN
Immune dysfunction with T-cell inactivation due to calcium entry defect
1 1 ORAI1
Amyloidosis, primary localized cutaneous 1 OSMR
Osteopetrosis, autosomal recessive 5 1 OSTM1
Deafness, autosomal recessive 22 1 OTOA
Auditory neuropathy, autosomal recessive, 1 1 OTOF
Deafness, autosomal recessive 9 1 OTOF
Microphthalmia, syndromic 5 1 OTX2
Bleeding disorder due to P2 Y12 defect 1 P2RY12
Hypotrichosis, localized, autosomal recessive, 3 1 P2RY5
Lissencephaly-1 1 PAFAH1B1
Phenylketonuria 1 PAH
Plasminogen activator inhibitor, type 1 1 PAI1
Mental retardation, X-linked 30 1 PAK3
Fanconi anemia, complementation group N 1 PALB2
HARP syndrome 1 PANK2
Neurodegeneration with brain iron accumulation 1 1 PANK2
SEMD, Pakistani type 1 PAPSS2
Optic nerve coloboma with renal disease 1 PAX2
Rhabdomyosarcoma 2, alveolar 2 PAX3
Diabetes mellitus, ketosis-prone 1 PAX4
Diabetes mellitus, type 2 1 PAX4
Maturity-onset diabetes of the young, type IX 1 PAX4
Aniridia 1 PAX6
Cataract with late-onset corneal dystrophy 1 PAX6
Coloboma of optic nerve 1 PAX6
Coloboma, ocular 2 PAX6
Gillespie syndrome 1 PAX6
Keratitis 1 PAX6
Optic nerve hypoplasia 1 PAX6
Rhabdomyosarcoma 2, alveolar 2 PAX7
Hypothyroidism, congenital, due to thyroid dysgenesis or hypoplasia 1 PAX8
Tooth agenesis, selective, 3 1 PAX9
Hyperphenylalaninemia, BH4-deficient, D 1 PCBD
Deafness, autosomal recessive 23 1 PCDH15
Epilepsy, female-restricted, with mental retardation 1 PCDH19
Thyroid carcinoma, papillary 1 PCM1
Obesity with impaired prohormone processing 1 PCSK1
Hypercholesterolemia, familial, 3 1 PCSK9
35
Table IX-21781338.1 Paget disease of bone 3 PDB4
Cerebral cavernous malformations 3 1 PDCD10
Nephrotic syndrome, type 2 1 PDCN
Pigmented nodular adrenocortical disease, primary, 2 1 PDE11A
Night blindness, congenital stationary, autosomal dominant 2 1 PDE6B
Cone dystrophy 4 1 PDE6C
Retinal cone dystrophy 3 1 PDE6H
Striatal degeneration, autosomal dominant 1 PDE8B
Hypereosinophilic syndrome, idiopathic, resistant to imatinib 1 PDGFRA
Myeloproliferative disorder with eosinophilia 1 PDGFRB
Colorectal cancer 6 PDGFRL
Hepatocellular cancer 1 PDGFRL
Leigh syndrome, X-linked 1 PDHA1
Pyruvate dehydrogenase deficiency 1 PDHA1
Coenzyme Q10 deficiency 6 PDSS1
Coenzyme Q10 deficiency 6 PDSS2
Lacticacidemia due to PDX1 deficiency 1 PDX1
Advanced sleep phase syndrome, familial 1 PER2
Adrenoleukodystrophy, neonatal 5 PEX1
Refsum disease, infantile 1 PEX1
Adrenoleukodystrophy, neonatal 5 PEX10
Adrenoleukodystrophy, neonatal 5 PEX13
Adrenoleukodystrophy, neonatal 5 PEX26
Refsum disease, infantile form 2 PEX26
Adrenoleukodystrophy, neonatal 5 PEX5
Properdin deficiency, X-linked 1 PFC
Phosphoglycerate kinase 1 deficiency 1 PGK1
Spastic paraplegia-7 1 PGN
Progesterone resistance 1 PGR
Hypophosphatemia, X-linked 1 PHEX
Mental retardation syndrome, X-linked, Siderius type 1 PHF8
Phosphoglycerate dehydrogenase deficiency 1 PHGDH
Muscle glycogenosis 1 PHKA1
Phosphorylase kinase deficiency of liver and muscle, autosomal
recessive 1 PHKB
Fibrosis of extraocular muscles, congenital, 2 1 PHOX2A
Leukemia, acute myeloid 14 PICALM
Glycosylphosphatidylinositol deficiency 1 PIGM
Breast cancer, somatic 4 PIK3CA
Colorectal cancer, somatic 6 PIK3CA
Gastric cancer, somatic 8 PIK3CA
36
Table IX-21781338.1 Hepatocellular carcinoma, somatic 3 PIK3CA
Nevus, epidermal 1 PIK3CA
Nonsmall cell lung cancer, somatic 2 PIK3CA
Ovarian cancer, somatic 2 PIK3CA
Corneal fleck dystrophy 1 PIKFYVE
Parkinson disease 6, early onset 1 PINK1
Clubfoot, congenital 1 PITX1
Iridogoniodysgenesis, type 2 1 PITX2
Anterior segment mesenchymal dysgenesis 2 PITX3
Cataract, posterior polar, 4 1 PITX3
Cataract, posterior polar, 4, syndromic 1 PITX3
Deafness, autosomal recessive 59 1 PJVK
Polycystic kidney disease, adult type 1 1 PKD1
Polycystic kidney disease 2 1 PKD2
Adenosine triphosphate, elevated, of erythrocytes 1 PKLR
Pyruvate kinase deficiency 1 PKLR
Ectodermal dysplasia/skin fragility syndrome 1 PKP1
• Arrhythmogenic right ventricular dysplasia, familial, 9 1 PKP2
Dystonia-parkinsonism, adult-onset 1 PLA2G6
Karak syndrome 1 PLA2G6
Neurodegeneration with brain iron accumulation 2B 1 PLA2G6
Adenomas, salivary gland pleomorphic 1 PLAG1
Hyperfibrinolysis, familial, due to increased release of PLAT 1 PLAT
Thrombophilia, familial, due to decreased release of PLAT 1 PLAT
Nephrotic syndrome, type 3 1 PLCE1
Muscular dystrophy with epidermolysis bullosa simplex 1 PLEC1
Spinal muscular atrophy, distal, autosomal recessive, 4 1 PLEKHG5
Osteopetrosis, autosomal recessive 6 1 PLEKHM1
Conjunctivitis, ligneous 1 PLG
Alpha-2-plasmin inhibitor deficiency 1 PLI
Cardiomyopathy, dilated, IP 1 PLN
Nevo syndrome 1 PLOD
Lysyl hydroxylase 3 deficiency 1 PLOD3
Spastic paraplegia-2 1 PLP1
Congenital disorder of glycosylation, type la 1 PMM2
Neuropathy, recurrent, with pressure palsies 1 PMP22
Mismatch repair cancer syndrome 1 PMS2
Central hypoventilation syndrome, congenital 5 PMX2B
Hirschsprung disease, short-segment 1 PMX2B
Microcephaly, seizures, and developmental delaty 1 PNKP
Hypertension, essential 3 PNMT
37
Table IX-21781338.1 Immunodeficiency due to purine nucleoside phosphorylase deficiency 1 PNP
Spastic paraplegia 39 1 PNPLA6
N syndrome 1 POLA
Progressive external ophthalmoplegia, autosomal dominant, with or
without hypogonadism 1 POLG
Progressive external ophthalmoplegia, autosomal recessive 1 POLG
Progressive external ophthalmoplegia with mitochondrial DNA
deletions, autosomal dominant 4 1 POLG 2
Obesity, adrenal insufficiency, and red hair due to POMC deficiency 1 POMC
Adrenal hyperplasia, congenital, due to combined P450C17 and
P450C21 deficiency 1 POR
Antley-Bixler syndrome-like with disordered steroidogenesis 1 POR
Disordered steroidogenesis, isolated 1 POR
POR deficiency 1 POR
Pituitary hormone deficiency, combined, 1 1 POU1F1
Deafness, X-linked 2 1 POU3F4
Deafness, autosomal dominant 15 1 POU4F3
Carotid intimal medial thickness 1 1 PPARG
Insulin resistance, severe, digenic 1 PPARG
Lipodystrophy, familial partial, type 3 1 ■PPARG
Obesity, severe 2 PPARG
Breast cancer 3 PPM1D
Porphyria variegata 1 PPOX
Insulin resistance, severe, digenic 1 PPP1R3A
Lung cancer 5 PPP2R1B
Spinocerebellar ataxia 12 1 PPP2R2B
Ceroid lipofuscinosis, neuronal 1, infantile 1 PPT1
Renpenning syndrome 1 PQBP1
Renal cell carcinoma, papillary, 1 2 PRCC
Hemophagocytic lymphohistiocytosis, familial, 2 1 PRF1
Lymphoma, non-Hodgkin 1 PRF1
Epilepsy, progressive myoclonic IB 1 PRICKLEl
Cardiomyopathy, hypertrophic 6, with WPW 1 PRKAG2
( Wolff-Parkinson-White syndrome 1 PRKAG2
Myxoma, intracardiac 1 PRKAR1A -
Pigmented adrenocortical disease, primary, 1 1 PRKAR1A
Thyroid carcinoma, papillary • 6 PRKAR1A
Spinocerebellar ataxia 14 1 PRKCG
Polycystic liver disease 2 PRKCSH
Adenocarcinoma of lung, somatic 3 PRKN
Adenocarcinoma, ovarian, somatic 1 PRKN
Parkinson disease, juvenile, type 2 1 PRKN
38
Table IX-21781338.1 Creutzfeldt-Jakob disease 1 PRNP
Gerstmann-Straussler disease 1 PRNP
Insomnia, fatal familial 1 PRNP
Prion disease with protracted course 1 PRNP
Thrombophilia due to protein C deficiency, autosomal dominant 1 PROC
Thrombophilia due to protein C deficiency, autosomal recessive 1 PROC
Hypogonadism, hypogonadotropic 1 PROK2
Macular dystrophy, retinal, 2 1 PROM1
Pituitary hormone deficiency, combined, 2 1 PROP1
Thrombophilia due to protein S deficiency 1 PROS1
Choriodal dystrophy, central areolar 2, 1 PRPH2
Foveomacular dystrophy, adult-onset, with choroidal neovascularization 1 PRPH2
Macular dystrophy, patterned ' 1 PRPH2
Macular dystrophy, vitelliform 1 PRPH2
Arts syndrome 1 PRPS1
Charcot-Marie-Tooth disease, X-linked recessive, 5 1 PRPS1
Deafness, X-linked 1 1 PRPS1
Gout, P PS-related 1 PRPS1
Pancreatitis, hereditary 1 PRSS1
Mental retardation, autosomal recessive 1 1 PRSS12
Enterokinase deficiency 1 PRSS7
Combined SAP deficiency 1 PSAP
Gaucher disease, atypical 1 PSAP
Krabbe disease, atypical 1 PSAP
Alzheimer disease, type 3 3 PSEN1
Alzheimer disease, type 3, with spastic paraparesis and apraxia 3 PSEN1
Alzheimer disease, type 3, with spastic paraparesis and unusual plaques 3 PSEN1
Dementia, frontotemporal 1 PSEN1
Pick disease 2 PSEN1
Alzheimer disease-4 1 PSEN2
Pyogenic sterile arthritis, pyoderma gangrenosum, and acne 1 PSTPIP1
Basal cell carcinoma, somatic 3 PTC HI
Basal cell nevus syndrome 1 PTCH1
Holoprosencephaly-7 1 PTCH1
Basal cell carcinoma, somatic 3 PTCH2
Medulloblastoma 1 PTCH2
Cowden disease 1 PTEN
VATER association with macrocephaly and ventriculomegaly 1 PTEN
Diabetes mellitus, permanent neonatal, with cerebellar agenesis 1 PTF1A
Hypertension, essential 3 PTGIS
Hypoparathyroidism, autosomal dominant 1 PTH
39
Table IX-21781338.1 Hypoparathyroidism, autosomal recessive 1 PTH
Failure of tooth eruption, primary 1 PTHR1
Leopard syndrome 1 PTPN11
Leukemia, juvenile myelomonocytic 3 PTPN11
Severe combined immunodeficiency, T cell-negative, B-cell/natural
killer-cell positive 3 PTPRC
Colon cancer, somatic 1 PTPRJ
Deafness, autosomal recessive 84 1 PTPRQ
Lipodystrophy, congenital generalized, type 4 1 PTRF
Hyperphenylalaninemia, BH4-deficient, A 1 PTS
Ectodermal dysplasia-syndactyly syndrome 1 1 PVRL4
Refsum disease, infantile form 2 PXMP3
Cutis laxa, autosomal recessive, type MB 1 PYCR1
Mental retardation, joint hypermobility and skin laxity, with or without
metabolic abnormalities 1 PYCS
McArdle disease 1 PYGM
Hyperphenylalaninemia, BH4-deficient, C 1 QDPR
Mental retardation, X-linked-72 1 RAB39B
Warburg micro syndrome 1 1 RAB3GAP1
Martsolf syndrome 1 RAB3GAP2
Charcot-Marie-Tooth disease type 2B 1 RAB7
Fanconi anemia, complementation group 0 1 RAD51C
Breast cancer, invasive ductal 1 RAD54L
Lymphoma, non-Hodgkin, somatic 1 RAD54L
LEOPARD syndrome 2 1 RAF1
Alpha/beta T-cell lymphopenia with gamma/delta T-cell expansion,
severe cytomegalovirus infection, and autoimmunity 1 RAG1
Combined cellular and humoral immune defects with granulomas 2 RAG1
Severe combined immunodeficiency, B cell-negative 2 RAG1
Combined cellular and humoral immune defects with granulomas 2 RAG 2
Severe combined immunodeficiency, B cell-negative 2 RAG 2
Myasthenic syndrome, congenital, associated with acetylcholine
receptor deficiency 4 RAPSN
Myasthenic syndrome, congenital, associated with facial dysmorphism
and acetylcholine receptor deficiency 1 RAPSN
Leukemia, acute promyelocytic 1 RARA
Basal cell carcinoma, somatic 3 RASA1
Capillary malformation-arteriovenous malformation 1 RASA1
Lung cancer 5 RASSF1
Microphthalmia, isolated 3 1 RAX
Bladder cancer 3 RBI
Osteosarcoma 3 RBI
40
Table IX-21781338.1 Breast cancer, somatic 4 RB1CC1
Cardiomyopathy, dilated, 1DD 1 RBM20
Alopecia, neurologic defects, and endocrinopathy syndrome 1 RBM28
Deafness, autosomal recessive, 24 1 RDX
Bloom syndrome 1 RECQL3
Spastic paraplegia 31 1 REEP1
Hyperuricemic nephropathy, familial juvenile 2 1 REN
Central hypoventilation syndrome, congenital 5 RET
Medullary thyroid carcinoma 1 RET
Multiple endocrine neoplasia IIA 1 RET
Multiple endocrine neoplasia IIB 1 RET
Pheochromocytoma 5 RET
Renal agenesis 1 RET
Congenital disorder of glycosylation, type In 1 RFT1
Bare lymphocyte syndrome, type II, complementation group C 1 RFX5
Bare lymphocyte syndrome, type II, complementation group E 1 RFX5
MHC class II deficiency, complementation group B 1 RFXANK
Bare lymphocyte syndrome, type II, complementation group D 1 RFXAP
Anemia, hemolytic, Rh-null, regulator type 1 RHAG
Night blindness, congenital stationery, autosomal dominant 1 1 RHO
Macrocephaly, alopecia, cutis laxa, and scoliosis 1 RIN2
Newfoundland. rod-cone dystrophy 1 RLBP1
Rippling muscle disease-1 1 RMD1
Anauxetic dysplasia 1 RMRP
Metaphyseal dysplasia without hypotrichosis 1 RMRP
Prostate cancer 1, 176807 1 RNASEL
Leukoencephalopathy, cystic, without megalencephaly 1 RNASET2
Renal cell carcinoma 2 RNF139
RIDDLE syndrome 1 RNF168
Recombination rate QTL 1 1 RNF212
Esophageal carcinoma, somatic 1 RNF6
Vesicoureteral reflux 2 1 ROB02
Gaze palsy, horizontal, with progressive scoliosis 1 ROB03
Robinow syndrome, autosomal recessive 1 ROR2 .
COACH syndrome 3 RPGRIP1L
Ribose-5-phosphate isomerase deficiency 1 RPIA
Diamond-Blackfan anemia 7 1 RPL11
Diamond-Blackfan anemia 5 1 RPL35A
Diamond-Blackfan anemia 6 1 RPL5
Diamond-Blackfan anemia 9 1 RPS10
Macrocytic anemia, refractory, due to 5q deletion, somatic 1 RPS14
41
Table IX-21781338.1 Diamond-Blackfan anemia 4 1 RPS17
Diamond-Blackfan anemia 1 1 RPS19
Diamond-blackfan anemia 1 RP524
Diamond-Blackfan anemia 10 1 RPS26
Diamond-Blackfan anemia 8 1 RPS7
Mitochondrial DNA depletion syndrome, encephalomyopathic form,
with renal tubulopathy 1 RRM2B
Progressive external ophthalmoplegia with mitochondrial DNA
deletions, autosomal dominant, 5 1 RRM2B
Ciliary dyskinesia, primary, 11 1 RSPH4A
Ciliary dyskinesia, primary, 12 1 RSPH9
Palmoplantar hyperkeratosis and true hermaphroditism 1 RSPOl
Anonychia congenita 1 RSP04
Leukemia, acute myeloid 14 RUNX1
Platelet disorder, familial, with associated myeloid malignancy 1 RUNX1
Minicore myopathy with external ophthalmoplegia 1 RYR1
Neuromuscular disease, congenital, with uniform type 1 fiber 1 RYR1
Arrhythmogenic right ventricular dysplasia 2 1 RYR2
Ventricular tachycardia, catecholaminergic polymorphic, 1 1 RYR2
Spastic ataxia, Charlevoix-Saguenay type 1 SACS
Tumoral calcinosis, familial, normophosphatemic 1 SAMD9
Porokeratosis, disseminated superficial actinic, 1 1 SART3
Cleft palate and mental retardation 1 SATB2
Epilepsy, generalized, with febrile seizures plus, type 2 1 SCN1A
Epilepsy, severe myoclonic, of infancy 1 SCN1A
Febrile convulsions, familial, 3A 1 SCN1A
Migraine, familial hemiplegic, 3 1 SCN1A
Brugada syndrome 5 1 SCN1B
Cardiac conduction defect, nonspecific 1 SCN1B
Generalized epilepsy with febrile seizures plus 1 SCN1B
Convulsions, benign familial infantile, 3 1 SCN2A1
Brugada syndrome 7 1 SCN3B
Myotonia congenita, atypical, acetazolamide-responsive 1 SCN4A
Paramyotonia congenita 1 SCN4A
Long QT syndrome-10 1 SCN4B
Brugada syndrome 1 1 SCN5A
Cardiomyopathy, dilated, IE 1 SCN5A
Heart block, nonprogressive 1 SCN5A
Heart block, progressive, type IA 1 SCN5A
Long QT syndrome-3 1 SCN5A
Ventricular fibrillation, familial, 1 1 SCN5A
42
Table IX-21781338.1 Erythermalgia, primary 1 SCN9A
Febrile convulsions, familial, 3B 1 SCN9A
Insensitivity to pain, channelopathy-associated 1 SCN9A
Bronchiectasis with or without elevated sweat chloride 2 1 SCNN1A
Bronchiectasis with or without elevated sweat chloride 1 1 SCNN1B
Bronchiectasis with or without elevated sweat chloride 3 1 SCNN1G
Cardioencephalomyopathy, fatal infantile, due to cytochrome c oxidase
deficiency 1 SC02
Geroderma osteodysplasticum 1 SCYL1BP1
Cardiomyopathy, dilated, 1GG 1 SDHA
Mitochondrial respiratory chain complex II deficiency 1 SDHA
Mitochondrial complex II deficiency 1 SDHAF1
Paragangliomas 2 1 SDHAF2
Cowden-like syndrome 2 SDHB
Paraganglioma and gastric stromal sarcoma 3 SDHB
Paraganglioma, familial chromaffin, 4 1 SDHB
Pheochromocytoma 5 SDHB
Paraganglioma and gastric stromal sarcoma 3 SDHC
Paragangliomas, familial nonchromaffin, 3 1 SDHC
Cowden-like syndrome 2 SDHD
Paraganglioma and gastric stromal sarcoma 3 SDHD
Paragangliomas, familial nonchromaffin, 1, with or without deafness 1 SDHD
Pheochromocytoma 5 SDHD
Anemia, dyserythropoietic congenital, type II 1 SEC23B
Polycystic liver disease 2 SEC63
Thyroid hormone metabolism, abnormal 1 SECISBP2
Muscular dystrophy, rigid spine, 1 1 SEPN1
Emphysema due to AAT deficiency 1 SERPINA1
Emphysema-cirrhosis, due to AAT deficiency 1 SERPINA1
Deafness, autosomal recessive 91 1 SERPINB6
Encephalopathy, familial, with neuroserpin inclusion bodies 1 SERPINI1
Ataxia-ocular apraxia-2 1 SETX
Pulmonary fibrosis, idiopathic 1 SFTPA2
Surfactant metabolism dysfunction, pulmonary, 1 1 SFTPB
Surfactant metabolism dysfunction, pulmonary, 2 1 SFTPC
Muscular dystrophy, limb-girdle, type 2D 1 SGCA
Muscular dystrophy, limb-girdle, type 2E 1 SGCB
Cardiomyopathy, dilated, 1L 1 SGCD
Muscular dystrophy, limb-girdle, type 2F 1 SGCD
Dystonia-11, myoclonic 1 SGCE
Muscular dystrophy, limb-girdle, type 2C 1 SGCG
43
Table IX-21781338.1 Lymphoproliferative syndrome, X-linked 1 SH2D1A
Leukemia, acute myeloid 14 SH3GL1
Mononeuropathy of the median nerve, mild 1 SH3TC2
Chromosome 22ql3.3 deletion syndrome 1 SHANK3
Coloboma, ocular 2 SHH
Holoprosencephaly-3 1 SHH
Microphthalmia, isolated, with coloboma 5 1 SHH
Leri-Weill dyschondrosteosis 2 SHOX
Short stature, idiopathic familial 2 SHOX
Langer mesomelic dysplasia 2 SHOXY
Leri-Weill dyschondrosteosis 2 SHOXY
Short stature, idiopathic familial 2 SHOXY
Stocco dos Santos X-linked mental retardation syndrome 1 SHROOM4
Amish infantile epilepsy syndrome 1 SIAT9
Obesity, severe 2 SIM1
Brachiootic syndrome 3 1 SIX1
Deafness, autosomal dominant 23 1 SIX1
Holoprosencephaly-2 1 SIX3
Branchiootorenal syndrome 2 1 SIX5
Microphthalmia, isolated, with cataract 2 1 SIX6
Bile acid malabsorption, primary 1 SLC10A2
Bartter syndrome, type 1 1 SLC12A1
Agenesis of the corpus callosum with peripheral neuropathy 1 SLC12A6
Erythrocyte lactate transporter defect 1 SLC16A1
Hyperinsulinemic hypoglycemia, familial, 7 1 SLC16A1
Cataract, juvenile, with microcornea and glucosuria 1 SLC16A12
Sialic acid storage disorder, infantile 1 SLC17A5
Deafness, autosomal dominant 25 1 SLC17A8
Basal ganglia disease, biotin-responsive 1 SLC19A3
Dicarboxylicaminoaciduria 1 SLC1A1
Episodic ataxia, type 6 1 S LCI A3
Hypouricemia, renal 1 SLC22A12
Breast cancer 3 SLC22A1L
Lung cancer 5 SLC22A1L
Rhabdomyosarcoma 1 SLC22A1L
Carnitine deficiency, systemic primary 1 SLC22A5
Hypomyelination, global cerebral 1 SLC25A12
Microcephaly, Amish type 1 SLC25A19
Epilepsy, neonatal myoclonic, with suppression-burst pattern 1 SLC25A22
Micochondrial phosphate carrier deficiency 1 SLC25A3
Anemia, sideroblastic, pyridoxine-refractory, autosomal recessive 1 SLC25A38
44
Table IX-21781338.1 Cardiomyopathy, familial hypertrophic 2 SLC25A4
Progressive external ophthalmoplegia with mitochondrial DNA deletions
3 1 SLC25A4
Achondrogenesis lb 1 SLC26A2
De la Chapelle dysplasia 1 SLC26A2
Diastrophic dysplasia, broad bone-platyspondylic variant 1 SLC26A2
Epiphyseal dysplasia, multiple, 4 1 SLC26A2
Chloride diarrhea, congenital, Finnish type 1 SLC26A3
Enlarged vestibular aqueduct 2 SLC26A4
Hyperpigmentation, cutaneous, with hypertrichosis,
hepatosplenomegaly, heart anomalies, hearing loss, and hypogonadism 1 SLC29A3
GLUT1 deficiency syndrome 1 1 SLC2A1
GLUT1 deficiency syndrome 2 1 SLC2A1
Fanconi-Bickel syndrome 1 SLC2A2
Hypouricemia, renal, 2 1 SLC2A9
Spastic paraplegia-42 1 SLC33A1
Fanconi renotubular syndrome 2 1 SLC34A1
Nephrolithiasis/osteoporosis, hypophosphatemia 1 1 SLC34A1
Pulmonary alveolar microlithiasis 1 SLC34A2
Testicular microlithiasis 1 SLC34A2
Hypophosphatemic rickets with hypercalciuria 1 SLC34A3
Congenital disorder of glycosylation, type llf 1 SLC35A1
Congenital disorder of glycosylation type lie 1 SLC35C1
Hyperglycinuria 3 SLC36A2
Spondylocheirodysplasia, Ehlers-Danlos syndrome-like 1 SLC39A13
Folate malabsorption, hereditary 1 SLC46A1
Renal tubular acidosis, distal, AD 1 SLC4A1
Renal tubular acidosis, distal, AR 1 SLC4A1
Spherocytosis, type 4 1 SLC4A1
Corneal dystrophy, Fuchs endothelial, 4 1 SLC4A11
Corneal endothelial dystrophy 2 1 SLC4A11
Corneal endothelial dystrophy and perceptive deafness 1 SLC4A11
Renal tubular acidosis, proximal, with ocular abnormalities 1 SLC4A4
Thyroid dyshormonogenesis 1 1 SLC5A5
Hyperglycinuria 3 SLC6A19
Orthostatic intolerance 1 SLC6A2
Hyperglycinuria 3 SLC6A20
Parkinsonism-dystonia, infantile 1 SLC6A3
Anxiety-related personality traits 1 SLC6A4
Hyperekplexia 2 SLC6A5
Creatine deficiency syndrome, X-linked 1 SLC6A8
AS
Table IX-21781338.1 Nephrolithiasis/osteoporosis, hypophosphatemia 2 1 SLC9A3R1
Mental retardation, X-linked syndromic, Christianson type 1 SLC9A6
Tourette syndrome 1 SLITRK1
Trichotillomania 1 SLITRK1
Meleda disease 1 SLURP1
Rhabdoid tumor predisposition syndrome 2 1 SMARCA4
Rhabdoid predisposition syndrome 1 1 SMARCB1
Spinal muscular atrophy-1 1 SMN1
Spinal muscular atrophy-2 1 SMN1
Spinal muscular atrophy-3 1 SMN1
Spinal muscular atrophy-4 1 SMN1
Mental retardation, X-linked, Snyder-Robinson type 1 SMS
Cerebral dysgenesis, neuropathy, ichthyosis, and palmoplantar
keratoderma syndrome 1 SNAP29
Dementia, Lewy body 1 SNCA
Parkinson disease 4 1 SNCA
Dementia, Lewy body 1 SNCB
Long QT syndrome 12 1 SNT1
Fibromatosis, gingival 1 SOS1
Van Buchem disease 1 SOST
PCWH syndrome 1 SOX10
Hypotrichosis-lymphedema-telangiectasia syndrome 1 SOX 18
Microphthalmia, syndromic 3 1 SOX2
Optic nerve hypoplasia and abnormalities of the central nervous system 1 SOX2
Mental retardation, X-linked, with isolated growth hormone deficiency 1 SOX3
Acampomelic campomelic dysplasia 1 • SOX9
Campomelic dysplasia with autosomal sex reversal 1 SOX9
Spastic paraplegia-4 1 S AST
Globozoospermia 2 SPATA16
Spastic paraplegia-11 1 SPG11
Troyer syndrome 1 SPG20
Spastic paraplegia-3A 1 SPG3A
Pancreatitis, hereditary 1 SPINK1
Tropical calcific pancreatitis 1 SPINK1
Atopy 1 SPINK5
Diarrhea 3, secretory sodium, congenital, syndromic 1 SPINT2
Dystonia, dopa-responsive, due to sepiapterin reductase deficiency 1 SPR
Legius syndrome 1 SPRED1
Elliptocytosis-2 1 SPTA1
Pyropoikilocytosis 1 SPTA1
Spherocytosis, type 3 1 SPTA1
16
Table IX-21781338.1 Epileptic encephalopathy, early infantile, 5 1 SPTAN1
Neuropathy, hereditary sensory and autonomic, type 1 1 SPTLC1
Neuropathy, hereditary sensory/autonomic, type IC 1 SPTLC2
Paget disease of bone 3 SQSTM1
Pseudovaginal perineoscrotal hypospadias 1 SRD5A2
Congenital disorder of glycosylation, type Ip 1 SRD5A3
Somatostatin analog, resistance to 1 SSTR5
Ichthyosis with hypotrichosis 1 ST14
Lipoid adrenal hyperplasia 1 STAR
Mycobacterial infection, atypical, familial disseminated 2 STAT1
Hyper-lgE recurrent infection syndrome 1 STAT3
Growth hormone insensitivity with immunodeficiency 1 STAT5B
Microcephaly, primary autosomal recessive, 7 1 STIL
Immune dysfunction, with T-cell inactivation due to calcium entry
defect 2 1 STIM1 ·
Testicular tumor, sporadic 1 STK11
Male infertility with large-headed, multiflagellar, polyploid spermatozoa 1 STK13
Microphthalmia, syndromic 9 1 STRA6
Polyhydramnios, megalencephaly, and symptomatic epilepsy 1 STRADA
Deafness, autosomal recessive 16 1 STRC
Ichthyosis, x-linked 1 STS
Hemophagocytic lymphohistiocytosis, familial, 4 1 STX11
Epileptic encephalopathy, early infantile, 4 1 STXBP1
Mitochondrial DNA depletion syndrome, encephalomyopathic form,
with methylmalonic aciduria 1 SUCLA2
Lactic acidosis, fatal infantile 1 SUCLG1
Azoospermia due to perturbations of meiosis 1 SYCP3
Epilepsy, X-linked, with variable learning disabilities and behavior
disorders 1 SYN1
Emery-Dreifuss muscular dystrophy 4 1 SYNE1
Emery-Dreifuss muscular dystrophy 5 1 SYNE2
Mental retardation, autosomal dominant 5 1 SYNGAP
Mental retardation, X-linked, with or without epilepsy 1 SYP
Congenital heart disease, nonsyndromic, 2 1 TAB2
Hypogonadotropic hypogonadism 6 TAC3
Hypogonadotropic hypogonadism 6 TACR3
Corneal dystrophy, gelatinous drop-like 1 TACSTD2
Dystonia-Parkinsonism, X-linked 1 TAF1
Chondrosarcoma, extraskeletal myxoid 3 TAF15
Bare lymphocyte syndrome, type 1 2 TAP1
Bare lymphocyte syndrome, type 1, due to TAP2 deficiency 1 TAP2
Bare lymphocyte syndrome, type 1 2 TAPBP
47
Table IX-21781338.1 Frontotemporal lobar degeneration, TARDBP-related 1 TARDBP
Barth syndrome 1 TAZ
Cardiomyopathy, dilated, 3A 1 TAZ
Hypoparathyroidism-retardation-dysmorphism syndrome 1 TBCE
Kenny-Caffey syndrome-1 1 TBCE
Spinocerebellar ataxia 17 1 TBP
Adrenocorticotropic hormone deficiency 1 TBS19
Conotruncal anomaly face syndrome 1 TBX1
DiGeorge syndrome 1 TBX1
Velocardiofacial syndrome 1 TBX1
Cousin syndrome 1 TBX15
Asthma and nasal polyps 1 TBX21
Cleft palate with ankyloglossia 1 TBX22
Small patella syndrome 1 TBX4
Cardiomyopathy, dilated, IN 1 TCAP
Muscular dystrophy, limb-girdle, type 2G 1 TCAP
Osteopetrosis, recessive 1 1 TCIRG1
Treacher Collins mandibulofacial dysostosis 1 TCOF1
Sveinsson choreoretinal atrophy 1 TEAD1
Deafness, autosomal dominant 8/12 1 TECTA
Deafness, autosomal recessive 21 1 TECTA
Venous malformations, multiple cutaneous and mucosal 1 TEK
Aplastic anemia 1 TERC
Atransferrinemia · 1 TF
Renal cell carcinoma, papillary, 1 2 TFE3
Chondrosarcoma, extraskeletal myxoid 3 TFG
Thyroid dyshormonogenesis 3 1 TG
Arrhythmogenic right ventricular dysplasia 1 1 TGFB3
Corneal dystrophy, Avellino type 1 TGFBI
Corneal dystrophy, epithelial basement membrane 1 TGFBI
Corneal dystrophy, Groenouw type 1 1 TGFBI
Corneal dystrophy, lattice type 1 1 TGFBI
Corneal dystrophy, lattice type IMA 1 TGFBI
Corneal dystrophy, Reis-Bucklers type 1 TGFBI
Corneal dystrophy, Thiel-Behnke type 1 TGFBI
Esophageal cancer, somatic 1 TGFBR2
Holoprosencephaly-4 1 TGIF lchthyosiform erythroderma, congenital 1 TGM1
Ichthyosis, lamellar, autosomal recessive 1 TGM1
Peeling skin syndrome, acral type 1 TGM6
Segawa syndrome, recessive 1 TH
48
Table IX-21781338.1 Dystonia 6, torsion 1 THAP1
Thrombocythemia, essential 3 THPO
Thyroid hormone resistance 1 TH B
Thyroid hormone resistance, autosomal recessive . 1 THRB
Thyroid hormone resistance, selective pituitary 1 THRB
Jensen syndrome 1 TIMM8A
Revesz syndrome 1 TINF2
Hypercholanemia, familial 3 TJP2
Mental retardation, X-linked 58 1 TM4SF2
Deafness, autosomal dominant 36 1 TMC1
Deafness, autosomal recessive 7 1 TMC1
Optic atrophy-7 1 TMEM126A
Congenital disorder of glycosylation, type Im 1 TMEM15
Arrhythmogenic right ventricular dysplasia, familial, 5 1 TMEM43
COACH syndrome 3 TMEM67
Encephalocardiomyopathy, neonatal, mitochondrial, due to ATP
synthase deficiency 1 TMEM70
Deafness, autosomal recessive 6 1 TMIE
Deafness, autosomal recessive 10, congenital 1 TMPRSS3
Deafness, autosomal recessive 8, childhood onset 1 TMPRSS3
Iron-refractory iron deficiency anemia 1 TMPRSS6
Osteolysis, familial expansile 1 TNFRSF11A
Osteopetrosis, autosomal recessive 7 1 TNFRSFllA
Paget disease of bone 3 TNFRSF11A
Paget disease, juvenile 1 TNFRSF11B
Immunodeficiency, common variable, 2 1 TNFRSF13B
Immunoglobulin A deficiency 2 1 TNFRSF13B
Immunodeficiency, common variable, 4 1 TNFRSF13C
Periodic fever, familial 1 TNFRSF1A
Immunodeficiency with hyper-lgM, type 3 1 TNFRSF5
Autoimmune lymphoproliferative syndrome, type IA 1 TNFRSF6
Osteopetrosis, autosomal recessive 2 1 TNFSF11
Immunodeficiency, X-linked, with hyper-lgM 1 TNFSF5
Cardiomyopathy, dilated, 1Z 1 TNNC1
Cardiomyopathy, familial hypertrophic, 13 1 TNNC1
Arthrogryposis multiplex congenita, distal type 2B 1 TNNI2
Cardiomyopathy, dilated, IFF 1 TNNI3
Cardiomyopathy, dilated, 2A 1 TNNI3
Cardiomyopathy, familial restrictive 1 TNNI3
Nemaline Myopathy, Amish Type 1 TNNT1
Cardiomyopathy, dilated, ID 1 TNNT2
49
Table IX-21781338.1 Cardiomyopathy, familial hypertrophic, 2 1 TNNT2
Cardiomyopathy, familial restrictive, 3 1 TNNT2
Arthyrgryposis, distal, type 2B 1 T NT3
Adrenal cortical carcinoma 1 TP53
Breast cancer 3 TP53
Choroid plexus papilloma 1 TP53
Colorectal cancer 6 TP53
Hepatocellular carcinoma 2 TP53
Osteosarcoma 3 TP53
Pancreatic cancer 2 TP53
ADULT syndrome 1 TP63
Ectrodactyly, ectodermal dysplasia, and cleft lip/palate syndrome 3 1 TP63
Hay-Wells syndrome 1 TP63
Orofacial cleft 8 1 TP63
Cardiomyopathy, dilated, 1Y 1 TPM1
Cardiomyopathy, familial hypertrophic, 3 1 TPM1
Arthrogryposis multiplex congenita, distal type 1 1 TPM2
Arthrogryposis, distal, type 2B 2 TPM2
Nemaline myopathy 1 TPM2
Nemaline myopathy 1, autosomal dominant 1 TPM3
6-mercaptopurine sensitivity 1 T MT
Thyroid dyshormonogenesis 2A 1 TPO
Ceroid-lipofuscinosis, neuronal 2, classic late infantile 1 TPP1
Deafness, autosomal recessive 79 1 TP N
Mental retardation, autosomal recessive 13 1 TRAPPC9
Trehalase deficiency 1 TREH
Nasu-Hakola disease 2 TREM2
Vasculopathy, retinal, with cerebral leukodystrophy 1 TREX1
Thyroid carcinoma, papillary 6 TRIM24
Muscular dystrophy, limb-girdle, type 2H 1 TRIM32
Thyroid carcinoma, papillary 6 TRIM33
Deafness, autosomal recessive 28 1 TRIOBP
Liver failure, acute infantile 1 TRMU
Glomerulosclerosis, focal segmental, 2 1 TRPC6
Night blindness, congenital stationary, type IC 1 TRPM1
Progressive familial heart block, type IB 1 TRPM4
Hypomagnesemia with secondary hypocalcemia 1 TRPM6
Hereditary motor and sensory neuropathy, type lie 1 TRPV4
Focal cortical dysplasia, Taylor balloon cell type 1 TSC1
Combined oxidative phosphorylation deficiency 3 1 TSFM
Hypothryoidism, congenital, nongoitrous 4 1 TSHB
50
Table IX-21781338.1 Hyperthyroidism, familial gestational 1 TSHR
Hyperthyroidism, nonautoimmune 1 TSHR
Hypothyroidism, congenital, nongoitrous 1 TSHR
Exudative vitreoretinopathy 5 1 TSPAN12
Muscular dystrophy, limb-girdle, type 1A . 1 TTID
Myopathy, spheroid body 1 TTID
Myotilinopathy 1 TTID
Cardiomyopathy, dilated, 1G 1 TTN
Muscular dystrophy, limb-girdle, type 2J 1 TTN
Myopathy, early-onset, with fatal cardiomyopathy 1 TTN
Myopathy, proximal, with early respiratory muscle involvement 1 TTN
Amyloidosis, hereditary, transthyretin-related 1 TTR
Carpal tunnel syndrome, familial 1 TTR
Lissencephaly 3 1 TUBA1A
Macrothrombocytopenia, autosomal dominant, TUBBl-related 1 TUBB1
Fibrosis of extraocular muscles, congenital, 3A 1 TUBB3
Combined oxidative phosphorylation deficiency 4 1 TUFM
Mental retardation, autosomal recessive 7 1 TUSC3
Craniosynostosis, type 1 1 TWIST1
Saethre-Chotzen syndrome 2 TWIST1
Saethre-Chotzen syndrome with eyelid anomalies 1 TWISTl
Ciliary dyskinesia, primary, 6 1 TXNDC3
Tyrosine kinase 2 deficiency 1 TYK2
Albinism, oculocutaneous, type IA 1 TYR
Albinism, oculocutaneous, type IB 1 TYR
Nasu-Hakola disease 2 TYROBP
Albinism, brown 1 TYRP1
Albinism, rufous 1 TYRP1
Cleft palate, isolated 1 UBB
Spinal muscular atrophy, X-linked 2, infantile 1 UBE1
Corneal dystrophy, crystalline, of Schnyder 1 UBIAD1
Hyperbilirubinemia, familial transcient neonatal 1 UGT1A1
Glomerulocystic kidney disease with hyperuricemia and isosthenuria 1 UMOD
Hyperuricemic nephropathy, familial juvenile 1 1 UMOD
Medullary cystic kidney disease 2 1 UMOD
Hemophagocytic lymphohistiocytosis, familial, 3 1 UNC13D
Immunodeficiency with hyper IgM, type 4 1 UNG
Mental retardation, X-linked, syndromic 14 1 UPF3B
Renal adysplasia 1 UPK3A
Mitochondrial complex III deficiency 1 UQCRB
Mitochondrial complex III deficiency 1 UQCRQ
51
Table IX-21781338.1 Porphyria, hepatoerythropoietic 1 UROD
Porphyria, congenital erythropoietic 1 UROS
Deafness, autosomal recessive 18 1 USH1C
Azoospermia 1 USP9Y
Caudal regression syndrome 1 VANGL1
Neural tube defects 1 VANGL1
Spinal muscular atrophy, late-onset, Finkel type 1 VAPB
Cardiomyopathy, dilated, 1W 1 VCL
Cardiomyopathy, familial hypertrophic, 15 1 VCL
Osteoporosis, involutional 1 VDR
Rickets, vitamin D-resistant, type HA 1 VDR
Pheochromocytoma 5 VHL
Polycythemia, benign familial 1 VHL
Renal cell carcinoma, somatic 1 VHL
Arthrogryposis, renal dysfunction, and cholestasis 2 1 VIPAR
Vitamin K-dependent clotting factors, combined deficiency of, 2 1 VKORC1
Warfarin resistance 1 VKORC1
Cerebellar hypoplasia and mental retardation with or without
quadrupedal locomotion 1 1 VLDLR
Arthrogryposis, renal dysfunction, and cholestasis 1 1 VPS33B
Corneal dystrophy, hereditary polymorphous posterior 1 VSX1
Keratoconus 1 VSX1
Neutropenia, severe congenital, X-linked 1 WAS
Thrombocytopenia, X-linked 1 WAS
Thrombocytopenia, X-linked, intermittent 1 WAS
Glaucoma 1, open angle, G 1 WDR36
Amelogenesis imperfecta, hypomaturation type, IIA3 1 WDR72
Hearing loss, low-frequency sensorineural 1 WFS1
Wolfram syndrome 1 WFS1
Wolfram-like syndrome, autosomal dominant 1 WFS1
Deafness, autosomal recessive 31 1 WHRN
Leukemia, acute myeloid 14 WHSC1L1
Arthropathy, progressive pseudorheumatoid, of childhood 1 WISP3
Spondyloepiphyseal dysplasia tarda with progressive arthropathy 1 WISP3
Neuropathy, hereditary sensory and autonomic, type II 1 WNK1
Mullerian aplasia and hyperandrogenism 1 WNT4
Ulna and fibula, absence of, with sever limb deficiency 1 WNT7A
Nephrotic syndrome, type 4 1 WT1
Wilms tumor, type 1 1 WT1
Esophageal squamous cell carcinoma 2 WWOX
Xanthinuria, type 1 1 XDH
52
Table IX-21781338.1 X-inactivation, familial skewed 1 XIC
Charcot-Marie-Tooth disease, dominant intermediate C 1 YARS
Skeletal defects, genital hypoplasia, and mental retardation 1 ZBTB16
Mental retardation, X-linked-91 1 ZDHHC15
Mental retardation, X-linked, ZDHHC9-related 1 ZDHHC9
Corneal dystrophy, Fuchs endothelial, 6 1 ZEB1
Corneal dystrophy, posterior polymorphous, 3 1 ZEB1
Mowat-Wilson syndrome 1 ZEB2
Ptosis, congenital 1 ZFHX4
Diabetes mellitus, transient neonatal, 1 1 ZFP57
Diaphragmatic hernia 3 1 ZFPM2
Spastic paraplegia 15 1 ZFYVE26
Spastic paraplegia 33 1 ZFYVE27
Dandy-Walker malformation 1 ZIC1
Holoprosencephaly-5 1 ZIC2
Congenital heart defects, nonsyndromic, 1, X-linked 1 ZIC3
Heterotaxy, visceral, 1, S-linke 1 ZIC3
Dandy-Walker malformation 1 ZIC4
Restrictive dermopathy, lethal 1 ZMPSTE24
Sertoli-cell-only syndrome 1 ZNF148
Mental retardation, X-linked 45 1 ZNF81
Myotonic dystrophy, type 2 1 ZNF9
Triple A syndrome 1 AAAS
Punctate palmoplanar keratoderma type 1 2 AAGAB
Autosomal dominant Charcot-Marie-Tooth disease type 2N 1 AARS
Combined oxidative phosphorylation defect type 8 1 AARS2
Hyperlysinemia 1 AASS
Saccharopinuria 1 AASS
Gamma aminobutyric acid transaminase deficiency 1 ABAT
Apolipoprotein A-l deficiency 2 ABCA1
Tangier disease 1 ABCA1
Congenital nonbullous ichthyosiform erythroderma 6 ABCA12
Harlequin ichthyosis 1 ABCA12
Lamellar ichthyosis 6 ABCA12
Congenital pulmonary alveolar proteinosis 5 ABCA3
Neonatal acute respiratory distress with surfactant metabolism deficiency 2 ABCA3
Retinitis pigmentosa 61 ABCA4
Stargardt disease 3 ABCA4
Benign recurrent intrahepatic cholestasis type 2 1 ABCB11
Intrahepatic cholestasis of pregnancy 4 ABCB11
Progressive familial intrahepatic cholestasis type 2 1 ABCB11
53
Table IX-21781338.1 Intrahepatic cholestasis of pregnancy 4 ABCB4
Low phospholipid associated cholelithiasis 1 ABCB4
Progressive familial intrahepatic cholestasis type 3 1 ABCB4
Colobomatous microphthalmia 8 ABCB6
X-linked sideroblastic anemia - ' ataxia 1 ABCB7
Dubin-Johnson syndrome 1 ABCC2
Generalized arterial calcification of infancy 2 ABCC6
Pseudoxanthoma elasticum 1 ABCC6
Autosomal dominant hyperinsulinism due to SURl deficiency 1 ABCC8
Autosomal recessive hyperinsulinism due to SURl deficiency 1 ABCC8
MODY syndrome 12 ABCC8
Permanent neonatal diabetes mellitus 5 ABCC8
Transient neonatal diabetes mellitus . 5 ABCC8
Acromegaloid facial appearance syndrome 1 ABCC9
Cantu syndrome 1 ABCC9
Familial atrial fibrillation 14 ABCC9
Familial isolated dilated cardiomyopathy 38 ABCC9
Hypertrichosis with acromegaloid facial appearence 1 ABCC9
Adrenomyeloneuropathy 1 ABCD1
X-linked cerebral adrenoleukodystrophy 1 ABCD1
Methylmalonic acidemia with homocystinuria, type cblF 2 ABCD4
Sitosterolemia 2 ABCG5
Sitosterolemia 2 ABCG8
Polyneuropathy - hearing loss - ataxia - retinitis pigmentosa - cataract 1 ABHD12
Dorfman-Chanarin disease 1 ABHD5
Chronic myeloid leukemia 3 ABL1
Precursor B-cell acute lymphoblastic leukemia 13 ABL1
Precursor T-cell acute lymphoblastic leukemia 19 ABL1
Isobutyryl-CoA dehydrogenase deficiency 1 ACAD8
Acyl-CoA dehydrogenase 9 deficiency 1 ACAD9
Isolated NADH-CoQ reductase deficiency 25 ACAD9
Long chain acyl-CoA dehydrogenase deficiency 1 ACADL
Medium chain acyl-CoA dehydrogenase deficiency 1 ACADM
Short chain acyl-CoA dehydrogenase deficiency 1 ACADS
2-methylbutyryl-CoA dehydrogenase deficiency 1 ACADSB
Very long chain acyl-CoA dehydrogenase deficiency 1 ACADVL
Familial osteochondritis dissecans 1 ACAN
Spondyloepimetaphyseal dysplasia, aggrecan type 1 ACAN
Spondyloepiphyseal dysplasia, Kimberley type 1 ACAN
Ketoacidosis due to betaketothiolase deficiency 1 ACAT1
Renal tubular dysgenesis of genetic origin 4 ACE
54
Table IX-21781338.1 Infantile cerebellar-retinal degeneration 1 AC02
Peroxisomal acyl-CoA oxidase deficiency 1 ACOX1
Acid phosphatase deficiency \ 1 ACP2
Spondyloenchondrodysplasia 1 ACP5
Combined malonic and methylmalonic acidemia 1 ACSF3
X-linked nonsyndromic intellectual deficit 24 ACSL4
Childhood-onset nemaline myopathy 5 ACTA1
Congenital myopathy with excess of thin filaments 1 ACTA1
Intermediate nemaline myopathy 3 ACTA1
Severe congenital nemaline myopathy 3 ACTA1
Typical nemaline myopathy 4 ACTA1
Familial thoracic aortic aneurysm and aortic dissection 8 ACTA2 oyamoya disease 2 ACTA 2
Baraitser-Winter syndrome 2 ACTB
Developmental malformations - deafness - dystonia 1 ACTB
Atrial septal defect, ostium secundum type 8 ACTC1
Familial isolated dilated cardiomyopathy 38 ACTC1
Left ventricular noncompaction 11 ACTC1
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 ACTG1
Baraitser-Winter syndrome 2 ACTG1
Familial visceral myopathy 1 ACTG2
Autosomal dominant macrothrombocytopenia 3 ACTN1
Familial isolated dilated cardiomyopathy 38 ACT 2
Familial idiopathic steroid-resistant nephrotic syndrome with focal
segmental hyalinosis 11 ACTN4
Fibrodysplasia ossificans progressiva 1 ACVR1
Situs ambiguus 6 ACVR2B
Heritable pulmonary arterial hypertension 5 ACVRL1
Rendu-Osler-Weber disease 3 ACVRL1
Neurological conditions associated with aminoacylase 1 deficiency 1 ACY1
Omenn syndrome 9 ADA
Severe combined immunodeficiency due to adenosine deaminase
deficiency 1 ADA
Reticulate acropigmentation of Kitamura 1 ADAM10
Neonatal inflammatory skin and bowel disease 1 ADAM17
Cone rod dystrophy 22 ADAM9
Weill-Marchesani syndrome 4 ADAMTS10
Congenital thrombotic thrombocytopenic purpura due to ADAMTS-13
deficiency 1 ADAMTS13
Weill-Marchesani syndrome 4 ADAMTS17
Ehlers-Danlos syndrome, dermatosparaxis type 1 ADAMTS2
Geleophysic dysplasia 2 ADAMTSL2
55
Table IX-21781338.1 Ectopia lentis syndrome 2 ADAMTSL4
Aicardi-Goutieres syndrome 6 ADAR
Dyschromatosis symmetrica hereditaria 1 ADAR
Autosomal recessive ataxia due to ubiquinone deficiency 1 ADCK3
Idiopathic hypercalciuria 1 ADCY10
Familial dyskinesia and facial myokymia 1 ADCY5
Hypermethioninemia encephalopathy due to adenosine kinase deficiency 1 ADK
Adenylosuccinate lyase deficiency 1 ADSL
Precursor B-cell acute lymphoblastic leukemia 13 AFF1
F AXE intellectual deficit 2 AFF2
Early-onset spastic ataxia-neuropathy syndrome 1 AFG3L2
Spinocerebellar ataxia type 28 1 AFG3L2
Congenital deficiency in alpha-fetoprotein 1 AFP
Hereditary persistence of alpha-fetoprotein 1 AFP
Aspartylglucosaminuria 1 AGA
Klippel-Trenaunay syndrome 1 AGGF1
Congenital cataract - hypertrophic cardiomyopathy - mitochondrial
myopathy 2 AGK
Berardinelli-Seip congenital lipodystrophy 3 AG AT2
Rhizomelic chondrodysplasia punctata type 3 1 AG PS
Postsynaptic congenital myasthenic syndromes 9 AGRN
Renal tubular dysgenesis of genetic origin 4 AGT
Renal tubular dysgenesis of genetic origin 4 AGTR1
X-linked nonsyndromic intellectual deficit 24 AGTR2
Primary hyperoxaluria type 1 1 AG XT
Psychomotor retardation due to S-adenosylhomocysteine hydrolase
deficiency 1 AHCY
Joubert syndrome with ocular defect 5 AHI1
Hyper-lgM syndrome type 2 1 AICDA
Severe X-linked mitochondrial encephalomyopathy 1 AIFM1
X-linked Charcot-Marie-Tooth disease type 4 1 AIFM1
Pelizaeus-Merzbacher-like due to AIMP1 mutation 1 AIMP1
Acromegaly 2 AIP
Familial isolated pituitary adenoma 1 AIP
Cone rod dystrophy 22 AIPL1
Leber congenital amaurosis 18 AIPL1
Retinitis pigmentosa 61 AIPL1
Autoimmune polyendocrinopathy type 1 1 AIRE
Hemolytic anemia due to adenylate kinase deficiency 1 AK1
Reticular dysgenesis 1 AK2
Romano-Ward syndrome 13 AKAP9
56
Table IX-21781338.1 46,XY disorder of sex development due to isolated 17, 20 lyase deficiency 4 AKR1C4
Congenital bile acid synthesis defect type 2 1 AKR1D1
Cowden syndrome 7 AKT1
Proteus syndrome 2 AKT1
Familial partial lipodystrophy due to AKT2 mutations 1 AKT2
Hypoinsulinemic hypoglycemia and body hemihypertrophy 1 AKT2
Hemimegalencephaly 2 AKT3
Megalencephaly - polymicrogyria - post-axial Polydactyly - hydrocephalus 2 AKT3
Porphyria due to ALA dehydratase deficiency 1 ALAD
Erythropoietic protoporphyria 2 ALAS2
X-linked sideroblastic anemia 1 ALAS2
Congenital analbuminemia 1 ALB
ALDH18Al-related DeBarsy syndrome 1 ALDH18A1
Isolated anophthalmia - microphthalmia 5 ALDH1A3
Sjogren-Larsson syndrome 1 ALDH3A2
Hyperprolinemia type II 1 ALDH4A1
Developmental delay due to methylmalonate semialdehyde
dehydrogenase deficiency 1 ALDH6A1
Pyridoxine-dependent epilepsy 1 ALDH7A1
Glycogen storage disease due to aldolase A deficiency 1 ALDOA
Hereditary fructose intolerance 1 ALDOB
ALG1-CDG syndrome 1 ALG1
Romano-Ward syndrome 13 ALG10
ALG11-CDG syndrome 1 ALG11
ALG12-CD.G syndrome 1 ALG12
ALG13-CDG syndrome 1 ALG13
Congenital myasthenic syndromes with glycosylation defect 4 ALG14
ALG2-CDG syndrome 1 ALG2
Congenital myasthenic syndromes with glycosylation defect 4 ALG2
ALG3-CDG syndrome 1 ALG3
ALG6-CDG syndrome 1 ALG6
ALG8-CDG syndrome 1 ALG8
ALG9-CDG syndrome 1 ALG9
ALK-positive anaplastic large cell lymphoma 1 ALK
Inflammatory myofibroblastic tumor 6 ALK
Neuroblastoma 7 ALK
Alstrom syndrome 1 ALMS1
Congenital nonbullous ichthyosiform erythroderma 6 ALOX12B
Lamellar ichthyosis 6 ALOX12B
Self-healing collodion baby 3 ALOX12B
Congenital nonbullous ichthyosiform erythroderma 6 ALOXE3
57
Table IX-21781338.1 Self-healing collodion baby 3 ALOXE3
Adult hypophosphatasia 1 ALPL
Childhood-onset hypophosphatasia 1 ALPL
Infantile hypophosphatasia 1 ALPL
Odontohypophosphatasia 1 ALPL
Perinatal lethal hypophosphatasia 1 ALPL
Infantile-onset ascending hereditary spastic paralysis 1 ALS2
Juvenile amyotrophic lateral sclerosis 3 ALS2
Juvenile primary lateral sclerosis 2 ALS2
Frontonasal dysplasia-severe microphthalmia-severe facial defting
syndrome 1 ALX1
Frontonasal dysplasia 2 ALX4
Frontonasal dysplasia with alopecia and genital anomaly 1 ALX4
Isolated scaphocephaly 3 ALX4
Parietal foramina 2 ALX4
Potocki-Shaffer syndrome 3 ALX4
Congenital bile acid synthesis defect type 4 1 AMACR
Hypomaturation amelogenesis imperfecta 5 AMELX
Osteopathia striata - cranial sclerosis 1 AMER1
Persistent Mullerian duct syndrome 2 AMH
Persistent Mullerian duct syndrome 2 AMHR2
Alport syndrome - intellectual deficit - midface hypoplasia - elliptocytosis 3 AMMECR1
Grasbeck-lmerslund disease 2 AMN
Adenosine monophosphate deaminase deficiency 2 AMPD1
Adenosine monophosphate deaminase deficiency 2 AMPD3
Atypical glycine encephalopathy 3 AMT
Infantile glycine encephalopathy 3 AMT
Neonatal glycine encephalopathy 3 AMT
Amyotrophic lateral sclerosis 23 ANG
8pll.2 deletion syndrome 1 ANK1
Hereditary spherocytosis 5 ANK1
Romano-Ward syndrome 13 ANK2
Intellectual deficiency - hypotonia - spasticity - sleep disorder 1 ANK3
Craniometaphyseal dysplasia . 1 ANKH
Familial articular chondrocalcinosis 1 ANKH
16q24.3 microdeletion syndrome 1 ANKRD11
KBG syndrome 1 ANKRD11
Autosomal thrombocytopenia with normal platelets 3 ANKRD26
Juvenile rheumatoid factor-negative polyarthritis 9 ANKRD55
Oligoarticular juvenile arthritis 9 ANKRD55
Adult-onset autosomal recessive cerebellar ataxia 1 ANO10
58
Table IX-21781338.1 Cervical dystonia 2 AN03
Autosomal recessive limb-girdle muscular dystrophy type 2L 1 AN05
Miyoshi myopathy 2 AN05
Scott syndrome 1 AN06
Familial capillary hemangioma 2 ANTX 1
GAPO syndrome 1 ANTXR1
Infantile systemic hyalinosis 1 ANTXR2
Juvenile hyaline fibromatosis 1 ANTXR2
MEDNIK syndrome 1 AP1S1
Fried syndrome 1 AP1S2
Familial hypocalciuric hypercalcemia type 3 1 AP2S1
Hermansky-Pudlak syndrome with neutropenia 1 AP3B1
Severe intellectual deficit and progressive spastic paraplegia 4 AP4B1
Severe intellectual deficit and progressive spastic paraplegia 4 AP4E1
Severe intellectual deficit and progressive spastic paraplegia 4 AP4M1
Severe intellectual deficit and progressive spastic paraplegia 4 AP4S1
Autosomal recessive spastic paraplegia type 48 1 AP5Z1
APC-related attenuated familial adenomatous polyposis 1 APC
Desmoid disease 2 APC
Familial adenomatous polyposis due to 5q22.2 microdeletion 1 APC
Gardner syndrome 1 APC
Turcot syndrome with polyposis 1 APC
Hypotrichosis simplex 6 APCDD1
Apolipoprotein A-l deficiency 2 APOA1
Familial renal amyloidosis due to Apolipoprotein Al variant 1 APOA1
Primary systemic amyloidosis 1 APOA1
Familial renal amyloidosis due to Apolipoprotein All variant 1 APOA2
Hyperlipoproteinemia type 4 2 APOA5
Hyperlipoproteinemia type 5 4 APOA5
Abetalipoproteinemia 2 APOB
Familial apolipoprotein C-ll deficiency 1 APOC2
Cholesterol-ester transfer protein deficiency 2 APOC3
Hyperlipidemia type 3 1 APOE
Lipoprotein glomerulopathy 1 APOE
Sea-blue histiocytosis 1 APOE
Sporadic idiopathic steroid-resistant nephrotic syndrome with focal
segmental hyalinosis 2 APOL1
Early-onset autosomal dominant Alzheimer disease 4 APP
Hereditary cerebral hemorrhage with amyloidosis, Arctic type 1 APP
Hereditary cerebral hemorrhage with amyloidosis, Dutch type 1 APP
Hereditary cerebral hemorrhage with amyloidosis, Flemish type 1 APP
59
Table IX-21781338.1 Hereditary cerebral hemorrhage with amyloidosis, Iowa type 1 APP
Hereditary cerebral hemorrhage with amyloidosis, Italian type 1 APP
Hereditary cerebral hemorrhage with amyloidosis, Piedmont type 1 APP
Adenine phosphoribosyltransferase deficiency 1 APRT
Ataxia - oculomotor apraxia type 1 1 APTX
Nephrogenic diabetes insipidus 2 AQP2
Complete androgen insensitivity syndrome 1 AR
Familial hypospadias 2 AR
Kennedy disease 1 AR
Partial androgen insensitivity syndrome 1 AR
Periventricular nodular heterotopia 2 ARFGEF2
Argininemia 1 ARG1
Familial idiopathic steroid-resistant nephrotic syndrome with focal
segmental hyalinosis 11 ARHGAP24
Adams-Oliver syndrome 4 ARHGAP31
Familial idiopathic steroid-resistant nephrotic syndrome with diffuse
mesangial sclerosis 4 ARHGDIA
Autosomal dominant slowed nerve conduction velocity 1 ARHGEF10
X-linked nonsyndromic intellectual deficit 24 ARHGEF6
Hyperekplexia - epilepsy 1 ARHGEF9
Coff in-Siris syndrome 4 ARID1A
6q25 microdeletion syndrome 1 ARID1B
Coffin-Siris syndrome 4 ARID1B
B-cell chronic lymphocytic leukemia 7 ARL11
Joubert syndrome 9 ARL13B
Bardet-Biedl syndrome 17 ARL6
Retinitis pigmentosa 61 ARL6
Metachromatic leukodystrophy, adult form 2 ARSA
Metachromatic leukodystrophy, juvenile form 2 AR5A
Metachromatic leukodystrophy, late infantile form 2 ARSA
Mucopolysaccharidosis type 6, rapidly progressing 1 ARSB
Brachytelephalangic chondrodysplasia punctata 1 ARSE
22qll.2 deletion syndrome 7 ARVCF
Early infantile epileptic encephalopathy 9 ARX
Micrencephaly - corpus callosum agenesis - abnormal genitalia 1 ARX
Partington syndrome 1 ARX
Spasticity - intellectual deficit - X-linked epilepsy 1 ARX
West syndrome 4 ARX
X-linked lissencephaly with abnormal genitalia 1 ARX
X-linked nonsyndromic intellectual deficit 24 ARX
Farber lipogranulomatosis 1 ASAH1
60
Table IX-21781338.1 Hereditary myoclonus - progressive distal muscular atrophy 1 ASAH1
Haddad syndrome 3 ASCL1
Ondine syndrome . 5 ASCL1
Argininosuccinic aciduria 1 ASL
Mild Canavan disease 1 AS PA
Severe Canavan disease 1 AS PA
Autosomal recessive primary microcephaly 11 ASPM
Alveolar soft-part sarcoma 2 ASPSCR1
Translocation renal cell carcinoma 7 ASPSCR1
Acute neonatal citrullinemia type 1 1 ASS1
Adult-onset citrullinemia type 1 1 ASS1
Bohring-Opitz syndrome 1 ASXL1
Severe feeding difficulties - failure to thrive - microcephaly due to ASXL3
deficiency 1 ASXL3
Cerebellar ataxia, Cayman type 1 ATCAY
Melanoma of soft part 3 ATF1
AICA-ribosiduria 1 ATIC
Autosomal dominant spastic paraplegia type 3 1 ATL1
Hereditary sensory and autonomic neuropathy type 1 4 ATL1
Ataxia-telangiectasia 1 ATM
B-cell chronic lymphocytic leukemia 7 ATM
Mantle cell lymphoma 3 ATM
Dentatorubral-pallidoluysian atrophy 1 ATN1 .
Congenital blindness due to retinal nonattachment 1 ATOH7
Congenital cataract microcornea with corneal opacity 2 ATOH7
Persistent hyperplastic primary vitreous 3 ATOH7
Angelman syndrome 6 ATP10A
Idiopathic pulmonary fibrosis 11 ATP11A
Kufor- akeb syndrome 1 ATP13A2
Parkinsonim due to ATP13A2 deficiency 1 ATP13A2
Alternating hemiplegia of childhood 4 ATP1A2
Familial or sporadic hemiplegic migraine 4 ATP1A2
Alternating hemiplegia of childhood 4 AT PI A3
Rapid-onset dystonia-parkinsonism 1 AT PI A3
Brody myopathy 1 ATP2A1
Acrokeratosis verruciformis of Hopf 1 ATP2A2
Darier disease 1 ATP2A2
X-linked non progressive cerebellar ataxia 1 ATP2B3
Familial benign chronic pemphigus 1 ATP2C1
Isolated ATP synthase deficiency 4 ATP5A1
Isolated ATP synthase deficiency 4 ATP5E
61
Table IX-21781338.1 Autosomal recessive cutis laxa type 2, classic type 1 ATP6V0A2
Wrinkly skin syndrome 1 ATP6V0A2
Autosomal recessive distal renal tubular acidosis without deafness 1 ATP6V0A4
Autosomal recessive distal renal tubular acidosis with deafness 1 ATP6V1B1
Menkes disease 1 ATP7A
Occipital horn syndrome 1 ATP7A
X-linked distal spinal muscular atrophy 1 ATP7A
Wilson disease 1 ATP7B
Dysequilibrium syndrome 4 ATP8A2
Intrahepatic cholestasis of pregnancy 4 ATP8B1
Progressive familial intrahepatic cholestasis type 1 1 ATP8B1
Isolated ATP synthase deficiency 4 ATPAF2
Familial cutaneous telangiectasia and oropharyngeal predisposition cancer
syndrome 1 AT
Seckel syndrome 6 ATR
Seckel syndrome 6 ATRIP
Alpha thalassemia - X-linked intellectual deficit syndrome 1 ATRX
Alpha-thalassemia - myelodysplastic syndrome 1 ATRX
Carpenter-Waziri syndrome 1 ATRX .
Chudley-Lowry-Hoar syndrome 1 ATRX
Holmes-Gang syndrome 1 ATRX
Juberg-Marsidi syndrome 1 ATRX
Smith-Fineman-Myers syndrome 1 ATRX
Vasquez-Hurst-Sotos syndrome 1 ATRX
Spinocerebellar ataxia type 1 1 ATXN1
Joubert syndrome with renal defect 5 ATXN10
Spinocerebellar ataxia type 10 1 ATX 10
Amyotrophic lateral sclerosis 23 ATXN2
Spinocerebellar ataxia type 2 1 ATXN2
Machado-Joseph disease type 1 1 ATXN3
Machado-Joseph disease type 2 1 ATXN3
Machado-Joseph disease type 3 1 ATXN3
Spinocerebellar ataxia type 7 1 ATXN7
Spinocerebellar ataxia type 8 2 ATXN8
Spinocerebellar ataxia type 8 2 ATXN80S
3-methylglutaconic aciduria type 1 1 AUH
X-linked hereditary sensory and autonomic neuropathy with deafness 1 AUNX1
Male infertility associated with large-headed multiflagellar polyploid
spermatozoa 1 AURKC
Hereditary central diabetes insipidus / 1 AVP
Nephrogenic diabetes insipidus 2 AVPR2
62
Table IX-21781338.1 Nephrogenic syndrome of inappropriate antidiuresis 1 AVP 2
Oligodontia 10 AXIN2
Oligodontia - cancer predisposition syndrome 1 AXIN2
Autosomal dominant beta2-microglobulinic amyloidosis 1 B2M
Muscle eye brain disease 7 B3GALNT2
Walker-Warburg syndrome 13 B3GALNT2
Ehlers-Danlos syndrome, progeroid type 2 B3GALT6
Spondyloepimetaphyseal dysplasia with joint laxity 1 B3GALT6
Peters-plus syndrome 1 B3GALTL
Larsen-like syndrome, B3GAT3 type 1 B3GAT3
Walker-Warburg syndrome 13 B3GNT1
Autosomal recessive spastic paraplegia type 26 1 B4GALNT1
B4GALT1-CDG syndrome 1 B4GALT1
Ehlers-Danlos syndrome, progeroid type 2 B4GALT7
Meckel syndrome 13 B9D1
Meckel syndrome \ 13 B9D2
Familial hypercholanemia 3 BAAT
Familial isolated dilated cardiomyopathy 38 BAG 3
Muscular dystrophy, Selcen type 1 BAG3
Nestor-Guillermo progeria syndrome 1 BANF1
BAPl-related tumor predisposition syndrome 1 BAP1
Hereditary breast and ovarian cancer syndrome 13 BARD1
Williams syndrome 17 BAZ1B
Bardet-Biedl syndrome 17 BBS1
Bardet-Biedl syndrome 17 BBS10
Bardet-Biedl syndrome 17 BBS12
Bardet-Biedl syndrome 17 BBS2
Bardet-Biedl syndrome 17 BBS4
Bardet-Biedl syndrome 17 BBS5
Bardet-Biedl syndrome 17 BBS7
Bardet-Biedl syndrome 17 BBS9
Butyrylcholinesterase deficiency 1 BCHE
Classic maple syrup urine disease 4 BCKDHA
Intermediate maple syrup urine disease 5 BCKDHA
Intermittent maple syrup urine disease 4 BCKDHA
Classic maple syrup urine disease 4 BCKDHB
Intermediate maple syrup urine disease 5 BCKDHB
Intermittent maple syrup urine disease 4 BCKDHB
Thiamin-responsive maple syrup urine disease 4 BCKDHB
Autism-epilepsy syndrome due to branched chain ketoacid dehydrogenase
kinase deficiency 1 BCKDK
63
Table IX-21781338.1 Hereditary persistence of fetal hemoglobin - beta-thalassemia 5 BCL11A
Follicular lymphoma 4 BCL2
Intravascular large B-cell lymphoma 2 BCL2
Follicular lymphoma 4 BCL6
Intravascular large B-cell lymphoma 2 BCL6
Primary, mediastinal large B-cell lymphoma 1 BCL6
Williams syndrome 17 BCL7B
Hereditary hypercarotenemia and vitamin A deficiency 1 BCMOl
Microphthalmia, Lenz type 1 BCOR
Oculofaciocardiodental syndrome 1 BCOR
Chronic myeloid leukemia 3 BCR
Distal 22qll.2 microdeletion syndrome 3 BCR
Precursor B-cell acute lymphoblastic leukemia 13 BCR
Precursor T-cell acute lymphoblastic leukemia 19 BCR
Bjornstad syndrome 1 BCS1L
GRACILE syndrome 1 BCS1L
Isolated CoQ-cytochrome C reductase deficiency 6 BCS1L
Leigh syndrome with nephrotic syndrome 4 BCS1L
Renal tubulopathy - encephalopathy - liver failure 1 BCS1L
Ondine syndrome 5 BDNF
WAGR syndrome 3 BDNF
Spinocerebellar ataxia type 31 1 BEA 1
Adult-onset foveomacular vitelliform dystrophy 2 BEST1
Autosomal dominant vitreoretinochoroidopathy 1 BEST1
Best disease 1 BEST1
MRCS syndrome 1 BEST1
Retinitis pigmentosa 61 BEST1
Retinopathy, Burgess-Black type 1 BEST1
Autosomal recessive childhood-onset cortical cataract 1 BFSP1
Partial congenital cataract 1 BFSP2
Tibial aplasia - ectrodactyly 1 BHLHA9
Bilateral multicystic renal dysplasia 1 BICC1
Autosomal recessive centronuclear myopathy 1 BIN1
MALT lymphoma 4 BIRC3
MODY syndrome 12 BLK
Autosomal agammaglobulinemia 7 BLNK
Hermansky-Pudlak syndrome type 8 1 BLOC1S3
Hyperbiliverdinemia 1 BLVRA
Osteogenesis imperfecta type 3 9 BMP1
46,XX gonadal dysgenesis 4 BMP15
Brachydactyly type A2 3 BMP2
64
Table IX-Z178 J 338.1 14q22q23 microdeletion syndrome 2 BMP4
Microphthalmia with brain and digit anomalies 1 BMP4
Diaphanospondylodysostosis 1 BMPER
Generalized juvenile polyposis/juvenile polyposis coli 3 BMPR1A
Hereditary mixed polyposis syndrome 2 BMPR1A
Hereditary nonpolyposis colon cancer 11 BMPR1A
Juvenile polyposis of infancy 2 BMPR1A
Brachydactyly type A2 3 BMPR1B
Brachydactyly type C 2 BMPR1B
Heritable pulmonary arterial hypertension 5 BMPR2
Pulmonary venoocclusive disease 1 BMPR2
Circumscribed cutaneous aplasia of the vertex 1 BMS1
Fatal multiple mitochondrial dysfunction syndrome 2 BOLA3
Hemolytic anemia due to diphosphoglycerate mutase deficiency 1 BPGM
Cardiofaciocutaneous syndrome 4 BRAF
Hairy cell leukemia 1 BRAF
Hashimoto-Pritzker syndrome 1 BRAF
LEOPARD syndrome 3 BRAF
Noonan syndrome 10 BRAF
Pilocytic astrocytoma 2 BRAF
Familial pancreatic carcinoma 8 • BRCA1
Familial prostate cancer 14 BRCA1
Hereditary breast and ovarian cancer syndrome 13 BRCA1
Hereditary breast cancer 4 BRCA1
Hereditary site-specific ovarian cancer syndrome 2 BRCA1
Primary peritoneal carcinoma 1 BRCA1
Familial pancreatic carcinoma 8 BRCA2
Familial prostate cancer 14 BRCA2
Fanconi anemia 16 BRCA2
Hereditary breast and ovarian cancer syndrome 13 BRCA2
Hereditary breast cancer 4 BRCA2
Hereditary site-specific ovarian cancer syndrome 2 BRCA2
Inherited cancer-predisposing syndrome due to biallelic BRCA2 mutations 1 BRCA2
Moyamoya disease - short stature - facial dysmorphism - hypergonadotropic hypogonadism 1 BRCC3
Fanconi anemia 16 BRIP1
Hereditary breast and ovarian cancer syndrome 13 BRIP1
Autosomal dominant spastic paraplegia type 17 1 BSCL2
Berardinelli-Seip congenital lipodystrophy 3 BSCL2
Distal hereditary motor neuropathy type 5 3 BSCL2
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 BSND
65
Table IX-21781338.1 Infantile Bartter syndrome with deafness 3 BSND
Biotinidase deficiency 1 BTD
Short stature due to isolated growth hormone deficiency with X-linked
hypogammaglobulinemia 2 BTK
X-linked agammaglobulinemia 1 BTK
Sarcoidosis 2 BTNL2
Mosaic variegated aneuploidy syndrome 2 BUB1B
Oculocutaneous albinism type 5 1 ClOORFll
Autosomal dominant progressive external ophthalmoplegia 5 C10ORF2
Infantile onset spinocerebellar ataxia 1 C10ORF2
Sensory ataxic neuropathy - dysarthria - ophthalmoparesis 2 C10ORF2
Temtamy syndrome 1 C120RF57
Autosomal recessive spastic paraplegia type 55 1 ' C120RF65
Combined oxidative phosphorylation defect type 7 1 C120RF65
Dyskeratosis congenita 10 C160RF57
Poikiloderma with neutropenia 1 C160RF57
Neurodegeneration with brain iron accumulation due to C19orfl2
mutation 1 C190RF12
Immunodeficiency due to an early component of complement deficiency 8 C1QA
Immunodeficiency due to an early component of complement deficiency 8 C1QB
Immunodeficiency due to an early component of complement deficiency 8 C1QC
Late-onset retinal degeneration 1 C1QTNF5
Immunodeficiency due to an early component of complement deficiency 8 CIS
Immunodeficiency due to an early component of complement deficiency 8 C2
Retinitis pigmentosa 61 C20RF71
Atypical hemolytic uremic syndrome with C3 anomaly 1 C3
Complement component 3 deficiency 1 C3
Immunodeficiency due to an early component of complement deficiency 8 C4A
Immunodeficiency due to an early component of complement deficiency 8 C4B
Hypomaturation amelogenesis imperfecta 5 C40RF26
Immunodeficiency due to a late component of complements deficiency 7 C5
Joubert syndrome 9 C50RF42
Monomelic amyotrophy 2 C50RF42
Immunodeficiency due to a late component of complements deficiency 7 C6
Immunodeficiency due to a late component of complements deficiency 7 C7
Glutaric acidemia type 3 1 C7ORF10
Immunodeficiency due to a late component of complements deficiency 7 C8A
Immunodeficiency due to a late component of complements deficiency 7 C8B
Immunodeficiency due to a late component of complements deficiency 7 C8G
Cone rod dystrophy 22 C80RF37
Retinitis pigmentosa 61 C80RF37
66
Table IX-21781338.1 Leigh syndrome with leukodystrophy 13 C80RF38
Immunodeficiency due to a late component of complements deficiency 7 C9
Amyotrophic lateral sclerosis 23 C90RF72
Frontotemporal dementia with motor neuron disease 3 C90RF72
Osteopetrosis with renal tubular acidosis • 1 CA2
Retinitis pigmentosa ' 61 CA4
Dysequilibrium syndrome 4 CA8
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 CABP2
Congenital stationary night blindness 12 CABP4
Alternating hemiplegia of childhood 4 CACNA1A
Benign paroxysmal torticollis of infancy 1 CACNA1A
Familial or sporadic hemiplegic migraine 4 CACNA1A
Familial paroxysmal ataxia 1 CACNA1A
Spinocerebellar ataxia type 6 1 CACNA1A
Brugada syndrome 12 CACNA1C
Timothy syndrome 1 CACNA1C
Sinoatrial node dysfunction and deafness 1 CACNA1D
Aland Island eye disease 1 CACNA1F
Cone rod dystrophy 22 CACNA1F
Congenital stationary night blindness 12 CACNA1F
Childhood absence epilepsy 6 CACNA1H
Hypokalemic periodic paralysis 3 CACNA1S
Malignant hyperthermia 2 CACNA1S
Thyrotoxic periodic paralysis 3 CACNA1S
Familial short QT syndrome 4 CACNA2D1
Early infantile epileptic encephalopathy 9 CACNA2D2
Cone rod dystrophy 22 CACNA2D4
Brugada syndrome 12 CACNB2
Episodic ataxia type 5 1 CACNB4
Juvenile myoclonic epilepsy 7 CACNB4
Autosomal dominant nonsyndromic intellectual deficit 15 CACNG2
Catecholaminergic polymorphic ventricular tachycardia ' 4 CALM1
Familial long QT syndrome 2 CALM1
Familial long QT syndrome 2 CALM2
2p21 microdeletion syndrome 4 CAMKMT
Atypical hypotonia - cystinuria syndrome 3 CAMKMT
Non progressive cerebellar ataxia with intellectual deficit 1 CAMTA1
Autosomal recessive limb girdle muscular dystrophy type 2A 1 CAPN3
Autosomal dominant neovascular inflammatory vitreoretinopathy 1 CAPN5
Persistent polyclonal B-cell lymphocytosis 1 CARD11
Severe combined immunodeficiency due to CARD11 deficiency 1 CARD11
67
Table IX-21781338.1 Pityriasis rubra pilaris 1 CARD14
Chronic mucocutaneous candidiasis 6 CARD9
Inflammatory myofibroblastic tumor 6 CARS
Autosomal recessive primary microcephaly 11 CASC5
X-linked intellectual deficit, Najm type 1 CASK
Autoimmune lymphoproliferative syndrome 6 CASP10
Autoimmune lymphoproliferative syndrome with recurrent infections 1 CASP8
Catecholaminergic polymorphic ventricular tachycardia 4 CASQ2
Autosomal dominant hypocalcemia 1 CASR
Bartter syndrome with hypocalcemia 1 CASR
Familial hypocalciuric hypercalcemia type 1 1 CASR
Familial isolated hypoparathyroidism due to impaired PTH secretion 2 CASR
Neonatal severe primary hyperparathyroidism 1 CASR
Acatalasemia 1 CAT
CATSPERl-related non syndromic male infertility 1 CATSPER1
Deafness-infertility syndrome 2 CATSPER2
Berardinelli-Seip congenital lipodystrophy' 3 CAV1
Diffuse cutaneous systemic sclerosis 4 CAV1
Heritable pulmonary arterial hypertension 5 CAV1
Limited cutaneous systemic sclerosis 4 CAV1
Autosomal dominant limb-girdle muscular dystrophy type 1C 1 CAV3
Rippling muscle disease 1 CAV3
Romano-Ward syndrome 13 CAV3
Acute megakaryoblastic leukemia without Down syndrome 2 CBFA2T3
Acute myeloid leukemia with abnormal bone marrow eosinophils
inv(16)(pl3q22) or t(16;16)(pl3;q22) 2 CBFB
Noonan syndrome 10 CBL
Heritable pulmonary arterial hypertension 5 CBLN2
Classical homocystinuria 1 CBS
46,XY complete gonadal dysgenesis 8 CBX2
Autosomal recessive nonsyndromic intellectual deficit 15 CC2D1A
Joubert syndrome with hepatic defect 3 CC2D2A
Joubert syndrome with oculorenal defect 6 CC2D2A
Meckel syndrome 13 CC2D2A
Hennekam syndrome 1 CCBE1
Primary ciliary dyskinesia 21 CCDC103
Situs ambiguus 6 CCDC11
Situs inversus totalis 3 CCDC11
Primary ciliary dyskinesia 21 CCDC114
Primary ciliary dyskinesia 21 CCDC39
Primary ciliary dyskinesia 21 CCDC40
68
Table IX-21781338.1 Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 CCDC50
Papillary or follicular thyroid carcinoma 14 CCDC6
Congenital myopathy with internal nuclei and atypical cores 1 CCDC78
3M syndrome 3 CCDC8
Congenital non-communicating hydrocephalus 1 CCDC88C
Hereditary cerebral cavernous malformation 3 CCM2
B-cell chronic lymphocytic leukemia 7 CCND1
Mantle cell lymphoma 3 CCND1
Multiple myeloma 1 CCND1
Hereditary sensory and autonomic neuropathy with spastic paraplegia 1 CCT5
Fetal and neonatal alloimmune thrombocytopenia 6 CD109
Nephrotic syndrome-deafness-pretibial epidermolysis bullosa syndrome 1 CD151
Common variable immunodeficiency 10 CD19
Juvenile rheumatoid factor-negative polyarthritis 9 CD247
Oligoarticular juvenile arthritis 9 CD247
T-B+ severe combined immunodeficiency due to
CD3delta/CD3epsilon/CD3zeta 3 CD247
Autosomal recessive lymphoproliferative disease 2 CD27
Familial idiopathic steroid-resistant nephrotic syndrome with focal
segmental hyalinosis 11 CD2AP
Methylmalonic aciduria due to transcobalamin receptor defect 1 CD320
T-B+ severe combined immunodeficiency due to
CD3delta/CD3epsilon/CD3zeta 3 CD3D
T-B+ severe combined immunodeficiency due to
CD3delta/CD3epsilon/CD3zeta 3 CD3E
Combined immunodeficiency due to CD3gamma deficiency 1 CD3G
Hyper-lgM syndrome type 3 1 CD40
X-linked hyper-lgM syndrome 1 CD40LG
Atypical hemolytic uremic syndrome with MCP/CD46 anomaly 1 CD46
Primary CD59 deficiency 1 CD59
Autosomal agammaglobulinemia 7 CD79A
Autosomal agammaglobulinemia 7 CD79B
Common variable immunodeficiency 10 CD81
Susceptibility to respiratory infections associated with CD8alpha chain
mutation 1 CD8A
C syndrome 1 CD96
Ear-patella-short stature syndrome 5 CDC6
Familial isolated hyperparathyroidism 3 CDC73
Familial parathyroid adenoma 2 CDC73
Hyperparathyroidism - jaw tumor syndrome 1 CDC73
Parathyroid carcinoma 2 CDC73
Familial gastric cancer 2 CDH1
69
Table IX-21781338.1 Gastric linitis plastica 1 CDH1
Autosomal dominant nonsyndromic intellectual deficit 15 CDH15
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 CDH23
Usher syndrome type 1 9 CDH23
EEM syndrome 1 CDH3
Hypotrichosis with juvenile macular degeneration 1 CDH3
Cone rod dystrophy 22 CDHR1
Retinitis pigmentosa 61 CDHR1
Dedifferentiated liposarcoma 3 CDK4
Familial melanoma 6 CDK4
Well-differentiated liposarcoma 3 CDK4
Autosomal recessive primary microcephaly 11 CDK5RAP2
Atypical Rett syndrome 4 CDKL5
West syndrome 4 CDKL5
Multiple endocrine neoplasia type 1 5 CDKN1A
Multiple endocrine neoplasia type 1 5 CDKN1B
Multiple endocrine neoplasia type 4 1 CDKN1B
Beckwith-Wiedemann syndrome due to CDKN1C mutation 1 CDKN1C
IMAGe syndrome 1 CDKN1C
Familial melanoma 6 CDKN2A
Familial pancreatic carcinoma 8 CDKN2A
Melanoma neural system tumor syndrome 1 CDKN2A
Melanoma-pancreatic cancer syndrome 1 CDKN2A
Precursor B-cell acute lymphoblastic leukemia 13 CDKN2A
Precursor T-cell acute lymphoblastic leukemia 19 CDKN2A
Familial melanoma 6 CDKN2B
Multiple endocrine neoplasia type 1 5 CDKN2B
Multiple endocrine neoplasia type 1 5 CDKN2C
Familial melanoma 6 CDKN2D
Alobar holoprosencephaly 14 CDON
Lobar holoprosencephaly 14 CDON
Microform holoprosencephaly 14 CDON
Midline interhemispheric variant of holoprosencephaly 14 CDON
Semilobar holoprosencephaly 14 CDON
Septopreoptic holoprosencephaly 14 CDON
Generalized peeling skin syndrome type B 1 CDSN
Hypotrichosis simplex of the scalp 2 CDSN
Ear-patella-short stature syndrome 5 CDT1
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 CEACAM16
Acute myeloid leukemia with CEBPA somatic mutations 1 CEBPA
Acute myeloid leukemia with t(8;21)(q22;q22) translocation 3 CEBPA
70
Table IX-21781338.1 Inherited acute myeloid leukemia 1 CEBPA
Recurrent infection due to specific granule deficiency 1 CEBPE
Cat-eye syndrome 2 CECR1
Cat-eye syndrome 2 CECR2
MODY syndrome 12 CEL
Autosomal recessive primary microcephaly 11 CENPJ
Seckel syndrome 6 CENPJ
Autosomal recessive primary microcephaly 11 CEP152
Seckel syndrome 6 CEP152
Senior-Loken syndrome 8 CEP164
Bardet-Biedl syndrome 17 CEP290
Joubert syndrome with oculorenal defect 6 CEP290
Leber congenital amaurosis 18 CEP290
Meckel syndrome 13 CEP290
Senior-Loken syndrome 8 CEP290
Joubert syndrome 9 CEP41
Joubert syndrome with ocular defect 5 CEP41
Mosaic variegated aneuploidy syndrome 2 CEP57
Autosomal recessive primary microcephaly 11 CEP63
Isolated cytochrome C oxidase deficiency 9 CEP89
Retinitis pigmentosa 61 CERKL
Cholesterol-ester transfer protein deficiency 2 CETP
Atypical hemolytic uremic syndrome with B factor anomaly 1 CFB
Biliary atresia with splenic malformation syndrome 1 CFC1
Double outlet right ventricle 3 CFC1
Situs ambiguus 6 CFC1
Recurrent Neisseria infections due to factor D deficiency 1 CFD
Dense deposit disease 2 CFH
Familial drusen 2 CFH
Immunodeficiency with factor H anomaly 1 CFH
Immunoglobulin-mediated membranoproliferative glomerulonephritis 2 CFH
Atypical hemolytic uremic syndrome with anti-factor H antibodies 3 CFHR1
C3 glomerulonephritis 2 CFHR1
Dense deposit disease 2 CFHR1
Atypical hemolytic uremic syndrome with anti-factor H antibodies 3 CFHR3
C3 glomerulonephritis 2 CFHR5
Atypical hemolytic uremic syndrome with 1 factor anomaly 1 CFI
Immunodeficiency with factor 1 anomaly 1 CFI
Typical nemaline myopathy 4 CFL2
Properdin deficiency 1 CFP
Congenital bilateral absence of vas deferens 1 CFTR
71
Table IX-21781338.1 Cystic fibrosis 4 CFTR
Hereditary chronic pancreatitis 6 CFTR
Idiopathic bronchiectasis 4 CFTR
CHARGE syndrome 2 CHD7
Kallmann syndrome 19 CHD7
Normosmic congenital hypogonadotropic hypogonadism 18 CHD7
Omenn syndrome 9 CHD7
Familial prostate cancer 14 CHEK2
Hereditary breast and ovarian cancer syndrome 13 CHEK2
Li-Fraumeni syndrome 2 CHEK2
Osteosarcoma, somatic 1 CHEK2
Amyotrophic lateral sclerosis 23 CHGB
Congenital muscular dystrophy due to phosphatidyl choline biosynthesis
defect 1 CHKB
Distal monosomy 3p 3 CHL1
Choroideremia 2 CHM
Pontocerebellar hypoplasia type 8 1 CHMP1A
Amyotrophic lateral sclerosis 23 CHMP2B
Behavioral variant of frontotemporal dementia 3 CHMP2B
Posterior polar cataract 5 CHMP4B
Duane syndrome 2 CHN1
Isolated congenital megalocornea 1 CHRDL1
Prune belly syndrome 1 CHRM3
Lethal multiple pterygium syndrome 4 CHRNA1
Postsynaptic congenital myasthenic syndromes 9 CHRNA1
Nocturnal frontal lobe epilepsy 5 CHRNA2
Nocturnal frontal lobe epilepsy 5 CHRNA4
15ql3.3 microdeletion syndrome 1 CHRNA7
Postsynaptic congenital myasthenic syndromes 9 CHRNB1
Nocturnal frontal lobe epilepsy 5 CHRNB2
Lethal multiple pterygium syndrome 4 CHRND
Postsynaptic congenital myasthenic syndromes 9 CHRND
Postsynaptic congenital myasthenic syndromes 9 CHRNE
Autosomal recessive multiple pterygium syndrome 1 CHRNG
Lethal multiple pterygium syndrome 4 CHRNG
Ehlers-Danlos syndrome, musculocontractural type 2 CHST14
CHST3-related skeletal dysplasia 1 CHST3
Macular corneal dystrophy 1 CHST6
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 CIB2
Usher syndrome type 1 9 CIB2
Immunodeficiency by defective expression of HLA class 2 5 CIITA
72
Table IX-21781338.1 Wolfram syndrome 2 CISD2
Atrial septal defect, ostium secundum type 8 CITED2
Atrial septal defect, sinus venosus type 1 CITED2
Single ventricular septal defect 1 CITED2
Situs inversus totalis 3 CITED2
Tetralogy of Fallot 8 CITED2
Ventricular septal defect 4 CITED2
Cystic fibrosis 4 CLCA4
Cold-induced sweating syndrome 2 CLCF1
Thomsen and Becker disease 1 CLCN1
Juvenile myoclonic epilepsy 7 CLCN2
Dent disease type 1 1 CLCN5
Albers-Schonberg osteopetrosis 1 CLCN7
Autosomal recessive malignant osteopetrosis 5 CLCN7
Intermediate osteopetrosis 2 CLCN7
Infantile Bartter syndrome with deafness 3 CLCNKA
Classic Bartter syndrome 1 CLCNKB
Gitelman syndrome 2 CLCNKB
Gitelman syndrome 2 CLCNKB
Infantile Bartter syndrome with deafness 3 CLCNKB
Ichthyosis - hypotrichosis - sclerosing cholangitis 1 CLDN1
Autosomal recessive no.nsyndromic sensorineural deafness type DFNB 52 CLDN14
Familial hypomagnesemia - hypercalciuria - nephrocalcinosis 1 CLDN16
Familial hypomagnesemia - hypercalciuria - nephrocalcinosis - severe
ocular involvement 1 CLDN19
Chronic mucocutaneous candidiasis 6 CLEC7A
X-linked intellectual deficit - cardiomegaly - congestive heart failure 1 CLIC2
Williams syndrome 17 CLIP2
Congenital short bowel syndrome 2 CLMP
CLN3 disease 1 CLN3
CLN5 disease 1 CLN5
CLN4A disease 1 CLN6
CLN6 disease 1 CLN6
CLN8 disease 1 CLN8
Progressive epilepsy - intellectual deficit, Finnish type 1 CLN8
Perrault syndrome 4 CLP
Retinitis pigmentosa 61 CLRN1
Usher syndrome type 3 3 CLRN1
Inflammatory myofibroblastic tumor 6 CLTC
Translocation renal cell carcinoma 7 CLTC
Proximal myotonic myopathy 1 CNBP
73
Table IX-21781338.1 Retinitis pigmentosa 61 CNGA1
Achromatopsia 6 CNGA3
Retinitis pigmentosa 61 CNGB1
Achromatopsia 6 CNGB3
Progressive cone dystrophy 4 CNGB3
Familial primary hypomagnesemia with normocalcuria and normocalcemia 2 CNNM2
Jalili syndrome 1 CNNM4
Precursor T-cell acute lymphoblastic leukemia 19 CNOT3
Congenital lethal myopathy, Compton-North type 1 CNTN1
Benign adult familial myoclonic epilepsy 1 CNTN2
Distal monosomy 3p 3 CNTN4
Cortical dysplasia - focal epilepsy syndrome 1 CNTNAP2
Pitt-Hopkins-like syndrome 2 CNTNAP2
Isolated cytochrome C oxidase deficiency 9 COA5
Leigh syndrome with cardiomyopathy 8 COA5
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 COCH
Meniere disease 1 COCH
COG1-CDG syndrome 1 COG1
COG4-CDG syndrome 1 COG4
COG5-CDG syndrome 1 COG5
COG7-CDG syndrome 1 COG7
COG8-CDG syndrome 1 COG8
Metaphyseal chondrodysplasia, Schmid type 1 COL10A1
Fibrochondrogenesis 2 COLllAl
Marshall syndrome 1. COL11A1
Stickler syndrome type 2 1 COLllAl
Autosomal dominant nonsyndromic sensorineural deafness type DFNA . 30 COL11A2
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 COL11A2
Fibrochondrogenesis 2 COL11A2
Otospondylomegaepiphyseal dysplasia 2 COL11A2
Stickler syndrome type 3 1 COL11A2
Weissenbacher- Zweymuller syndrome 1 COL11A2
Punctate palmoplantar keratoderma type Γ 2 COL14A1
Generalized junctional epidermolysis bullosa, non-Herlitz type 5 COL17A1
Late-onset junctional epidermolysis bullosa 1 COL17A1
Localized junctional epidermolysis bullosa, non-Herlitz type 2 COL17A1
Caffey disease 1 COL1A1
Dermatofibrosarcoma protuberans 2 COL1A1
Ehlers-Danlos syndrome type 1 3 COL1A1
Ehlers-Danlos syndrome type 7A 1 COL1A1
Ehlers-Danlos syndrome, vascular-like type 1 COL1A1
74
Table IX-21781338.1 Ehlers-Danlos/osteogenesis imperfecta syndrome 2 COL1A1
High bone mass osteogenesis imperfecta 2 COL1A1
Osteogenesis imperfecta type 1 2 COL1A1
Osteogenesis imperfecta type 2 5 COL1A1
Osteogenesis imperfecta type 3 9 COL1A1
Osteogenesis imperfecta type 4 7 COL1A1
Eh!ers-Danlos syndrome type 7B 1 COL1A2
Ehlers-Danlos syndrome, cardiac valvular type 1 COL1A2
Ehlers-Danlos/osteogenesis imperfecta syndrome 2 COL1A2
High bone mass osteogenesis imperfecta 2 COL1A2
Osteogenesis imperfecta type 1 2 COL1A2
Osteogenesis imperfecta type 2 5 COL1A2
Osteogenesis imperfecta type 3 9 COL1A2
Osteogenesis imperfecta type 4 7 COL1A2
Achondrogenesis type 2 1 COL2A1
Autosomal dominant rhegmatogenous retinal detachment 1 COL2A1
Czech dysplasia, metatarsal type 1 COL2A1
Familial avascular necrosis of femoral head 1 COL2A1
Hypochondrogenesis 1 COL2A1
Kniest dysplasia 1 COL2A1
Legg-Calve-Perthes disease 1 COL2A1
Mild spondyloepiphyseal dysplasia due to COL2A1 mutation with early- onset osteoarthritis 1 COL2A1
Multiple epiphyseal dysplasia, Beighton type 1 COL2A1
Otospondylomegaepiphyseal dysplasia 2 COL2A1
Platyspondylic dysplasia, Torrance type 1 COL2A1
Spondyloepimetaphyseal dysplasia congenita, Strudwick type 1 COL2A1
Spondyloepiphyseal dysplasia congenita 1 COL2A1
Spondylometaphyseal dysplasia, 'corner fracture' type 1 COL2A1
Spondyloperipheral dysplasia - short ulna 1 COL2A1
Stickler syndrome type 1 1 COL2A1
Acrogeria 1 COL3A1
Ehlers-Danlos syndrome, vascular type 1 COL3A1
Familial abdominal aortic aneurysm 1 COL3A1
Autosomal dominant familial hematuria - retinal arteriolar tortuosity - contractures 1 COL4A1
Familial vascular leukoencephalopathy 1 COL4A1
Schizencephaly 4 COL4A1
Walker-Warburg syndrome 13 COL4A1
Familial porencephaly 2 COL4A2
Autosomal dominant Alport syndrome 2 COL4A3
75
Table IX-21781338.1 Autosomal recessive Alport syndrome 2 COL4A4
X-linked Alport syndrome 1 COL4A5
X-linked diffuse leiomyomatosis - Alport syndrome 2 COL4A5
X-linked diffuse leiomyomatosis - Alport syndrome 2 COL4A6
Ehlers-Danlos syndrome type 1 3 COL5A1
Ehlers-Danlos syndrome type 2 2 COL5A1
Ehlers-Danlos syndrome type 1 3 COL5A2
Ehlers-Danlos syndrome type 2 2 COL5A2
Bethlem myopathy 3 COL6A1
Congenital muscular dystrophy, Ullrich type 3 COL6A1
Bethlem myopathy 3 COL6A2
Congenital muscular dystrophy, Ullrich type 3 COL6A2
Myosclerosis 1 COL6A2
Bethlem myopathy 3 COL6A3
Congenital muscular dystrophy, Ullrich type 3 COL6A3
Acral dystrophic epidermolysis bullosa 1 COL7A1
Centripetalis recessive dystrophic epidermolysis bullosa 1 COL7A1
Dystrophic epidermolysis bullosa pruriginosa 1 COL7A1
Dystrophic epidermolysis bullosa, nails only 1 COL7A1
Epidermolysis bullosa simplex superficialis 1 COL7A1
Generalized dominant dystrophic epidermolysis bullosa 1 COL7A1
Pretibial dystrophic epidermolysis bullosa ■ 1 COL7A1
Recessive dystrophic epidermolysis bullosa inversa 1 COL7A1
Recessive dystrophic epidermolysis bullosa-generalized other 1 COL7A1
Severe generalized recessive dystrophic epidermolysis bullosa 2 COL7A1
Transient bullous dermolysis of the newborn 1 COL7A1
Fuchs endothelial corneal dystrophy 4 COL8A2
Posterior polymorphous corneal dystrophy 3 COL8A2
Autosomal recessive Stickler syndrome 2 COL9A1
Multiple epiphyseal dysplasia due to collagen 9 anomaly 3 COL9A1
Autosomal recessive Stickler syndrome 2 COL9A2
Multiple epiphyseal dysplasia due to collagen 9 anomaly 3 COL9A2
Multiple epiphyseal dysplasia due to collagen 9 anomaly 3 COL9A3
Craniofacial-ulnar-renal syndrome 2 COLEC11
Synaptic congenital myasthenic syndromes 2 COLQ
Multiple epiphyseal dysplasia type 1 1 COMP
Pseudoachondroplasia 1 COMP
22qll.2 deletion syndrome 7 COMT
Leigh syndrome with nephrotic syndrome 4 COQ2
Familial steroid-resistant nephrotic syndrome with sensorineural deafness 1 COQ6
Encephalopathy - hypertrophic cardiomyopathy - renal tubular disease 1 COQ9
76
Table IX-21781338.1 Severe combined immunodeficiency due to COROIA deficiency 1 COROIA
Fatal infantile cytochrome C oxidase deficiency 5 COX10
Leigh syndrome with cardiomyopathy 8 COX10
Leigh syndrome with leukodystrophy 13 COX10
Isolated cytochrome C oxidase deficiency 9 COX 14
Fatal infantile cytochrome C oxidase deficiency 5l COX15
Leigh syndrome with cardiomyopathy 8 COX15
Isolated cytochrome C oxidase deficiency 9 COX20
Pancreatic insufficiency - anemia - hyperostosis 1 COX4I2
Isolated cytochrome C oxidase deficiency 9 COX6B1
Leigh syndrome with cardiomyopathy 8 COX6B1
Microphthalmia with linear skin defects syndromes 2 COX7B
Aceruloplasminemia 1 CP
Duane syndrome 2 CPA6
Familial mesial temporal lobe epilepsy with febrile seizures 1 CPA6
Hereditary coproporphyria 1 CPOX
Carbamoylphosphate synthetase deficiency 1 CPS1
Carnitine palmitoyi transferase 1A deficiency 1 CPT1A
Acute necrotizing encephalopathy of childhood 2 CPT2
Carnitine palmitoyi transferase II deficiency, myopathic form 1 CPT2
Carnitine palmitoyi transferase II deficiency, neonatal form 1 CPT2
Carnitine palmitoyi transferase II deficiency, severe infantile form 1 CPT2
Common variable immunodeficiency 10 CR2
Autosomal recessive nonsyndromic intellectual deficit 15 CRADD
Leber congenital amaurosis 18 CRB1
Pigmented paravenous retinochoroidal atrophy 1 CRB1
Retinitis pigmentosa 61 CRB1
Autosomal recessive nonsyndromic intellectual deficit 15 CRBN
Distal monosomy 3p 3 CRBN
Melanoma of soft part 3 CREB1
Myxofibrosarcoma 3 CREB3L1
Hyperlipoproteinemia type 5 4 CREB3L3
Rubinstein-Taybi syndrome due to CREBBP mutations 1 CREBBP
Complete atrioventricular canal 3 CRELD1
Partial atrioventricular canal 3 CRELD1
Distal 22qll.2 microdeletion syndrome 3 CRKL
Crisponi syndrome 1 CRLF1
Osteogenesis imperfecta type 2 5 CRTAP
Osteogenesis imperfecta type 3 9 CRTAP
Osteogenesis imperfecta type 4 7 CRTAP
Cone rod dystrophy 22 CRX
77
Table IX-21781338.1 Leber congenital amaurosis 18 CRX
Retinitis pigmentosa 61 CRX
Cataract-microcomea syndrome 8 CRYAA
Nuclear cataract 9 CRYAA
Zonular cataract 9 CRYAA
Alpha-crystallinopathy 1 CRYAB
Familial isolated dilated cardiomyopathy 38 CRYAB
Fatal infantile hypertonic myofibrillar myopathy 1 CRYAB
Posterior polar cataract 5 CRYAB
Zonular cataract 9 CRYAB
Cataract with Y-shaped suture opacities 3 CRYBA1
Zonular cataract 9 CRYBA1
Cataract-microcornea syndrome 8 CRYBA4
Microphthalmia - cataract 4 CRYBA4
Zonular cataract 9 CRYBA4
Cataract-microcornea syndrome 8 CRYBB1
Nuclear cataract 9 CRYBB1
Pulverulent cataract 7 CRYBB1
Cataract with Y-shaped suture opacities 3 CRYBB2
Cataract, Coppock-like 4 CRYBB2
Cataract-microcornea syndrome 8 CRYBB2
Cerulean cataract . 4 CRYBB2
Nuclear cataract 9 CRYBB2
Total congenital cataract 5 CRYBB2
Nuclear cataract 9 CRYBB3
Anterior polar cataract 2 CRYGB
Total congenital cataract 5 CRYGB
Zonular cataract 9 CRYGB
Cataract, Coppock-like 4 CRYGC
Cataract-microcornea syndrome 8 CRYGC
Pulverulent cataract 7 CRYGC
Zonular cataract 9 CRYGC
Cataract, Coppock-like 4 CRYGD
Cataract-microcornea syndrome 8 CRYGD
Cerulean cataract 4 CRYGD
Coralliform cataract 1 CRYGD
Nuclear cataract 9 CRYGD
Zonular cataract 9 CRYGD
Autosomal dominant childhood-onset cortical cataract 1 CRYGS
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 CRYM
Adult-onset leukoencephalopathy with axonal spheroids and pigmented 1 CSF1R
78
Table IX-21781338.1 glia
Congenital pulmonary alveolar proteinosis 5 CSF2RA
Congenital pulmonary alveolar proteinosis 5 CSF2RB
Atypical chronic myeloid leukemia 1 CSF3R
Chronic neutrophilic leukemia 1 CSF3R
Hereditary neutrophilia 1 CSF3R
Familial advanced sleep-phase syndrome 2 CSNK1D
Familial isolated dilated cardiomyopathy 38 CSRP3
Hereditary cerebral hemorrhage with amyloidosis, Icelandic type 1 CST3
Exfoliative ichthyosis 1 CSTA
Unverricht-Lundborg disease 3 CSTB
Anterior polar cataract 2 CTAA1
Coats plus syndrome 1 CTC1
Dyskeratosis congenita 10 CTC1
Congenital cataracts - facial dysmorphism - neuropathy 1 CTDP1
Diffuse cutaneous systemic sclerosis 4 CTGF
Limited cutaneous systemic sclerosis 4 CTGF
Cystathioninuria 1 CTH
Wegener granulomatosis 3 CTLA4
Autosomal dominant nonsyndromic intellectual deficit 15 CTNNB1
Desmoid disease 2 CTNNB1
Hepatocellular carcinoma, childhood-onset 2 CTNNB1
Pilomatrixoma 1 CTNNB1
Monosomy 5p 2 CTNND2
Hereditary chronic pancreatitis 6 CTRC
Galactosialidosis 1 CTSA
Haim-Munk syndrome 1 CTSC
Papillon-Lefevre syndrome 1 CTSC
CLN10 disease 1 CTSD
CLN13 disease 1 CTSF
Pycnodysostosis 1 CTSK
Grasbeck-lmerslund disease 2 CUBN
Pseudohypoaldosteronism type 2E 1 CUL3
Cabezas syndrome 1 CUL4B
3M syndrome 3 CUL7
WHIM syndrome 1 CXCR4
46,XY disorder of sex development due to isolated 17, 20 lyase deficiency 4 CYB5A
Recessive hereditary methemoglobinemia type 1 1 CYB5R3
Recessive hereditary methemoglobinemia type 2 1 CYB5R3 '
X-linked mendelian susceptibility to mycobacterial diseases due to CYBB
deficiency . 1 CYBB
79
Table IX-21781338.1 Autosomal thrombocytopenia with normal platelets 3 CYCS
Angelman syndrome 6 CYFIP1
Familial cylindromatosis 1 CYLD
Familial multiple trichoepithelioma 1 CYLD
46,XY disorder of sex development - adrenal insufficiency due to CYP11A1
deficiency 2 CYP11A1
Inherited isolated adrenal insufficiency due to CYP11A1 deficiency 1 CYP11A1
Congenital adrenal hyperplasia due to 11-beta-hydroxylase deficiency 1 CYP11B1
Familial hyperaldosteronism type 1 2 CYP11B1
Familial hyperaldosteronism type 1 2 CYP11B2
Familial hyperaldosteronism type 1 2 CYP11B2
Familial hyperreninemic hypoaldosteronism type 1 1 CYP11B2
46,XY disorder of sex development due to isolated 17, 20 lyase deficiency 4 CYP17A1
Congenital adrenal hyperplasia due to 17-alpha-hydroxylase deficiency 1 CYP17A1
Aromatase deficiency 1 CYP19A1
Aromatase excess syndrome 1 CYP19A1
Congenital glaucoma 3 CYP1B1
Juvenile glaucoma 2 CYP1B1
Peters anomaly 6 CYP1B1
Classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency,
salt wasting form 1 CYP21A2
Classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency,
simple virilizing form 1 CYP21A2
Nonclassic congenital adrenal hyperplasia due to 21-hydroxylase
deficiency 1 CYP21A2
Autosomal recessive infantile hypercalcemia 1 CYP24A1
Lethal occipital encephalocele-skeletal dysplasia syndrome 1 CYP26B1
Focal facial dermal dysplasia 2 CYP26C1
Cerebrotendinous xanthomatosis 1 CYP27A1
Hypocalcemic vitamin D dependent rickets 2 CYP27B1
Hypocalcemia vitamin D dependent rickets 2 CYP2R1
Autosomal recessive spastic paraplegia type 56 1 CYP2U1
Lamellar ichthyosis 6 CYP4F22
Bietti crystalline dystrophy 1 CYP4V2
Retinitis pigmentosa 61 CYP4V2
Hypercholesterolemia due to cholesterol 7alpha-hydroxylase deficiency . 1 CYP7A1
Autosomal recessive spastic paraplegia type 5A 1 CYP7B1
Congenital bile acid synthesis defect type 3 1 CYP7B1
D-2-hydroxyglutaric aciduria 2 D2HGDH
Craniorachischisis 1 DACT1
Occipital encephalocele 1 DACT1
Autosomal recessive limb-girdle muscular dystrophy - dystroglycanopathy 1 DAG1
80
Table IX-21781338.1 type C7
Amyotrophic lateral sclerosis 23 DAO
Leukoencephalopathy with brain stem and spinal cord involvement - lactate elevation 1 DARS2
Partial chromosome Y deletion 8 DAZ1
Partial chromosome Y deletion 8 DAZ2
Partial chromosome Y deletion 8 DAZ3
Partial chromosome Y deletion 8 DAZ4
Dopamine beta-hydroxylase deficiency 1 DBH
Classic maple syrup urine disease 4 DBT
Intermediate maple syrup urine disease 5 DBT
Intermittent maple syrup urine disease 4 DBT
Thiamin-responsive maple syrup urine disease 4 DBT
Woodhouse-Sakati syndrome 1 DCAF17
Familial congenital mirror movements 2 DCC
Omenn syndrome 9 DCLREIC
Severe combined immunodeficiency due to DCLREIC deficiency 1 DCLREIC
Congenital stromal corneal dystrophy 1 DCN
Amyotrophic lateral sclerosis 23 DCTNl
Distal hereditary motor neuropathy type 7 2 DCTN1
Perry syndrome 1 DCTNl
Cystic fibrosis 4 DCTN4
Lissencephaly type 1 due to doublecortin gene mutation 1 DCX
Subcortical band heterotopia 2 DCX
Pentosuria 1 DCXR
Xeroderma pigmentosum complementation group E 1 DDB2
Aromatic L-amino acid decarboxylase deficiency 1 DDC
Autosomal recessive spastic paraplegia type 28 1 DDHD1
Autosomal recessive spastic paraplegia type 54 1 DDHD2
Myxoid/round cell liposarcoma 2 DDIT3
DDOST-CDG syndrome 1 DDOST
Spondyloepimetaphyseal dysplasia - short limb - abnormal calcification 1 DDR2
Warsaw breakage syndrome 1 DDX11
Precursor T-cell acute lymphoblastic leukemia 19 DDX3X
Partial chromosome Y deletion 8 DDX3Y
Autosomal dominant epilepsy with auditory features 2 DEPDC5
Familial focal epilepsy with variable foci 1 DEPDC5
Nocturnal frontal lobe epilepsy 5 DEPDC5
Autosomal dominant limb-girdle muscular dystrophy type IE 1 DES
Desminopathy 1 DES
Familial isolated dilated cardiomyopathy 38 DES
81
Table IX-21781338.1 Scapuloperoneal amyotrophy 4 DES
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 DFNA5
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 DFNB31
Usher syndrome type 2 4 DFNB31
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 DFNB59
Congenital chronic diarrhea with protein-losing enteropathy 1 DGAT1
Atypical hemolytic uremic syndrome with DGKE deficiency 1 DGKE
Immunoglobulin-mediated membranoproliferative glomerulonephritis 2 DGKE
Adult-onset multiple mitochondrial DNA deletion syndrome due to DGUOK
deficiency 1 DGUOK
Adult-onset multiple mitochondrial DNA deletion syndrome due to DGUOK
deficiency 1 DGUOK
Mitochondrial DNA depletion syndrome, hepatocerebral form due to
DGUOK deficiency 1 DGUOK
Desmosterolosis . 1 DHCR24
Smith-Lemli-Opitz syndrome 1 DHCR7
Smith-Magenis syndrome 3 DHCR7
Retinitis pigmentosa 61 DHDDS
Constitutional megaloblastic anemia with severe neurologic disease 1 DHFR
46,XY complete gonadal dysgenesis 8 DHH
46, XY gonadal dysgenesis - motor and sensory neuropathy 1 DHH
Postaxial acrofacial dysostosis 1 DHODH
2-aminoadipic 2-oxoadipic aciduria 1 DHTKD1
Autosomal dominant Charcot-Marie-Tooth disease type 2Q 1 DHTKD1
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 DIABLO
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 DIAPH1
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 DIAPH3
Familial multinodular goiter 1 DICERl
Ovarian malignant Sertoli-Leydig cell tumor 1 DICERl
Pleuropulmonary blastoma family tumor susceptibility syndrome 1 DICERl
Familial renal cell carcinoma 6 DIRCl
Familial renal cell carcinoma 6 DIRC2
Familial renal cell carcinoma 6 DIRC3
Nephroblastoma 5 DIS3L2
Perlman syndrome 1 DIS3L2
Alobar holoprosencephaly 14 DISP1
Lobar holoprosencephaly 14 DISP1
Microform holoprosencephaly 14 DISP1
Midline interhemispheric variant of holoprosencephaly 14 DISP1
Semilobar holoprosencephaly 14 DISP1
Septopreoptic holoprosencephaly 14 DISP1
Dyskeratosis congenita 10 DKC1
82
Table IX-21781338.1 Hoyeraal-Hreidarsson syndrome 4 DKC1
Idiopathic juvenile osteoporosis 4 DKK1
Idiopathic juvenile osteoporosis 4 DKK1
Pyruvate dehydrogenase E2 deficiency 1 DLAT
Classic maple syrup urine disease 4 DLD
Intermediate maple syrup urine disease 5 DLD
Intermittent maple syrup urine disease 4 DLD
Leigh syndrome with nephrotic syndrome 4 DLD
Pyruvate dehydrogenase E3 deficiency 1 DLD
Thiamin-responsive maple syrup urine disease 4 DLD
X-linked nonsyndromic intellectual deficit 24 DLG3
Maternal uniparental disomy of chromosome 14 3 DLK1
Paternal uniparental disomy of chromosome 14 3 DLK1
Alobar holoprosencephaly 14 DLL1
Lobar holoprosencephaly 14 DLL1
Microform holoprosencephaly 14 DLL1
Midline interhemispheric variant of holoprosencephaly 14 DLL1
Semilobar holoprosencephaly 14 DLL1
Septopreoptic holoprosencephaly 14 DLL1
Autosomal recessive spondylocostal dysostosis 4 DLL3
Hypomaturation-hypoplastic amelogenesis imperfecta with taurodontism 1 DLX3
Tricho-dento-osseous syndrome 1 DLX3
Split hand - split foot - deafness 1 DLX5
Split hand - split foot - deafness 1 DLX5
Becker muscular dystrophy 1 DMD
Congenital stationary night blindness 12 DMD
Duchenne muscular dystrophy 2 DMD
Familial isolated dilated cardiomyopathy 38 DMD
Symptomatic form of muscular dystrophy of Duchenne and Becker in
female carriers 1 DMD
Dimethylglycine dehydrogenase deficiency 1 DMGDH
Autosomal recessive hypophosphatemic rickets 2 DMP1
Steinert myotonic dystrophy 1 DMPK
46,XY complete gonadal dysgenesis 8 DMRT1
46,XY complete gonadal dysgenesis 8 DMRT2
Mitochondrial DNA deletion syndrome with progressive myopathy 1 DNA2
Primary ciliary dyskinesia 21 DNAAF2
Primary ciliary dyskinesia 21 DNAAF3
Primary ciliary dyskinesia 21 DNAH11
Primary ciliary dyskinesia 21 DNAH5
Primary ciliary dyskinesia 21 DNAI1
83
Table IX-21781338.1 Primary ciliary dyskinesia 21 DNAI2
Young adult-onset distal hereditary motor neuropathy 1 DNAJB2
Autosomal dominant limb-girdle muscular dystrophy type ID 1 DNAJB6
Dilated cardiomyopathy with ataxia 1 DNAJC19
Williams syndrome 17 DNAJC30
CLN4B disease 1 DNAJC5
Juvenile parkinsonism with intellectual deficit due to DNAJC6 deficiency 1 DNAJC6
Levodopa-unresponsive juvenile parkinsonism 1 DNAJC6
Primary ciliary dyskinesia 21 DNAL1
Autosomal recessive systemic lupus erythematosus 1 DNASE1L3
Pediatric systemic lupus erythematosus 1 DNASE1L3
Lethal encephalopathy due to mitochondrial and peroxisomal fission
defect 1 DNM1L
Autosomal dominant centronuclear myopathy 4 DNM2
Autosomal dominant Charcot-Marie-Tooth disease type 2M 1 DNM2
Autosomal dominant Charcot-Marie-Tooth disease type 2M 1 DNM2
Autosomal dominant intermediate Charcot-Marie-Tooth disease type B 1 DNM2
Cerebellar ataxia-deafness-narcolepsy syndrome 1 DNMT1
Hereditary sensory and autonomic neuropathy type 1 4 DNMT1
ICF syndrome 2 DNMT3B
Adams-Oliver syndrome 4 DOCK6
Autosomal dominant nonsyndromic intellectual deficit 15 DOCK8
Autosomal recessive hyper IgE syndrome 1 DOCK8
Combined immunodeficiency due to DOCK8 deficiency 1 DOCK8
Fetal akinesia deformation sequence. 2 DOK7
Postsynaptic congenital myasthenic syndromes 9 DOK7
DK1-CDG syndrome 1 DOLK
Familial isolated dilated cardiomyopathy 38 DOLK
Congenital myasthenic syndromes with glycosylation defect 4 DPAGT1
DPAGT1-CDG syndrome 1 DPAGT1
Diffuse panbronchiolitis 2 DPC 1
DPM1-CDG syndrome 1 DPMI
DPM2-CDG syndrome 1 DPM2
DPM3-CDG syndrome 1 DPM3
Idiopathic ventricular fibrillation, not Brugada type 2 DPP6
Idiopathic pulmonary fibrosis 11 DPP9
Globozoospermia 2 DPY19L2
Dihydropyrimidine dehydrogenase deficiency 1 DPYD
Dihydropyrimidinuria 1 DPYS
Primary ciliary dyskinesia 21 DRC1
Myoclonic dystonia 11 3 DRD2
84
Table IX-21781338.1 Benign essential blepharospasm 1 D D5
Cervical dystonia 2 DRD5
Familial isolated arrhythmogenic ventricular dysplasia, biventricular form ' 10 DSC2
Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 DSC2
Familial isolated arrhythmogenic ventricular dysplasia, right dominant
form 10 DSC2
Hereditary hypotrichosis with recurrent skin vesicles 1 DSC3
Ehlers-Danlos syndrome, musculocontractural type 2 DSE
Keratosis palmoplantaris striata 3 DSG1
Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 DSG2
Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 DSG2
Familial isolated arrhythmogenic ventricular dysplasia, right dominant
form 10 DSG2
Familial isolated dilated cardiomyopathy 38 DSG2
Hypotrichosis simplex 6 DSG4
Monilethrix 4 DSG4
Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 DSP
Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 DSP
Familial isolated arrhythmogenic ventricular dysplasia, right dominant
form 10 DSP
Idiopathic pulmonary fibrosis 11 DSP
Keratosis palmoplantaris striata 3 DSP
Lethal acantholytic epidermolysis bullosa 2 DSP
Skin fragility-woolly hair-palmoplantar keratoderma syndrome 1 DSP
Woolly hair-palmoplantar keratoderma-dilated cardiomyopathy syndrome 1 DSP
Dentin dysplasia type 1 1 DSPP
Dentin dysplasia type II 1 DSPP
Dentinogenesis imperfecta type 2 1 DSPP
Dentinogenesis imperfecta type 3 1 DSPP
Autosomal recessive epidermolysis bullosa simplex 2 DST
Hereditary sensory and autonomic neuropathy type 6 1 DST
Left ventricular noncompactioh 11 DTNA
Hermansky-Pudlak syndrome type 7 1 DTNBP1
Familial thyroid dyshormonogenesis 6 DUOX2
Genetic transient congenital hypothyroidism 1 DUOX2
Familial thyroid dyshormonogenesis 6 DUOXA2
Kallmann syndrome 19 DUSP6
Normosmic congenital hypogonadotropic hypogonadism 18 DUSP6
Facioscapulohumeral dystrophy 4 DUX4
D ggve-Melchior-Clausen disease 1 DYM
Smith-McCort dysplasia 2 DYM
Autosomal dominant Charcot-Marie-Tooth disease type 20 1 DYNC1H1
85
Table IX-21781338.1 Autosomal dominant nonsyndromic intellectual deficit 15 DYNC1H1
Childhood-onset proximal spinal muscular atrophy, autosomal dominant 1 DYNC1H1
Jeune syndrome 6 DYNC2H1
Short rib-polydactyly syndrome, Majewski type 2 DYNC2H1
Short rib-polydactyly syndrome, Verma-Naumoff type 3 DYNC2H1
Autosomal dominant nonsyndromic intellectual deficit 15 DYRK1A
Autosomal recessive limb-girdle muscular dystrophy type 2B 1 DYSF
Congenital myopathy, Paradas type 1 DYSF
Distal myopathy with anterior tibial onset 1 DYSF
Miyoshi myopathy 2 DYSF
Primary dystonia, DYT13 type 1 DYT13
Myoclonic dystonia 15 1 DYT15
Primary dystonia, DYT2 type 2 DYT17
Primary dystonia, DYT2 type 2 DYT2
Primary dystonia, DYT21 type 1 DYT21
Autosomal dominant focal dystonia, DYT7 type 1 DYT7
Leukoencephalopathy - thalamus and brainstem anomalies - high lactate 1 EARS2
X-linked dominant chondrodysplasia punctata 1 EBP
Hirschsprung disease 7 ECE1
Distal arthrogryposis type 5D 1 ECEL1
Lipoid proteinosis 1 ECM1
Oligodontia 10 EDA
X-linked hypohidrotic ectodermal dysplasia 2 EDA
X-linked hypohidrotic ectodermal dysplasia 2 EDA2R
Autosomal dominant hypohidrotic ectodermal dysplasia 3 EDAR
Autosomal recessive hypohidrotic ectodermal dysplasia 3 EDAR
Autosomal dominant hypohidrotic ectodermal dysplasia 3 EDARADD
Autosomal recessive hypohidrotic ectodermal dysplasia 3 EDARADD
Oligodontia 10 EDARADD
Hirschsprung disease 7 EDN3
Ondine syndrome 5 EDN3
Waardenburg-Shah syndrome 3 EDN3
Hirschsprung disease 7 EDNRB
Waardenburg-Shah syndrome 3 EDNRB
Familial drusen 2 EFEMP1
Autosomal recessive cutis laxa type 1 2 EFEMP2
Lethal arteriopathy syndrome due to FBLN4 deficiency 1 EFEMP2
Juvenile absence epilepsy 1 EFHC1
Juvenile myoclonic epilepsy 7 EFHC1
Isolated plagiocephaly 3 EFNA4
Craniofrontonasal dysplasia 1 EFNB1
86
Table IX-21781338.1 Mandibulofacial dysostosis-microcephaly syndrome 1 EFTUD2
Adult hepatocellular carcinoma 1 EGF
Familial primary hypomagnesemia with normocalcuria and normocalcemia 2 EGF
Giant cell glioblastoma 10 EGFR
Gliosarcoma 10 EGFR
Autosomal dominant secondary polycythemia 2 EGLN1
Charcot-Marie-Tooth disease type ID 1 EGR2
Charcot-Marie-Tooth disease type 4E 1 EGR2
Dejerine-Sottas syndrome 4 EGR2
Bifunctional enzyme deficiency 2 EHHADH
Kleefstra syndrome due to 9q34 microdeletion 1 EHMT1
Kleefstra syndrome due to a point mutation 1 EHMT1
Wolcott-Rallison syndrome 1 EIF2AK3
Cree leukoencephalopathy 5 EIF2B1
Ovarioleukodystrophy 5 EIF2B1
Cree leukoencephalopathy 5 EIF2B2
Ovarioleukodystrophy . 5 EIF2B2
Cree leukoencephalopathy 5 EIF2B3
Ovarioleukodystrophy 5 EIF2B3
Cree leukoencephalopathy 5 EIF2B4
Ovarioleukodystrophy 5 EIF2B4
Cree leukoencephalopathy 5 EIF2B5
Ovarioleukodystrophy 5 EIF2B5
Young adult-onset Parkinsonism 13 EIF4G1
Williams syndrome 17 EIF4H
Familial prostate cancer 14 ELAC2
Autosomal dominant severe congenital neutropenia 2 ELANE
Cyclic neutropenia 1 ELANE
Short stature due to isolated growth hormone deficiency with X-linked
hypogammaglobulinemia 2 ELF4
Autosomal dominant cutis laxa 2 ELN
Supravalvular aortic stenosis 1 ELN
Williams syndrome 17 ELN
Congenital ichthyosis - intellectual deficit - spastic quadriplegia 1 ELOVL4
Stargardt disease 3 ELOVL4
X-linked Emery-Dreifuss muscular dystrophy 2 EMD
Bowen-Conradi syndrome 1 EMG1
Schizencephaly 4 EMX2
Hypoplastic amelogenesis imperfecta 1 ENAM
Generalized juvenile polyposis/juvenile polyposis coli 3 ENG
Rendu-Osler-Weber disease 3 ENG
87
Table IX-21781338.1 Glycogen storage disease due to muscle beta-enolase deficiency 1 EN03
Autosomal recessive hypophosphatemic rickets 2 ENPP1
Generalized arterial calcification of infancy 2 ENPP1
Adams-Oliver syndrome 4 EOGT
Microcephaly - polymicrogyria - corpus callosum agenesis 1 EOMES
Rubinstein-Taybi syndrome due to EP300 haploinsufficiency 1 EP300
Autosomal dominant secondary polycythemia 2 EPAS1
Multiple paragangliomas associated with polycythemia 1 EPAS1
Sporadic pheochromocytoma 1 EPAS1
Sporadic secreting paraganglioma 1 EPAS1
Hereditary elliptocytosis 3 EPB41
Autosomal dominant nonsyndromic intellectual deficit 15 EPB41L1
Hereditary spherocytosis 5 EPB42
Hereditary nonpolyposis colon cancer 11 EPCAM
Intestinal epithelial dysplasia 1 EPCAM
Vici syndrome 1 EPG5
Posterior polar cataract 5 EPHA2
Total congenital cataract 5 EPHA2
Familial prostate cancer 14 EPHB2
Familial hypercholanemia 3 EPHX1
Fetal hydantoin syndrome 1 EPHX1
Lafora disease 2 EPM2A
Primary familial polycythemia 1 EPOR
Primary familial polycythemia 1 EPOR
Marie Unna hereditary hypotrichosis 2 EPS8L3
Lethal congenital contracture syndrome type 2 1 ERBB3
Distal monosomy 12p 1 ERC1
Papillary or follicular thyroid carcinoma 14 ERC1
Cockayne syndrome type 2 3 ERCC1
COFS syndrome 4 ERCC1
Beta-thalassemia - trichothiodystrophy 1 ERCC2
COFS syndrome 4 ERCC2
IBIDS syndrome 3 ERCC2
PIBIDS syndrome 3 ERCC2
Xeroderma pigmentosum complementation group D 1 ERCC2
Xeroderma pigmentosum/Cockayne syndrome complex 3 ERCC2
IBIDS syndrome 3 ERCC3
PIBIDS syndrome 3 ERCC3
Xeroderma pigmentosum complementation group B 1 ERCC3
Xeroderma pigmentosum/Cockayne syndrome complex 3 ERCC3
Cockayne syndrome type 1 3 ERCC4
88
Table IX-21781338.1 Fanconi anemia 16 ERCC4
Xeroderma pigmentosum complementation group F 1 ERCC4
COFS syndrome 4 ERCC5
Xeroderma pigmentosum complementation group G 1 ERCC5
Xeroderma pigmentosum/Cockayne syndrome complex 3 ERCC5 .
Cockayne syndrome type 1 3 ERCC6
Cockayne syndrome type' 2 3 ERCC6
Cockayne syndrome type 3 2 ERCC6
COFS syndrome 4 ERCC6
UV-sensitive syndrome 3 ERCC6
Cockayne syndrome type 1 3 ERCC8
Cockayne syndrome type 2 3 ERCC8
Cockayne syndrome type 3 2 ERCC8
UV-sensitive syndrome 3 ERCC8
Crouzon disease 2 ERF
Isolated cloverleaf skull syndrome 2 ERF
Isolated scaphocephaly 3 ERF
Ewing sarcoma 5 ERG
Autosomal recessive spastic paraplegia type 18 1 ERLIN2
Juvenile primary lateral sclerosis 2 ERLIN2
Recessive intellectual disability - motor dysfunction - multiple joint
contractures 1 ERLIN2
Roberts syndrome 1 ESC02
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 ESPN
Estrogen resistance syndrome 1 ESR1
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 ESRRB
Glutaric acidemia type 2 3 ETFA
Glutaric acidemia type 2 3 ETFB
Glutaric acidemia type 2 3 ETFDH
Ethylmalonic encephalopathy 1 ETHE1
Ewing sarcoma 5 ETVl
Ewing sarcoma 5 ETV4
Chronic myelomonocytic leukemia 2 ETV6
Congenital mesoblastic nephroma 2 ETV6
Fibrosarcoma 2 ETV6
Precursor B-cell acute lymphoblastic leukemia 13 ETV6
Acrofacial dysostosis, Weyers type 2 EVC
Ellis Van Creveld syndrome 2 EVC
Acrofacial dysostosis, Weyers type 2 EVC2
Ellis Van Creveld syndrome 2 EVC2
Desmoplastic small round cell tumor 2 EWSR1
89
Table IX-21781338.1 Ewing sarcoma 5 EWSR1
Melanoma of soft part 3 EWSR1
Pontocerebellar hypoplasia type 1 < 4 EXOSC3
Chondrosarcoma 1 EXT1
Langer-Giedion syndrome 2 EXT1
Multiple osteochondromas 2 EXT1
Multiple osteochondromas 2 EXT2
Potocki-Shaffer syndrome 3 EXT2
BO syndrome 3 EYAl
Branchio-otic syndrome 2 EYA1
Otofaciocervical syndrome 1 EYAl
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 EYA4
Sensorineural deafness with dilated cardiomyopathy 1 EYA4
Retinitis pigmentosa 61 EYS
Weaver syndrome 2 EZH2
Congenital factor X deficiency 1 F10
Congenital factor XI deficiency 1 Fll
Congenital factor XII deficiency 1 F12
Hereditary angioedema type 3 1 F12
Congenital factor XIII deficiency 2 F13A1
Congenital factor XIII deficiency 2 F13B
Congenital factor II deficiency 1 F2
Budd-Chiari syndrome 2 F5
Congenital factor V deficiency 1 F5
Congenital factor VII deficiency 1 F7
Mild hemophilia A 1 F8
Moderately severe hemophilia A 1 F8
Severe hemophilia A 1 F8
Symptomatic form of hemophilia A in female carriers 1 F8
Mild hemophilia B 1 F9
Moderately severe hemophilia B 1 F9
Severe hemophilia B 1 F9
Symptomatic form of hemophilia B in female carriers 1 F9
Autosomal recessive spastic paraplegia type 35 1 FA2H
Fatty acid hydroxylase-associated neurodegeneration 1 FA2H
FADD-related immunodeficiency 1 FADD
Oculootodental syndrome 2 FADD
Tyrosinemia type 1 1 FAH
Autosomal dominant Kenny-Caffey syndrome 1 FAM111A
Osteocraniostenosis 1 FAM111A
Hypomyelination - congenital cataract . 1 FAM126A
90
Table IX-21781338.1 Hereditary sensory and autonomic neuropathy type 2 4 FAM134B
Idiopathic pulmonary fibrosis 11 FAM13A
Retinitis pigmentosa 61 FAM161A
Amelogenesis imperfecta - nephrocalcinosis 1 FAM20A
Amelogenesis imperfecta and gingival hyperplasia syndrome 1 FAM20A
Lethal osteosclerotic bone dysplasia 1 FAM20C
Syndactyly - telecanthus - anogenital and renal malformations 1 FAM58A
Hypocalcified amelogenesis imperfecta 2 FAM83H
Late-onset autosomal recessive medullary cystic kidney disease 3 FAN1
Fanconi anemia 16 - FANCA
Fanconi anemia 16 FANCB
VACTERL with hydrocephalus 1 FANCB
Fanconi anemia 16 FANCC
Fanconi anemia 16 FANCD2
Fanconi anemia 16 FANCE
Fanconi anemia 16 FANCF
Fanconi anemia 16 FANCG
Fanconi anemia 16 FANCI
Fanconi anemia 16 FANCL
Fanconi anemia 16 FAN CM
Combined oxidative phosphorylation defect type 14 1 FARS2
Autoimmune lymphoproliferative syndrome 6 FAS
Juvenile rheumatoid factor-negative polyarthritis 9 FAS
Oligoarticular juvenile arthritis 9 FAS
Autoimmune lymphoproliferative syndrome 6 FASLG
FASTKD2-related infantile mitochondrial encephalomyopathy 1 FASTKD2
Isolated cytochrome C oxidase deficiency 9 FASTKD2
Synpolydactyly type 2 1 FBLN1
Autosomal dominant cutis laxa 2 FBLN5
Autosomal recessive cutis laxa type 1 2 FBLN5
Hereditary sensorimotor neuropathy with hyperelastic skin 1 FBLN5
Acromicric dysplasia 1 FBN1
Acromicric dysplasia 1 FBN1
Ectopia lentis syndrome 2 FBN1
Familial thoracic aortic aneurysm and aortic dissection 8 FBN1
Geleophysic dysplasia 2 FBN1
Glaucoma - ectopia - microspherophakia - stiff joints - short stature 1 FBN1
Marfan syndrome type 1 1 FBN1
Neonatal Marfan syndrome 1 FBN1
Progeroid and marfanoid aspect-lipodystrophy syndrome 1 FBN1
Shprintzen-Goldberg syndrome 2 FBN1
91
Table IX-21781338.1 Stiff skin syndrome 1 FBN1
Weill-Marchesani syndrome 4 FBN1
Congenital contractural arachnodactyly 1 FBN2
Fructose-l,6-bisphosphatase deficiency 1 FBP1
Parkinsonian-pyramidal syndrome 2 FBX07
Split hand-split foot malformation 4 FBXW4
Immunodeficiency due to ficolin3 deficiency 1 FCN3
Erythropoietic protoporphyria 2 FECH
Hereditary acrokeratotic poikiloderma, Weary type 1 FE MT1
Kindler syndrome 2 FERMT1
Leukocyte adhesion deficiency type III 1 FERMT3
Familial afibrinogenemia 3 FGA
Familial dysfibrinogenemia 3 FGA
Familial hypodysfibrinogenemia 3 FGA
Familial hypofibrinogenemia 3 FGA
Familial renal amyloidosis due to fibrinogen A alpha-chain variant 1 FGA
Familial afibrinogenemia 3 FGB
Familial dysfibrinogenemia 3 FGB
Familial hypodysfibrinogenemia 3 FGB
Familial hypofibrinogenemia 3 FGB
Aarskog-Scott syndrome 1 FGD1
Charcot-Marie-Tooth disease type 4H 1 FGD4
Aplasia of lacrimal and salivary glands 1 FGF10
Lacrimo-auriculo-dento-digital syndrome 3 FGF10
Spinocerebellar ataxia type 27 1 FGF14
Kallmann syndrome 19 FGF17
Normosmic congenital hypogonadotropic hypogonadism 18 FGF17
Autosomal dominant hypophosphatemic rickets 1 FGF23
Autosomal dominant hypophosphatemic rickets 1 FGF23
Hypercalcemic tumoral calcinosis 3 FGF23
Deafness with labyrinthine aplasia, microtia, and microdontia 1 FGF3
Oculootodental syndrome 2 FGF3
Otodental syndrome 1 FGF3
Alobar holoprosencephaly 14 FGF8
Kallmann syndrome 19 FGF8
Lobar holoprosencephaly 14 FGF8
Microform holoprosencephaly 14 FGF8
Midline interhemispheric variant of holoprosencephaly 14 FGF8
Normosmic congenital hypogonadotropic hypogonadism 18 FGF8
Semilobar holoprosencephaly 14 FGF8
Septopreoptic holoprosencephaly 14 FGF8
92
Table IX-21781338.1 Multiple synostoses syndrome 3 FGF9
Giant cell glioblastoma 10 FGFRl .
Gliosarcoma 10 FGFRl
Isolated trigonocephaly 2 FGFRl
Kallmann syndrome 19 FGFRl
Myeloid neoplasm associated with FGFRl rearrangement 1 FGFRl
Normosmic congenital hypogonadotropic hypogonadism 18 FGFRl
Oligodontia 10 FGFRl
Osteoglophonic dwarfism 1 FGFRl Pfeiffer syndrome type 1 2 FGFRl
Septo-optic dysplasia 6 FGFRl
Antley-Bixler syndrome 1 FGFR2
Apert syndrome 1 FGFR2
Crouzon disease 2 FGFR2
Cutis gyrata - acanthosis nigricans - craniosynostosis 2 FGFR2
Familial scaphocephaly syndrome, McGillivray type 1 FGFR2
FGFR2-related bent bone dysplasia 1 FGFR2
Jackson-Weiss syndrome 2 FGFR2
Lacrimo-auriculo-dento-digital syndrome 3 FGFR2
Pfeiffer syndrome type 1 2 FGFR2
Pfeiffer syndrome type 2 1 FGFR2
Pfeiffer syndrome type 3 1 FGFR2
Saethre-Chotzen syndrome 4 FGFR2
Achondroplasia 1 FGFR3
Camptodactyly - tall stature - scoliosis - hearing loss 1 FGFR3
Crouzon syndrome - acanthosis nigricans 1 FGFR3
Cutis gyrata - acanthosis nigricans - craniosynostosis 2 FGFR3
Giant cell glioblastoma 10 FGFR3
Gliosarcoma 10 FGFR3
Hypochondroplasia 1 FGFR3
Isolated brachycephaly 3 FGFR3
Isolated cloverleaf skull syndrome 2 FGFR3
Isolated plagiocephaly 3 FGFR3
Lacrimo-auriculo-dento-digital syndrome 3 FGFR3
Muenke syndrome 1 FGFR3
Muenke syndrome 1 FGFR3
Saethre-Chotzen syndrome 4 FGFR3
Severe achondroplasia - developmental delay - acanthosis nigricans 1 FGFR3
Thanatophoric dwarfism type 1 1 FGFR3
Thanatophoric dwarfism type II 1 FGFR3
Familial afibrinogenemia 3 FGG
93
Table IX-21781338.1 Familial dysfibrinogenemia 3 FGG
Familial hypodysfibrinogenemia 3 FGG
Familial hypofibrinogenemia 3 FGG
Familial leiomyomatosis 1 FH
Fumaric aciduria 1 FH
Familial renal cell carcinoma 6 FHIT
Reducing body myopathy 1 FHL1
Scapuloperoneal amyotrophy 4 FHL1
X-linked Emery-Dreifuss muscular dystrophy 2 FHL1
X-linked myopathy with postural muscle atrophy 1 FHL1
Familial isolated dilated cardiomyopathy 38 FHL2
Amyotrophic lateral sclerosis 23 FIG4
Charcot-Marie-Tooth disease type 4J 1 FIG4
Primary lateral sclerosis 1 FIG4
Yunis-Varon syndrome 1 FIG4
Idiopathic hypereosinophilic syndrome 3 FIP1L1
Bruck syndrome 2 FKBP10
Osteogenesis imperfecta type 5 2 FKBP10
Ehlers-Danlos syndrome, kyphoscoliotic and deafness type 1 FKBP14
Williams syndrome 17 FKBP6
Autosomal recessive limb-girdle muscular dystrophy type 21 1 FKRP
Congenital muscular dystrophy type 1C 1 FKRP
Muscle eye brain disease 7 FKRP
Walker-Warburg syndrome 13 FKRP
Autosomal recessive limb-girdle muscular dystrophy type 2M 1 FKTN
Familial isolated dilated cardiomyopathy 38 FKTN
Muscle eye brain disease 7 FKTN
Walker-Warburg syndrome 13 FKTN
Birt-Hogg-Dube syndrome 1 FLCN
Familial spontaneous pneumothorax 1 FLCN
Ewing sarcoma 5 FLU
Paris-Trousseau thrombocytopenia 1 FLU
Smith-Magenis syndrome 3 FLU
Chronic intestinal pseudo-obstruction 1 FLNA
Congenital short bowel syndrome 2 FLNA
Congenital valvular dysplasia 1 FLNA
Ehlers-Danlos syndrome with periventricular heterotopia 1 FLNA
Frontometaphyseal dysplasia 1 FLNA
Osteodysplasty, Melnick-Needles type 1 FLNA
Otopalatodigital syndrome type 1 1 FLNA
Otopalatodigital syndrome type 2 1 FLNA
94
Table IX-21781338.1 Periventricular nodular heterotopia 2 FLNA
Terminal osseous dysplasia - pigmentary defects 1 FLNA
Atelosteogenesis 1 1 FLNB
Atelosteogenesis type III 1 FLNB
Autosomal dominant Larsen syndrome 1 FLNB
Boomerang dysplasia 1 FLNB
Synspondylism 1 FLNB
Distal myopathy with posterior leg and anterior hand involvement 1 FLNC
Muscle filaminopathy 1 FLNC
Kallmann syndrome 19 FLRT3
Acute biphenotypic leukemia 2 FLT3
Precursor B-cell acute lymphoblastic leukemia 13 FLT3
Precursor T-cell acute lymphoblastic leukemia 19 FLT3
Milroy disease 2 FLT4 -
Posterior column ataxia - retinitis pigmentosa 1 FLVCR1
Fowler syndrome 1 FLVCR2
Trimethylaminuria 1 FM03
Fragile X syndrome 1 FMR1
Fragile X-associated tremor/ataxia syndrome 1 FMR1
Xq27.3q28 duplication syndrome 1 FMR1
FRAXE intellectual deficit 2 FMR3
Fibronectin glomerulopathy 1 FN1
Neurodegenerative syndrome due to cerebral folate transport deficiency 1 FOLR1
Axenfeld-Rieger syndrome 2 FOXC1
Axenfeld's anomaly 2 FOXC1
Peters anomaly 6 FOXC1
Rieger's anomaly 2 FOXC1
Lymphedema - distichiasis 1 FOXC2
Yellow nail syndrome 1 FOXC2
Vitiligo-associated autoimmune disease 2 FOXD3
Athyreosis 5 FOXE1
Bamforth syndrome 1 FOX El
Papillary or follicular thyroid carcinoma 14 FOXE1
Thyroid hypoplasia 5 FOXE1
Congenital primary aphakia 1 FOXE3
Familial ocular anterior segment mesenchymal dysgenesis 2 FOXE3
Congenital alveolar capillary dysplasia 1 FOXF1
14qll.2 microduplication syndrome 1 FOXG1
14ql2 microdeletion syndrome 1 FOXG1
Atypical Rett syndrome 4 FOXG1
Alobar holoprosencephaly 14 FOXH1
95
Table IX-21781338.1 Lobar holoprosencephaly 14 FOXH1
Microform holoprosencephaly 14 FOXH1
Midline interhemispheric variant of holoprosencephaly 14 FOXH1
Semilobar holoprosencephaly 14 FOXH1
Septopreoptic holoprosencephaly 14 FOXH1
Pendred syndrome 3 FOXI1
Blepharophimosis - epicanthus inversus - ptosis due to a point mutation 1 FOXL2
Blepharophimosis - epicanthus inversus - ptosis, due to 3q23
microdeletion 1 FOXL2
Severe T-cell immunodeficiency - congenital alopecia - nail dystrophy 1 FOXN1
Alveolar rhabdomyosarcoma 4~ FOXOl
MALT lymphoma 4 FOXP1
7q31 microdeletion syndrome 1 FOXP2
Childhood apraxia of speech 1 FOXP2
X-linked immune dysregulation - polyendocrinopathy - enteropathy 1 FOXP3
Isolated NADH-CoQ reductase deficiency 25 FOXRED1
Fraser syndrome 3 FRAS1
BNAR syndrome 1 FREM1
Congenital diaphragmatic hernia 2 FREM1
Isolated trigonocephaly 2 FREM1
Oculotrichoanal syndrome 1 FREM1
Fraser syndrome 3 FREM2
Facioscapulohumeral dystrophy 4 FRG1
Retinitis pigmentosa 61 FSCN2
Isolated follicle stimulating hormone deficiency 1 FSHB
46,XX gonadal dysgenesis 4 FSHR
Ovarian hyperstimulation syndrome 1 FSHR
Formiminoglutamic aciduria 1 FTCD
FTHl-related iron overload 1 FTH1
Genetic hyperferritinemia without iron overload 1 FTL
Hereditary hyperferritinemia with congenital cataracts 1 FTL
Neuroferritinopathy 1 FTL
Lethal polymalformative syndrome, Boissel type 1 FTO
X-linked nonsyndromic intellectual deficit 24 FTSJl
Fucosidosis 1 FUCA1
Amyotrophic lateral sclerosis 23 FUS
Frontotemporal dementia with motor neuron disease 3 FUS
Juvenile amyotrophic lateral sclerosis 3 FUS
Myxofibrosarcoma 3 FUS
Myxoid/round cell liposarcoma 2 FUS
Arnold-Chiari malformation type II 1 FUZ
96
Table IX-21781338.1 Caudal regression sequence 2 FUZ
Cervical spina bifida aperta 6 FUZ
Cervical spina bifida cystica 6 FUZ
Cervicothoracic spina bifida aperta 6 FUZ
Cervicothoracic spina bifida cystica 6 FUZ
Lumbosacral spina bifida aperta 6 FUZ
Lumbosacral spina bifida cystica 6 FUZ
Thoracolumbosacral spina bifida aperta 6 FUZ
Thoracolumbosacral spina bifida cystica 6 FUZ
Total spina bifida aperta 6 FUZ
Total spina bifida cystica 6 FUZ
Upper thoracic spina bifida aperta 6 FUZ
Upper thoracic spina bifida cystica 6 FUZ
Friedreich ataxia 1 FXN
Autosomal dominant primary hypomagnesemia with hypocalcuria 2 FXYD2
Nuclear cataract 9 FYCOl
Familial exudative vitreoretinopathy 5 FZD4
Persistent hyperplastic primary vitreous 3 FZD4
Retinopathy of prematurity 3 FZD4
Autosomal recessive nail dysplasia 1 FZD6
Glycogen storage disease due to glucose-6-phosphatase deficiency type a 1 G6PC
Autosomal recessive severe congenital neutropenia due to G6PC3
deficiency 1 G6PC3
Glycogen storage disease due to acid maltase deficiency, adult onset 1 GAA
Glycogen storage disease due to acid maltase deficiency, juvenile onset 1 GAA
Childhood absence epilepsy 6 GABRA1
Juvenile myoclonic epilepsy 7 GABRA1
Thyrotoxic periodic paralysis 3 GABRA3
Childhood absence epilepsy 6 GABRB3 lp36 deletion syndrome 4 GABRD
Generalized epilepsy with febrile seizures-plus context 6 GABRD
Juvenile myoclonic epilepsy 7 GABRD
Childhood absence epilepsy 6 GABRG2
Dravet syndrome 4 GABRG2
Generalized epilepsy with febrile seizures-plus context 6 GABRG2
Inherited congenital spastic tetraplegia 2 GAD1
Adult Krabbe disease 1 GALC
Infantile Krabbe disease 2 GALC
Late-infantile/juvenile Krabbe disease 1 GALC
Erythrocyte galactose epimerase deficiency 1 GALE
Generalized galactose epimerase deficiency 1 GALE
97
Table IX-21781338.1 Galactokinase deficiency 1 GALK1
Mucopolysaccharidosis type 4A 1 GALNS
Hypercalcemic tumoral calcinosis 3 GALNT3
Classic galactosemia 1 GALT
Guanidinoacetate methyltransferase deficiency 1 GAMT
Giant axonal neuropathy' 1 GAN
Autosomal dominant Charcot-Marie-Tooth disease type 2D 1 GARS
Distal hereditary motor neuropathy type 5 3 GARS
Alobar holoprosencephaly 14 GAS1
Lobar holoprosencephaly 14 GAS1
Microform holoprosencephaly 14 GAS1
Midline interhemispheric variant of holoprosencephaly 14 GAS1
Semilobar holoprosencephaly 14 GAS1
Septopreoptic holoprosencephaly 14 GAS1
Acute basophilic leukemia 2 GATA1
Beta-thalassemia - X-linked thrombocytopenia 1 GATA1
Blackfan-Diamond anemia 11 GATA1
Congenital dyserythropoietic anemia with thrombocytopenia 1 GATA1
Congenital erythropoietic porphyria 2 GATA1
Acute myeloid leukemia 1 GATA2
Deafness - lymphedema - leukemia 1 GATA2
Monocytopenia with susceptibility to infections 1 GATA2
Myelodysplastic syndromes 2 GATA2
Hypoparathyroidism - deafness - renal disease 1 GATA3
46,XY partial gonadal dysgenesis 7 GATA4
8p23.1 microdeletion syndrome 1 GATA4
Atrial septal defect, ostium secundum type 8 GATA4
Complete atrioventricular canal 3 GATA4
Familial atrial fibrillation 14 GATA4
Partial atrioventricular canal 3 GATA4
Tetralogy of Fallot 8 GATA4
Ventricular septal defect 4 GATA4
Familial atrial fibrillation 14 GATA5
Ventricular septal defect 4 GATA5
Atrial septal defect, ostium secundum type 8 GATA6
Complete atrioventricular canal 3 GATA6
Familial atrial fibrillation 14 GATA6
Pancreatic hypoplasia - diabetes - congenital heart disease 1 GATA6
Partial atrioventricular canal 3 GATA6
Tetralogy of Fallot 8 GATA6
Familial isolated dilated cardiomyopathy 38 GATAD1
98
Table IX-21781338.1 Autosomal dominant nonsyndromic intellectual deficit 15 GATAD2B
Arginine:glycine amidinotransferase deficiency 1 GATM
Fetal Gaucher disease 1 GBA
Gaucher disease - ophthalmoplegia - cardiovascular calcification 1 GBA
Gaucher disease - ophthalmoplegia - cardiovascular calcification 1 GBA
Gaucher disease type 1 2 GBA
Gaucher disease type 2 1 GBA
Gaucher disease type 3 1 GBA
Young adult-onset Parkinsonism 13 GBA
Autosomal recessive cerebellar ataxia with late-onset spasticity 1 GBA2
Autosomal recessive cerebellar ataxia with late-onset spasticity 1 GBA2
Autosomal recessive spastic paraplegia type 46 1 GBA2
Adult polyglucosan body disease 1 GBE1
Glycogen storage disease due to glycogen branching enzyme deficiency,
adult neuromuscular form 1 GBE1
Glycogen storage disease due to glycogen branching enzyme deficiency,
childhood combined hepatic and myopathic form 1 GBE1
Glycogen storage disease due to glycogen branching enzyme deficiency,
childhood neuromuscular form 1 GBE1
Glycogen storage disease due to glycogen branching enzyme deficiency,
congenital neuromuscular form 1 GBE1
Glycogen storage disease due to glycogen branching enzyme deficiency,
fatal perinatal neuromuscular form 1 GBE1
Glycogen storage disease due to glycogen branching enzyme deficiency,
non progressive hepatic form 1 GBE1
Glycogen storage disease due to glycogen branching enzyme deficiency,
progressive hepatic form 1 GBE1
Glutaryl-CoA dehydrogenase deficiency 1 GCDH
Autosomal dominant dopa-responsive dystonia 1 GCH1
GTP cyclohydrolase 1 deficiency 1 GCH1
Hyperinsulinism due to glucokinase deficiency 1 GCK
MODY syndrome 12 GCK
Permanent neonatal diabetes mellitus 5 GCK
Gamma-glutamylcysteine synthetase deficiency 1 GCLC
Familial isolated hypoparathyroidism due to agenesis of parathyroid gland 1 GCM2
Atypical glycine encephalopathy 3 GCSH
Infantile glycine encephalopathy 3 GCSH
Neonatal glycine encephalopathy 3 GCSH
Autosomal dominant Charcot-Marie-Tooth disease type 2K 1 GDAP1
Autosomal recessive Charcot-Marie-Tooth disease with hoarseness 1 GDAP1
Autosomal recessive intermediate Charcot-Marie-Tooth disease type A 1 GDAP1
Charcot-Marie-Tooth disease type 2H 1 GDAP1
Charcot-Marie-Tooth disease type 4A 1 GDAP1
99
Table IX-21781338.1 Double outlet right ventricle 3 GDF1
Tetralogy of Fallot 8 GDF1
Colobomatous microphthalmia 8 GDF3
Isolated anophthalmia - microphthalmia 5 GDF3
Isolated Klippel-Feil syndrome 3 GDF3
Acromesomelic dysplasia, Grebe type i GDF5
Acromesomelic dysplasia, Hunter-Thomson type 1 GDF5
Angel-shaped phalango-epiphyseal dysplasia 1 GDF5
Brachydactyly type Al 2 GDF5
Brachydactyly type A2 3 GDF5
Brachydactyly type C 2 GDF5
Fibular aplasia - complex brachydactyly 1 GDF5
Multiple synostoses syndrome 3 GDF5
Proximal symphalangism 2 GDF5
Colobomatous microphthalmia 8 GDF6
Isolated Klippel-Feil syndrome 3 GDF6
Leber congenital amaurosis 18 GDF6
X-linked nonsyndromic intellectual deficit 24 GDI1
Hirschsprung disease 7 GDNF
# of Genes
per Gene
Disease Name Disease Name
Alexander disease 1 GFAP
Congenital cataract - progressive muscular hypotonia - hearing loss - developmental delay 1 GFER
Autosomal dominant severe congenital neutropenia 2 GFI1
Hepatoencephalopathy due to combined oxidative phosphorylation
deficiency type 1 1 GFM1
Congenital myasthenic syndromes with glycosylation defect 4 GFPT1
Body skin hyperlaxity due to vitamin K-dependent coagulation factor
deficiency 1 GGCX
Hereditary combined deficiency of vitamin K-dependent clotting factors 2 GGCX
Gamma-glutamyl transpeptidase deficiency 1 GGT1
Isolated growth hormone deficiency type IA 1 GH1
Isolated growth hormone deficiency type IB 2 GH1
Isolated growth hormone deficiency type II 1 GH1
Short stature due to growth hormone qualitative anomaly 1 GH1
Laron syndrome 1 GHR
•Short stature due to partial GH deficiency 1 GHR
Isolated growth hormone deficiency type IB 2 GHRHR
Short stature due to GHSR deficiency 1 GHSR
Congenital intrinsic factor deficiency 1 GIF
100
Table IX-21781338.1 Young adult-onset Parkinsonism 13 GIGYF2
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 GIPC3
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 GJA1
Hypoplastic left heart syndrome 2 GJA1
Oculodentodigital dysplasia 1 GJA1
Syndactyly type 3 1 GJA1
Cataract, Coppock-like 4 GJA3
Nuclear cataract 9 GJA3
Posterior polar cataract 5 GJA3
Pulverulent cataract 7 GJA3
Atrial stand still 2 GJA5
Familial atrial fibrillation 14 GJA5
Tetralogy of Fallot 8 GJA5
Cataract-microcornea syndrome 8 GJA8
Pulverulent cataract 7 GJA8
X-linked Charcot-Marie-Tooth disease type 1 1 GJB1
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 GJB2
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 GJB2
Hypotrichosis-deafness syndrome 1 GJB2
Keratoderma hereditarium mutilans 1 GJB2
KID syndrome 2 GJB2
Knuckle pads-leuconychia-sensorineural deafness-palmoplantar
hyperkeratosis syndrome 1 GJB2
Palmoplantar keratoderma-deafness syndrome 2 GJB2
Porokeratotic eccrine ostial and dermal duct nevus 1 GJB2
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 GJB3
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 GJB3
Erythrokeratodermia variabilis 2 GJB3
Neuropathy with hearing impairment 1 GJB3
Transgrediens et progrediens palmoplantar keratoderma 1 GJB3
Erythrokeratodermia variabilis 2 GJB4
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 GJB6
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 GJB6
Hidrotic ectodermal dysplasia 1 GJB6
KID syndrome 2 GJB6
Autosomal recessive spastic paraplegia type 44 1 GJC2
Autosomal recessive spastic paraplegia type 44 1 GJC2
Milroy disease 2 GJC2
Pelizaeus-Merzbacher-like due to GJC2 mutation 1 GJC2
Glycerol kinase deficiency, adult form 1 GK
Glycerol kinase deficiency, infantile form 1 GK
101
Table IX-21781338.1 Fabry disease 1 GLA
GM1 gangliosidosis type 2 1 GLB1
GM1 gangliosidosis type 3 1 GLB1
Mucopolysaccharidosis type 4B 1 GLB1
Atypical glycine encephalopathy 3 GLDC
Infantile glycine encephalopathy 3 GLDC
Neonatal glycine encephalopathy 3 GLDC
Lethal arthrogryposis - anterior horn cell disease 1 GLE1
Lethal congenital contracture syndrome type 1 1 GLE1
Alobar holoprosencephaly 14 GLI2
Combined pituitary hormone deficiencies, genetic forms 5 GLI2
Lobar holoprosencephaly 14 GLI2
Microform holoprosencephaly 14 GLI2
Midline interhemispheric variant of holoprosencephaly 14 GLI2
Semilobar holoprosencephaly 14 GLI2
Septopreoptic holoprosencephaly 14 GLI2
Acrocallosal syndrome 2 GLI3
Greig cephalopolysyndactyly syndrome 1 GLI3
Paliister-Hall syndrome 2 GLI3
Polysyndactyly, bilateral 1 GLI3
Polysyndactyly, unilateral 1 GLI3
Postaxial Polydactyly type A, bilateral 2 GLI3
Postaxial Polydactyly type A, unilateral 1 GLI3
Postaxial Polydactyly type B, bilateral 1 GLI3
Postaxial Polydactyly type B, unilateral 1 GLI3
Acute megakaryoblastic leukemia without Down syndrome 2 GLIS2
Juvenile autosomal recessive medullary cystic kidney disease 4 GLIS2
Neonatal diabetes - congenital hypothyroidism - congenital glaucoma - hepatic fibrosis - polycystic kidneys 1 GLIS3
Glomuvenous malformation 1 GLMN
Hereditary hyperekplexia 4 GL A1
Hereditary hyperekplexia 4 GLRB
Autosomal recessive pyridoxine-refractory sideroblastic anemia 2 GLRX5
Hyperinsulinism-hyperammonemia syndrome 1 GLUD1
Congenital brain dysgenesis due to glutamine synthetase deficiency 1 GLUL
D-glyceric aciduria 1 GLYCTK
GM2-gangliosidosis, AB variant 1 GM2A
Auriculo-condylar syndrome 2 GNAI3
Autosomal dominant focal dystonia, DYT25 1 GNAL
Nevi flammei 1 GNAQ
Sturge-Weber syndrome • 1 GNAQ
102
Table IX-21781338.1 Fibrous dysplasia of bone 1 GNAS
Mazabraud syndrome 1 GNAS
McCune-Albright syndrome 1 GNAS
Progressive osseous heteroplasia 1 GNAS
Pseudohypoparathyroidism type 1A 1 GNAS
Pseudohypoparathyroidism type 1A 1 GNAS
Pseudohypoparathyroidism type IB 2 GNAS
Pseudohypoparathyroidism type 1C 1 GNAS
Pseudopseudohypoparathyroidism 1 GNAS
Congenital stationary night blindness 12 GNAT1
Achromatopsia 6 GNAT2
Progressive cone dystrophy 4 GNAT2
Autosomal dominant intermediate Charcot-Marie-Tooth disease type F 1 GNB4
Distal myopathy, Nonaka type 1 GNE
Distal myopathy, Nonaka type 1 GNE
Sialuria 1 GNE
Hypermethioninemia due to glycine N-methyltransferase deficiency 1 GNMT
Rhizomelic chondrodysplasia punctata type 2 1 GNPAT
Mucolipidosis type 2 1 GNPTAB
Mucolipidosis type 3 2 GNPTAB
Mucolipidosis type 3 2 GNPTG
Normosmic congenital hypogonadotropic hypogonadism 18 GNRH1
Normosmic congenital hypogonadotropic hypogonadism 18 GNRHR
Sanfilippo syndrome type D 1 GNS
Papillary or follicular thyroid carcinoma 14 GOLGA5
Geroderma osteodysplastica 2 GORAB
Progressive myoclonic epilepsy type 6 1 GOSR2
Bernard-Soulier syndrome 3 GP1BA
Fetal and neonatal alloimmune thrombocytopenia 6 GP1BA
Von Willebrand disease, platelet type 1 GP1BA
22qll.2 deletion syndrome 7 GP1BB
Bernard-Soulier syndrome 3 GP1BB
Fetal and neonatal alloimmune thrombocytopenia 6 GP1BB
Bleeding diathesis due to glycoprotein VI deficiency 1 GP6
Bernard-Soulier syndrome 3 GP9
Biliary atresia 1 GPC1
Simpson-Golabi-Behmel syndrome 2 GPC3
Simpson-Golabi-Behmel syndrome 2 GPC4
Autosomal recessive omodysplasia 1 GPC6
Infantile regressive hypertriglyceridemia and hepatosteatosis 1 GPD1
Brugada syndrome 12 GPD1L
103
Table IX-21781338.1 Hereditary hyperekplexia 4 GPHN
Sulfite oxidase deficiency due to molybdenum cofactor deficiency type C 1 GPHN
Hemolytic anemia due to glucophosphate isomerase deficiency 1 GPI
Hyperlipoproteinemia type 1 2 GPIHBP1
Hyperlipoproteinemia type 5 4 GPIHBP1
X-linked recessive ocular albinism 1 GPR143
Congenital stationary night blindness 12 GPR179
Bilateral frontoparietal polymicrogyria 1 GPR56
Usher syndrome type 2 4 GPR98
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 GPSM2
Chudley-McCullough syndrome 1 GPSM2
Hereditary mixed polyposis syndrome 2 GREM1
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 GRHL2
Primary hyperoxaluria type 2 1 GRHPR
X-linked nonsyndromic intellectual deficit 24 GRIA3
Autosomal recessive nonsyndromic intellectual deficit 15 GRIK2
Autosomal dominant nonsyndromic intellectual deficit 15 GRIN1
Early-onset epileptic encephalopathy and intellectual deficit due to
GRIN2A mutation 1 GRIN2A
Autosomal dominant nonsyndromic intellectual deficit 15 GRIN2B
Fraser syndrome 3 GRIP1
Oguchi disease 2 GRK1
Autosomal recessive congenital cerebellar ataxia due to MGLUR1
deficiency 1 GRM1
Congenital stationary night blindness 12 GRM6
CLN11 disease 1 GRN
Progressive non-fluent aphasia 3 GRN
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 GRXCR1
Familial amyloidosis, Finnish type 1 GSN
Hemolytic anemia due to glutathione reductase deficiency 1 GSR
Hemolytic anemia due to glutathione reductase deficiency 1 GSR
Glutathione synthetase deficiency with 5-oxoprolinuria 1 GSS
Glutathione synthetase deficiency without 5-oxoprolinuria 1 GSS
Walker-Warburg syndrome 13 GTDC2
(BIDS syndrome 3 GTF2H5
PIBIDS syndrome 3 GTF2H5
Williams syndrome 17 GTF2I
Williams syndrome 17 GTF2IRD1
Cone rod dystrophy 22 GUCA1A
Progressive cone dystrophy 4 GUCA1A
Retinitis pigmentosa 61 GUCA1B
104
Table IX-21781338.1 Chronic diarrhea due to guanylate cyclase 2C overactivity 1 GUCY2C
Intestinal obstruction in the newborn due to guanylate cyclase 2C
deficiency 1 GUCY2C
Central areolar choroidal dystrophy 2 GUCY2D
Cone rod dystrophy 22 GUCY2D
Leber congenital amaurosis 18 GUCY2D
Mucopolysaccharidosis type 7 1 GUSB
Glycogen storage disease due to glycogenin deficiency 1 GYG1
Glycogen storage disease due to muscle and heart glycogen synthase
deficiency 1 GYS1
Glycogen storage disease due to hepatic glycogen synthase deficiency 1 GYS2
Beckwith-Wiedemann syndrome due to llpl5 microdeletion 2 H19
Beckwith-Wiedemann syndrome due to imprinting defect of llpl5 3 H19
Hemihypertrophy 3 H19
Nephroblastoma 5 H19
Silver-Russell syndrome due to llpl5 microduplication 2 H19
Silver-Russell syndrome due to llpl5 microduplication 2 H19
Silver-Russell syndrome due to imprinting defect of llpl5 2 H19
Hyperandrogenism due to cortisone reductase deficiency 2 H6PD
Neuroblastoma 7 HACE1
Hyperinsulinism due to 3-hydroxylacyl-CoA dehydrogenase deficiency 1 HADH
Acute fatty liver of pregnancy 1 HADHA
Long chain 3-hydroxyacyl-CoA dehydrogenase deficiency 1 HADHA
Mitochondrial trifunctional protein deficiency 2 HADHA
Mitochondrial trifunctional protein deficiency 2 HADHB
Histidinemia 1 HAL
Hemochromatosis type 2 2 HAMP
Usher syndrome type 3 3 HARS
Perrault syndrome 4 HARS2
Kostmann syndrome 1 HAX1
Alpha thalassemia - intellectual deficit syndrome linked to chromosome 16 2 HBA1
Autosomal dominant methemoglobinemia 3 HBA1
Hb Bart's Hydrops Fetalis 2 HBA1
Hemoglobin H disease 2 HBA1
Alpha thalassemia - intellectual deficit syndrome linked to chromosome 16 2 HBA2
Autosomal dominant methemoglobinemia 3 HBA2
Hb Bart's Hydrops Fetalis 2 HBA2
Hemoglobin H disease 2 HBA2
Autosomal dominant methemoglobinemia 3 HBB
Beta-thalassemia intermedia 1 HBB
Beta-thalassemia major 1 HBB
105
Table IX-21781338.1 Delta-beta thalassemia 3 HBB
Dominant beta-thalassemia 1 HBB
Heinz body anemia 1 HBB
Hemoglobin C - beta-thalassemia 1 HBB
Hemoglobin C disease 1 HBB
Hemoglobin D disease 1 HBB
Hemoglobin E - beta-thalassemia 1 HBB
Hemoglobin Lepore - beta-thalassemia 2 HBB
Hereditary persistence of fetal hemoglobin - beta-thalassemia 5 HBB
Hereditary persistence of fetal hemoglobin - sickle cell disease 4 HBB
Sickle cell - beta-thalassemia disease 1 HBB
Sickle cell - hemoglobin C disease 1 HBB
Sickle cell - hemoglobin D disease 1 HBB
Sickle cell - hemoglobin E disease 1 HBB
Sickle cell anemia 1 HBB
Delta-beta thalassemia 3 HBD
Hemoglobin Lepore - beta-thalassemia 2 HBD
Delta-beta thalassemia 3 HBG1
Hereditary persistence of fetal hemoglobin - beta-thalassemia 5 HBG1
Hereditary persistence of fetal hemoglobin - sickle cell disease 4 HBG1
Hemoglobinopathy Toms River 1 HBG2
Hereditary persistence of fetal hemoglobin - beta-thalassemia 5 HBG2
Hereditary persistence of fetal hemoglobin - sickle cell disease 4 HBG2
Microphthalmia with linear skin defects syndromes 2 HCCS
X-linked nonsyndromic intellectual deficit 24 HCFC1
Brugada syndrome 12 HCN4
Sick sinus syndrome 3 HCN4
Narcolepsy-cataplexy 4 HCRT
2q37 microdeletion syndrome 1 HDAC4
X-linked dominant chondrodysplasia, Chassaing-Lacombe type 1 HDAC6
Cornelia de Lange syndrome 5 HDAC8
Wilson-Turner syndrome 1 HDAC8
Peters anomaly 6 HDAC9
Primary ciliary dyskinesia 21 HEATR2
Megalencephalic leukoencephalopathy with subcortical cysts 2 HEPACAM
Developmental delay with autism spectrum disorder and gait instability 1 HERC2
Autosomal recessive spondylocostal dysostosis 4 HES7
Combined pituitary hormone deficiencies, genetic forms 5 HESX1
Hypothyroidism due to deficient transcription factors involved in pituitary
development or function 5 HESX1
Kallmann syndrome 19 HESX1
10f)
Table IX-21781338.1 Pituitary stalk interruption syndrome 2 HESX1
Septo-optic dysplasia 6 HESX1
Tay-Sachs disease, B variant, adult form 1 HEXA
Tay-Sachs disease, B variant, infantile form 1 HEXA
Tay-Sachs disease, B variant, juvenile form 1 HEXA
Tay-Sachs disease, Bl variant 1 HEXA
Sandhoff disease, adult form 1 HEXB
Sandhoff disease, adult form 1 HEXB
Sandhoff disease, infantile form 1 HEXB
Sandhoff disease, juvenile form 1 HEXB
Porphyria cutanea tarda 2 HFE
Porphyria variegata 2 HFE
Hemochromatosis type 2 2 HFE2
Alkaptonuria 1 HGD
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 HGF
Sanfilippo syndrome type C 1 HGSNAT
Neurodegeneration due to 3-hydroxyisobutyryl-CoA hydrolase deficiency 1 HIBCH
Miller-Dieker syndrome 3 HICl
Autosomal recessive axonal neuropathy with neuromyotonia 1 HINT1
22qll.2 deletion syndrome 7 HI A
Charcot-Marie-Tooth disease type 4G 1 HK1
Nonspherocytic hemolytic anemia due to hexokinase deficiency 1 HK1
Chorioretinopathy, Birdshot type 1 HLA-A
Behcet disease 9 HLA-B
Stevens-Johnson syndrome 1 HLA-B
Narcolepsy without cataplexy 1 HLA-DQB1
Narcolepsy-cataplexy 4 HLA-DQB1
Graham Little-Piccardi-Lassueur syndrome 1 HLA-DRA
Diffuse cutaneous systemic sclerosis 4 HLA-DRB1
Follicular lymphoma 4 HLA-DRB1
Limited cutaneous systemic sclerosis 4 HLA-DRB1
Limited systemic sclerosis 1 HLA-DRBl
Narcolepsy-cataplexy 4 HLA-DRB1
Pediatric systemic sclerosis 1 HLA-DRBl
Sarcoidosis 2 HLA-DRBl
Holocarboxylase synthetase deficiency 1 HLCS
Precursor B-cell acute lymphoblastic leukemia 13 HLF
Acute intermittent porphyria 1 HMBS
12ql4 microdeletion syndrome 2 HMGA2
Dedifferentiated liposarcoma 3 HMGA2
Well-differentiated liposarcoma 3 HMGA2
107
Table IX-21781338.1 3-hydroxy-3-methylglutaric aciduria 1 HMGCL
3-hydroxy 3-methylglutaryl-CoA synthase deficiency 1 HMGCS2
Oculoauricular syndrome, Schorderet type 1 HMX1
Hyperinsulinism due to HNF1A deficiency 1 HNF1A
MODY syndrome 12 HNF1A
17ql2 microdeletion syndrome 2 HNF1B
Autosomal dominant primary hypomagnesemia with hypocalcuria 2 HNF1B
Familial prostate cancer 14 HNF1B
Mullerian aplasia 1 HNF1B
Renal cysts and diabetes syndrome 1 HNF1B
Hyperinsulinism due to HNF4A deficiency 1 HNF4A
MODY syndrome 12 H F4A
Amyotrophic lateral sclerosis 23 HNRNPA1
Inclusion body myopathy with Paget disease of bone and frontotemporal
dementia 2 HNRNPA2B1
Precursor T-cell acute lymphoblastic leukemia 19 HNRNPH1
Primary hyperoxaluria type 3 1 HOGA1
Athabaskan brainstem dysgenesis syndrome 1 HOXA1
Bosley-Salih-Alorainy syndrome 1 HOXA1
Radio-ulnar synostosis - amegakaryocytic thrombocytopenia 1 HOXA11
Radio-ulnar synostosis - amegakaryocytic thrombocytopenia 1 HOXA11
Guttmacher syndrome 1 HOXA13
Hand-foot-genital syndrome 1 HOXA13
Anotia 1 HOXA2
Bilateral microtia - deafness - cleft palate 1 HOXA2
Congenital hereditary facial paralysis with variable hearing loss 1 HOXB1
Familial prostate cancer 14 HOXB13
Pure hair and hail ectodermal dysplasia 2 HOXC13
Congenital vertical talus, bilateral 1 HOXD10
Congenital vertical talus, unilateral 1 HOXD10
Brachydactyly type E 2 HOXD13
Brachydactyly-syndactyly, Zhao type 1 HOXD13
Synpolydactyly type 1 1 HOXD13
VACTERL/VATER association 1 HOXD13
Hawkinsinuria 1 HPD
Tyrosinemia type 3 1 HPD
Cranio-osteoarthropathy 1 HPGD
Isolated congenital digital clubbing 1 HPGD
Pachydermoperiostosis 2 HPGD
Kelley-Seegmiller syndrome 1 HPRT1
Lesch-Nyhan syndrome 1 HPRT1
108
Table IX-21781338.1 Hermansky-Pudlak syndrome with pulmonary fibrosis 2 HPS1
Hermansky-Pudlak syndrome without pulmonary fibrosis 3 HPS3
Hermansky-Pudlak syndrome with pulmonary fibrosis 2 HPS4
Hermansky-Pudlak syndrome without pulmonary fibrosis 3 HPS5
Hermansky-Pudlak syndrome without pulmonary fibrosis 3 HPS6
Ochoa syndrome 2 HPSE2
Alopecia universalis 1 HR
Atrichia with papular lesions 1 HR
Marie Unna hereditary hypotrichosis 2 HR
Costello syndrome 2 HRAS
Hereditary thrombophilia due to congenital histidine-rich (poly-L)
glycoprotein deficiency 1 HRG
Familial isolated hyperparathyroidism 3 HRPT1
Kallmann syndrome 19 HS6ST1
Normosmic congenital hypogonadotropic hypogonadism 18 HS6ST1
Hyperandrogenism due to cortisone reductase deficiency 2 HSD11B1
Apparent mineralocorticoid excess 1 HSD11B2
Intellectual deficit, X-linked - choreoathetosis - abnormal behavior 1 HSD17B10
Short chain 3-hydroxyacyl-CoA dehydrogenase deficiency 1 HSD17B10
46,XY disorder of sex development due to 17-beta-hydroxysteroid
dehydrogenase 3 deficiency 1 HSD17B3
Familial prostate cancer 14 HSD17B3
Bifunctional enzyme deficiency 2 HSD17B4
Perrault syndrome 4 HSD17B4
Congenital adrenal hyperplasia due to 3-beta-hydroxysteroid
dehydrogenase deficiency 1 HSD3B2 .
Familial prostate cancer 14 HSD3B2
Congenital bile acid synthesis defect type 1 1 HSD3B7
Zonular cataract 9 HSF4
Autosomal dominant Charcot-Marie-Tooth disease type 2F 1 HSPB1
Distal hereditary motor neuropathy type 2 3 HSPB1
Distal hereditary motor neuropathy type 2 3 HSPB3
Autosomal dominant Charcot-Marie-Tooth disease type 2L 1 HSPB8
Distal hereditary motor neuropathy type 2 3 HSPB8
Familial renal cell carcinoma 6 HSPBAP1
Autosomal dominant spastic paraplegia type 13 1 HSPD1
Pelizaeus-Merzbacher-like due to HSPD1 mutation 1 HSPD1
Dyssegmental dysplasia, Silverman-Handmaker type 1 HSPG2
Schwartz-Jampel syndrome 1 HSPG2
CARASIL 1 HTRA1
Young adult-onset Parkinsonism 13 HTRA2
Huntington disease 1 HTT
109
Table IX-21781338.1 Juvenile Huntington disease 1 HTT
Intellectual deficit, X-linked, Turner type 1 HUWE1
Hyaluronidase deficiency 1 HYAL1
Primary ciliary dyskinesia 21 HYDIN
Hydrolethalus 2 HYLS1
Paternal uniparental disomy of chromosome 6 2 HYMAI
Transient neonatal diabetes mellitus 5 HYMAI
Chronic mucocutaneous candidiasis 6 ICAM1
Endocrine-cerebro-osteodysplasia syndrome 1 ICK
Endocrine-cerebro-osteodysplasia syndrome 1 ICK
Common variable immunodeficiency 10 ICOS
Enchondromatosis 3 IDH1
Giant cell glioblastoma 10 IDH1
Gliosarcoma 10 IDH1
Maffucci syndrome 2 IDH1
Metaphyseal chondromatosis with D-2-hydroxyglutaric aciduria 1 IDH1
D-2-hydroxyglutaric aciduria 2 IDH2
Enchondromatosis 3 IDH2
Maffucci syndrome 2 IDH2
Retinitis pigmentosa 61 IDH3B
Mucopolysaccharidosis type 2A 1 IDS
Mucopolysaccharidosis type 2B 1 IDS
Hurler syndrome 1 IDUA
Hurler-Scheie syndrome 1 IDUA
Scheie syndrome 1 IDUA
Primary microcephaly-epilepsy-permanent neonatal diabetes syndrome 1 IER3IP1
Osteogenesis imperfecta type 5 2 IFITM5
Idiopathic aplastic anemia 5 IFNG
Autosomal dominant mendelian susceptibility to mycobacterial diseases
due to partial IFNgamma l deficiency 1 IFNGR1
Autosomal recessive mendelian susceptibility to mycobacterial diseases
due to partial IFNgammaRl deficiency 1 IFNGR1
Mendelian susceptibility to mycobacterial diseases due to complete
IFNgammaRl deficiency 1 IFNGR1
Autosomal dominant mendelian susceptibility to mycobacterial diseases
due to partial IFNgammaR2 deficiency 1 IFNGR2
Autosomal recessive mendelian susceptibility'to mycobacterial diseases
due to partial IFNgammaR2 deficiency 1 IFNGR2
Mendelian susceptibility to mycobacterial diseases due to complete
IFNgammaR2 deficiency 1 IFNGR2
Spinocerebellar ataxia type 18 1 IFRD1
Cranioectodermal dysplasia 4 IFT122
110
Table IX-21781338.1 Jeune syndrome 6 IFT140
Saldino-Mainzer syndrome 1 IFT140
Saldino-Mainzer syndrome 1 IFT140
Cranioectodermal dysplasia 4 IFT43
Jeune syndrome 6 IFT80
Short rib-polydactyly syndrome, Verma-Naumoff type 3 IFT80
Jeune syndrome 6 IFT88
Meckel syndrome 13 IFT88
Agenesis of the corpus callosum - intellectual deficit - coloboma - micrognathia 1 IGBP1
Growth delay due to insulin-like growth factor 1 deficiency 1 IGF1
Growth delay due to insulin-like growth factor 1 resistance 1 IGF1R
Beckwith-Wiedemann syndrome due to imprinting defect of llpl5 3 IGF2
Hemihypertrophy 3 IGF2
Silver-Russell syndrome due to llpl5 microduplication 2 IGF2
Silver-Russell syndrome due to imprinting defect of llplS 2 IGF2
Short stature due to primary acid-labile subunit deficiency 1 IGFALS
Familial retinal arterial macroaneurysm 1 IGFBP7 ,
Follicular lymphoma 4 IGH@
MALT lymphoma 4 IGH@
Mantle cell lymphoma 3 IGH@
Precursor B-cell acute lymphoblastic leukemia 13 IGH@
B-cell chronic lymphocytic leukemia 7 IGHG1
Recurrent infections associated with rare immunoglobulin isotypes
deficiency 2 IGHG2
Autosomal agammaglobulinemia 7 IGHM
Spinal muscular atrophy with respiratory distress 2 IGHMBP2
B-cell chronic lymphocytic leukemia 7 IGHV3-21
Hairy cell leukemia variant 1 IGHV4-34
Recurrent infections associated with rare immunoglobulin isotypes
deficiency 2 IGKC
Autosomal agammaglobulinemia 7 IGLL1
X-linked central congenital hypothyroidism with late-onset testicular
enlargement 1 IGSF1
X-linked central congenital hypothyroidism with late-onset testicular
enlargement 1 IGSF1
Acrocapitofemoral dysplasia 1 IHH
Brachydactyly type Al 2 IHH
Familial dysautonomia 1 IKBKAP
Anhidrotic ectodermal dysplasia - immunodeficiency - osteopetrosis - lymphedema 1 IKBKG
Hypohidrotic ectodermal dysplasia with immunodeficiency 2 IKBKG
111
Table IX-21781338.1 Incontinentia pigmenti 1 IKBKG
X-linked mendelian susceptibility to mycobacterial diseases due to IKBKG
deficiency 1 IKBKG
Pancytopenia due to IKZF1 mutations 1 IKZF1
Autosomal recessive early-onset inflammatory bowel disease 3 IL10
Behcet disease 9 IL10
Autosomal recessive early-onset inflammatory bowel disease 3 IL10RA
Autosomal recessive early-onset inflammatory bowel disease 3 IL10RB
Craniosynostosis and dental anomalies 1 IL11RA
Primary biliary cirrhosis 8 IL12A
Mendelian susceptibility to mycobacterial diseases due to complete IL12B
deficiency 1 IL12B
Mendelian susceptibility to mycobacterial diseases due to complete
IL12RB1 deficiency 1 IL12RB1
Primary biliary cirrhosis 8 IL12RB1
Behcet disease 9 IL12RB2
Chronic mucocutaneous candidiasis 6 IL17F
Chronic mucocutaneous candidiasis 6 IL17RA
Kallmann syndrome 19 IL17RD
X-linked nonsyndromic intellectual deficit 24 ILIRAPLI
Sterile multifocal osteomyelitis with periostitis and pustulosis 1 IL1RN
Cryptosporidiosis - chronic cholangitis - liver disease 1 IL21R
Behcet disease 9 IL23R
Immunodeficiency due to CD25 deficiency 1 IL2RA
Juvenile rheumatoid factor-negative polyarthritis 9 IL2RA
Oligoarticular juvenile arthritis 9 IL2RA
Juvenile rheumatoid factor-negative polyarthritis 9 IL2RB
Oligoarticular juvenile arthritis 9 IL2RB
Omenn syndrome 9 IL2RG
T-B+ severe combined immunodeficiency due to gamma chain deficiency 1 IL2RG
Familial primary localized cutaneous amyloidosis 2 IL31RA
Acrodermatitis continua suppurativa of Hallopeau 1 IL36RN
Generalized pustular psoriasis 1 IL36RN
Pustulosis palmaris et plantaris 1 IL36RN
Pustulosis palmaris et plantaris 1 IL36RN
Systemic-onset juvenile idiopathic arthritis 2 IL6
Omenn syndrome 9 IL7R
T-B+ severe combined immunodeficiency due to IL-7Ralpha deficiency 1 IL7R
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 ILDR1
Catel-Manzke syndrome 1 IMPAD1
Chondrodysplasia with joint dislocations, gPAPP type 1 IMPAD1
Leber congenital amaurosis 18 IMPDH1
112
Table IX-21781338.1 Retinitis pigmentosa 61 IMPDH1
Retinitis pigmentosa 61 IMPG2
Autosomal dominant intermediate Charcot-Marie-Tooth disease type E 1 INF2
Familial idiopathic steroid-resistant nephrotic syndrome with focal
segmental hyalinosis 11 INF2
Squamous cell carcinoma of head and neck 4 ING1
Squamous cell carcinoma of head and neck 4 ING3
Joubert syndrome 9 INPP5E
MORM syndrome 1 INPP5E
Opsismodysplasia 1 INPPL1
MODY syndrome 12 INS
Permanent neonatal diabetes mellitus 5 INS
Hyperinsulinism due to INSR deficiency 1 INSR
Insulin-resistance syndrome type A 1 INSR
Leprechaunism 1 INSR
Rabson-Mendenhall syndrome 1 INSR
Infantile autosomal recessive medullary cystic kidney disease 3 INVS
Senior-Loken syndrome 8 INVS
Leber congenital amaurosis 18 IQCB1
Senior-Loken syndrome 8 IQCB1
X-linked nonsyndromic intellectual deficit 24 IQSEC2
Immunodeficiency due to interleukin-1 receptor-associated kinase-4
deficiency 1 IRAK4
Diffuse cutaneous systemic sclerosis 4 IRF5
Limited cutaneous systemic sclerosis 4 IRF5
Primary biliary cirrhosis 8 IRF5
Autosomal dominant popliteal pterygium syndrome 1 IRF6
Oligodontia 10 IRF6
Van Der Woude syndrome 1 IRF6
Mendelian susceptibility to mycobacterial diseases due to partial IRF8
deficiency 1 IRF8
Craniofacial dysplasia-osteopenia syndrome 1 IRX5
Hereditary myopathy with lactic acidosis due to ISCU deficiency 1 ISCU
Mendelian susceptibility to mycobacterial diseases due to complete ISG15
deficiency 1 ISG15
Autosomal recessive limb-girdle muscular dystrophy due to ISPD
deficiency 1 ISPD
Autosomal recessive limb-girdle muscular dystrophy with cerebellar
involvement 1 ISPD
Walker-Warburg syndrome 13 ISPD
Syndromic multisystem autoimmune disease due to Itch deficiency 1 ITCH
Fetal and neonatal alloimmune thrombocytopenia 6 ITGA2
113
Table IX-21781338.1 Fetal and neonatal alloimmune thrombocytopenia 6 ITGA2B
Glanzmann thrombasthenia 2 ITGA2B
Congenital nephrotic syndrome-interstitial lung disease-epidermolysis
bullosa syndrome 1 ITGA3
Junctional epidermolysis bullosa - pyloric atresia 2 ITGA6
Congenital muscular dystrophy with integrin deficiency 1 ITGA7
Leukocyte adhesion deficiency type 1 1 ITGB2
Autosomal dominant macrothrombocytopenia 3 ITGB3
Fetal and neonatal alloimmune thrombocytopenia 6 ITGB3
Glanzmann thrombasthenia 2 ITGB3
Epidermolysis bullosa simplex with pyloric atresia 2 ITGB4
Generalized junctional epidermolysis bullosa, non-Herlitz type 5 ITGB4
Junctional epidermolysis bullosa - pyloric atresia 2 ITGB4
Localized junctional epidermolysis bullosa, non-Herlitz type 2 ITGB4
Autosomal recessive lymphoproliferative disease 2 ITK
Familial dementia, British type 1 IT 2B
Spinocerebellar ataxia type 15/16 1 ITPR1
Spinocerebellar ataxia type 29 1 ITPR1
Spinocerebellar ataxia type 29 1 ITPR1
Isovaleric acidemia 1 IVD
Familial thyroid dyshormonogenesis 6 IYD
Tetralogy of Fallot 8 JAG1
Budd-Chiari syndrome 2 JAK2
Essential thrombocythemia 4 JAK2
Familial thrombocytosis 3 JAK2
Myelofibrosis with myeloid metaplasia 3 JAK2
Polycythemia vera 3 JAK2
T-B+ severe combined immunodeficiency due to JAK3 deficiency 1 JAK3
Porencephaly-microcephaly-bilateral congenital cataract syndrome 1 JAM3
Huntington disease-like 2 1 JPH3
Childhood absence epilepsy 6 JRK
Juvenile myoclonic epilepsy 7 JRK
Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 JUP
Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 JUP
Familial isolated arrhythmogenic ventricular dysplasia, right dominant
form 10 JUP
Lethal acantholytic epidermolysis bullosa 2 JUP
Naxos disease 1 JUP
Kallmann syndrome 19 KALI
Normosmic congenital hypogonadotropic hypogonadism 18 KALI
Familial congenital palsy of trochlear nerve 1 KANK1
114
Table IX-21781338.1 Inherited congenital spastic tetraplegia 2 KANK1
17q21.31 microdeletion syndrome 1 KANSL1
Autosomal recessive intermediate Charcot-Marie-Tooth disease type B 1 KARS
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 KARS
Blepharophimosis-intellectual deficit syndrome, SBBYS type 1 KAT6B
Genitopatellar syndrome 1 KAT6B
Genitopatellar syndrome 1 KAT6B
Noonan syndrome 10 KAT6B
Childhood-onset nemaline myopathy 5 KBTBD13
Episodic ataxia type 1 1 KCNA1
Hereditary continuous muscle fiber activity 1 KCNA1
Isolated autosomal dominant hypomagnesemia, Glaudemans type 1 KCNA1
Familial atrial fibrillation 14 KCNA5 lp36 deletion syndrome 4 KCNAB2
Spinocerebellar ataxia type 13 1 KCNC3
Brugada syndrome 12 KCND3
Spinocerebellar ataxia type 19/22 1 KCND3
Familial atrial fibrillation 14 KCNE1
Jervell and Lange-Nielsen syndrome 2 KCNE1
Romano-Ward syndrome 13 KCNE1
Alport syndrome - intellectual deficit - midface hypoplasia - elliptocytosis 3 KCNE1L
Familial atrial fibrillation 14 KCNE2
Romano-Ward syndrome 13 KCNE2
Brugada syndrome 12 KCNE3
Hypokalemic periodic paralysis 3 KCNE3
Familial short QT syndrome 4 KCNH2
Romano-Ward syndrome 13 KCNH2
Antenatal Bartter syndrome 2 KCNJ1
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 KCNJ10
Pendred syndrome 3 KCNJ10
SeSAME syndrome 1 KCNJ10
Autosomal dominant hyperinsulinism due to Kir6.2 deficiency 1 KCNJ11
Autosomal recessive hyperinsulinism due to Kir6.2 deficiency 1 KCNJ11
DEND syndrome 1 KCNJ11
Diazoxide-resistant focal hyperinsulinism due to Kir6.2 deficiency 1 KCNJ11
MODY syndrome 12 KCNJ11
Permanent neonatal diabetes mellitus 5 KCNJ11
Transient neonatal diabetes mellitus 5 KCNJ11
Leber congenital amaurosis 18 KCNJ13
Snowflake vitreoretinal degeneration 1 KCNJ13
Thyrotoxic periodic paralysis 3 KCNJ18
115
Table IX-21781338.1 Cardiodysrhythmic potassium-sensitive periodic paralysis 1 KCNJ2
Familial atrial fibrillation 14 KCNJ2
Familial short QT syndrome 4 KCNJ2
Familial hyperaldosteronism type 3 1 KCNJ5
Romano-Ward syndrome 13 KCNJ5
Brugada syndrome 12 KCNJ8
Intellectual deficit, Birk-Barel type 1 KCNK9
Generalized epilepsy - paroxysmal dyskinesia 1 KCNMA1
Familial atrial fibrillation 14 KCNQ1
Familial short QT syndrome 4 KCNQ1
Jervell and Lange-Nielsen syndrome 2 KCNQ1
Romano-Ward syndrome 13 KCNQ1
Beckwith-Wiedemann syndrome due to llpl5 microdeletion 2 KCNQ10T1
Beckwith-Wiedemann syndrome due to imprinting defect of llpl5 3 KCNQIOTI
Hemihypertrophy 3 KCNQ10T1
Benign familial infantile seizures 4 KCNQ2
Benign familial neonatal seizures 2 KCNQ2
Benign familial neonatal-infantile seizures 2 KCNQ2
Early infantile epileptic encephalopathy 9 KCNQ2
Benign familial infantile seizures 4 KCNQ3
Benign familial neonatal seizures 2 KCNQ3
Juvenile myoclonic epilepsy 7 KCNQ3
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 KCNQ4
Malignant migrating partial seizures of infancy 4 KCNT1
Nocturnal frontal lobe epilepsy 5 KCNT1
Cone dystrophy with supernormal rod response 1 KCNV2
Scalp-ear-nipple syndrome 1 KCTD1
Progressive myoclonic epilepsy type 3 1 KCTD7
Syndromic X-linked intellectual deficit due to JARIDIC mutation 1 KDM5C
Kabuki syndrome 2 KDM6A
Familial capillary hemangioma 2 KDR
Congenital cornea plana 1 KERA
Complete hydatidiform mole 2 KHDC3L
Essential fructosuria 1 KHK
Autosomal dominant spastic paraplegia type 8 1 KIAA0196
Autosomal recessive nonsyndromic intellectual deficit 15 KIAA1033
Goldberg-Shprintzen megacolon syndrome 1 KIAA1279
Monomelic amyotrophy 2 KIAA1377
Pilocytic astrocytoma 2 KIAA1549
Intellectual deficit, X-linked, Cantagrel type 1 KIAA2022
Microcephaly - lymphedema - chorioretinopathy 1 KIF11
116
Table IX-21781338.1 Autosomal dominant nonsyndromic intellectual deficit 15 KIF1A
Autosomal recessive spastic paraplegia type 30 1 KIF1A
Hereditary sensory and autonomic neuropathy type 2 4 KIF1A
Autosomal dominant Charcot-Marie-Tooth disease type 2A1 1 IF1B
Congenital fibrosis of extraocular muscles 4 KIF21A
Spondyloepimetaphyseal dysplasia with multiple dislocations 1 KIF22
Congenital dyserythropoietic anemia type 3 1 KIF23
Autosomal dominant Charcot-Marie-Tooth disease type 2 due to KIF5A
mutation 1 KIF5A
Autosomal dominant spastic paraplegia type 10 1 KIF5A
Acrocallosal syndrome 2 KIF7
Hydrolethalus 2 KIF7
Joubert syndrome 9 KIF7
Joubert syndrome with ocular defect 5 KIF7
Joubert syndrome with orofaciodigital defect 4 KIF7
Multiple epiphyseal dysplasia, Al-Gazali type 1 KIF7
Autosomal dominant nonsyndromic intellectual deficit 15 KIRREL3
Normosmic congenital hypogonadotropic hypogonadism 18 KISS1
Kallmann syndrome 19 KISS1R
Normosmic congenital hypogonadotropic hypogonadism 18 KISS1R
Aggressive systemic mastocytosis 1 IT
Bullous diffuse cutaneous mastocytosis 1 KIT
Cutaneous mastocytoma 1 IT
Gastrointestinal stromal tumor 4 KIT
Indolent systemic mastocytosis 1 KIT
Maculopapular cutaneous mastocytosis 1 KIT
Mast cell leukemia 1 KIT
Piebaldism 2 KIT .
Pseudoxanthomatous diffuse cutaneous mastocytosis 1 KIT
Systemic mastocytosis with an associated clonal hematologic non-mast
cell lineage disease 1 KIT
Telangiectasia macularis eruptiva perstans 1 KIT
Familial progressive hyper- and hypopigmentation 1 KITLG
Familial progressive hyperpigmentation 1 KITLG
Hypercalcemic tumoral calcinosis 3 KL
Congenital dyserythropoietic anemia due to KLF1 mutation 1 KLF1
Hereditary persistence of fetal hemoglobin - beta-thalassemia 5 KLF1
Hereditary persistence of fetal hemoglobin - sickle cell disease 4 KLF1
MODY syndrome 12 KLF11
Pseudohypoaldosteronism type 2D 1 KLHL3
Severe congenital nemaline myopathy 3 KLHL40
Π7
Table IX-21781338.1 Retinitis pigmentosa 61 KLHL7
Hypomaturation amelogenesis imperfecta 5 KLK4
Congenital prekallikrein deficiency 1 KLKB1
Hereditary breast cancer 4 KLLN
Congenital high-molecular-weight kininogen deficiency 1 KNG1
Cardiofaciocutaneous syndrome 4 KRAS
Costello syndrome 2 KRAS
Familial pancreatic carcinoma 8 KRAS
Juvenile myelomonocytic leukemia 3 KRAS
Linear nevus sebaceus syndrome 2 KRAS
Linear nevus sebaceus syndrome 2 KRAS
Noonan syndrome 10 KRAS
Hereditary cerebral cavernous malformation 3 KRIT1
Annular epidermolytic ichthyosis 2 KRT1
Epidermolytic ichthyosis 2 KRT1
Epidermolytic palmoplantar keratoderma 4 KRT1
Ichthyosis hystrix of Curth-Macklin 1 KRT1
Keratosis palmoplantaris striata 3 KRT1
Annular epidermolytic ichthyosis 2 KRT10
Congenital reticular ichthyosiform erythroderma 1 KRT10
Epidermolytic ichthyosis 2 KRT10
Meesmann corneal dystrophy 2 KRT12
White sponge nevus 2 KRT13
Autosomal recessive epidermolysis bullosa simplex 2 KRT14
Dermatopathia pigmentosa reticularis 1 KRT14
Epidermolysis bullosa simplex with mottled pigmentation 2 KRT14
Epidermolysis bullosa simplex, Dowling-Meara type 2 KRT14
Localized epidermolysis bullosa simplex 2 KRT14
Naegeli-Franceschetti-Jadassohn syndrome 1 KRT14
Non-Dowling-Meara generalized epidermolysis bullosa simplex 2 KRT14
Epidermolytic palmoplantar keratoderma 4 KRT16
Pachyonychia congenita 4 KRT16
Pachyonychia congenita 4 KRT17
Sebocystomatosis 1 KRT17
Superficial epidermolytic ichthyosis 1 KRT2
Meesmann corneal dystrophy 2 KRT3
White sponge nevus • 2 KRT4
Dowling-Degos disease 2 KRT5
Epidermolysis bullosa simplex with circinate migratory erythema 1 KRT5
Epidermolysis bullosa simplex, Dowling-Meara type 2 KRT5
Localized epidermolysis bullosa simplex 2 KRT5
118
Table IX-21781338.1 Non-Dowling-Meara generalized epidermolysis bullosa simplex 2 K T5
Pachyonychia congenita 4 KRT6A
Pachyonychia congenita 4 K T6B
Epidermolytic palmoplanar keratoderma 4 KRT6C
Woolly hair 4 KRT71
Hypotrichosis simplex of the scalp 2 KRT74
Woolly hair 4 KRT74
Monilethrix 4 KRT81
Monilethrix 4 KRT83
Pure hair and nail ectodermal dysplasia 2 KRT85
Monilethrix 4 KRT86
Epidermolytic palmoplantar keratoderma 4 KRT9
Encephalopathy due to hydroxykynureninuria 1 KYNU
Hirschsprung disease 7 L1CAM
Hydrocephalus with stenosis of aqueduct of Sylvius 1 L1CAM
MASA syndrome 2 L1CAM
X-linked complicated corpus callosum dysgenesis 1 L1CAM
X-linked complicated spastic paraplegia type 1 1 L1CAM
L-2-hydroxyglutaric aciduria 1 L2HGDH
Congenital muscular dystrophy type 1A 1 LAMA2
Generalized junctional epidermolysis bullosa, non-Herlitz type 5 LAMA3
Junctional epidermolysis bullosa, Herlitz type 3 LAMA3
LOC syndrome 1 LAMA3
Familial isolated dilated cardiomyopathy 38 LAMA4
Cobblestone lissencephaly without muscular or ocular involvement 1 LAMB1
LAMB-2-related infantile-onset nephrotic syndrome 1 LAMB2
Pierson syndrome 1 LAMB2
Synaptic congenital myasthenic syndromes 2 LAMB2
Generalized junctional epidermolysis bullosa, non-Herlitz type 5 LAMB3
Junctional epidermolysis bullosa, Herlitz type 3 LAMB3
Generalized junctional epidermolysis bullosa, non-Herlitz type 5 LAMC2
Junctional epidermolysis bullosa inversa 1 LAMC2
Junctional epidermolysis bullosa, Herlitz type 3 LAMC2
Occipital pachygyria and polymicrogyria 1 LAMC3
Glycogen storage disease due to LAMP-2 deficiency 1 LAMP2
Primary immunodeficiency syndrome due to pl4 deficiency 1 LAMTOR2
Cobblestone lissencephaly type B 1 LARGE
Congenital muscular dystrophy type ID 1 LARGE
Muscle eye brain disease 7 LARGE
Walker-Warburg syndrome 13 LARGE
Microcephalic primordial dwarfism, Alazami type 1 LARP7
119
Table IX-21781338.1 Perrault syndrome 4 LARS2
Williams syndrome 17 LAT2
Greenberg dysplasia 1 LBR
Reynolds syndrome 1 LBR
Leber congenital amaurosis 18 LCA5
Familial LCAT deficiency 1 LCAT
Fish-eye disease 1 LCAT
Severe combined immunodeficiency due to LCK deficiency 1 LCK
Congenital lactase deficiency 1 LCT
Congenital lactase deficiency 1 LCT
Arrhythmogenic right ventricular dysplasia 1 LDB3
Familial isolated dilated cardiomyopathy 38 LDB3
Left ventricular noncompaction 11 LDB3
Tibial muscular dystrophy 2 LDB3
ZASP-related myofibrillar myopathy 1 LDB3
Glycogen storage disease due to lactate dehydrogenase M-subunit
deficiency 1 LDHA
Glycogen storage disease due to lactate dehydrogenase H-subunit
deficiency 1 LDHB
Situs ambiguus 6 LEFTY2
12ql4 microdeletion syndrome 2 LEMD3
Buschke-Ollendorff syndrome 1 LEMD3
Isolated osteopoikilosis 1 LEMD3
Melorheostosis with osteopoikilosis 1 LEMD3
Obesity due to congenital leptin deficiency 1 LEP
Obesity due to leptin receptor gene deficiency 1 LEPR
Osteogenesis imperfecta type 2 5 LEPRE1
Osteogenesis imperfecta type 3 9 LEPRE1
Rare isolated myopia 1 LEPREL1
Wolf-Hirschhorn syndrome 3 LETM1
Autosomal recessive spondylocostal dysostosis 4 LFNG
Autosomal dominant epilepsy with auditory features 2 LGI1
Leydig cell hypoplasia due to LHB deficiency 1 LHB
Leydig cell hypoplasia due to complete LH resistance 1 LHCGR
Testotoxicosis 1 LHCGR
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 LHFPL5
17ql2 microdeletion syndrome 2 LHX1
Classic Mayer-Rokitansky-Kuster-Hauser syndrome 1 LHX1
MURCS association 1 LHX1
Hypothyroidism due to deficient transcription factors involved in pituitary
development or function 5 LHX3
Nonacquired combined pituitary hormone deficiency with spine 1 LHX3
120
Table IX-21781338.1 abnormalities
Hypothyroidism due to deficient transcription factors involved in pituitary
development or function 5 LHX4
Pituitary stalk interruption syndrome 2 LHX4
Short stature - pituitary and cerebellar defects - small sella turcica 1 LHX4
Stiive-Wiedemann syndrome 1 LIFR
LIG4 syndrome 1 LIG4
Omenn syndrome 9 LIG4
Pulverulent cataract 7 LIM2
Williams syndrome 17 LIMK1
Neuroblastoma 7 LIN28B
Autosomal recessive nonsyndromic intellectual deficit 15 LINS
Cholesteryl ester storage disease 1 LI PA
Wolman disease 1 LI PA
Hyperlipidemia due to hepatic triglyceride lipase deficiency 1 LI PC
Hypotrichosis simplex 6 LIPH
Woolly hair 4 LIPH
Hyperlipoproteinemia type 4 2 LIPI
Lamellar ichthyosis 6 LIPN
Charcot-Marie-Tooth disease type 1C 1 LITAF
Combined deficiency of factor V and factor VIII 2 LMAN1
Adactyly of foot, bilateral 1 LMBR1
Adactyly of foot, unilateral 1 LMBR1
Hypoplastic tibiae - postaxial Polydactyly 2 LMBR1
Polydactyly of a triphalangeal thumb, bilateral 2 LMBR1
Polydactyly of a triphalangeal thumb, unilateral 2 LMBR1
Radial hemimelia, bilateral 2 LMBR1
Radial hemimelia, unilateral 2 LMBR1
Syndactyly type 4 2 LMBR1
Triphalangeal thumb - polysyndactyly syndrome 2 LMBR1
Methylmalonic acidemia with homocystinuria, type cblF 2 LMBRD1
Hyperlipoproteinemia type 1 2 LMF1
Atypical Werner syndrome 1 LMNA
Autosomal codominant severe lipodystrophic laminopathy 1 LMNA
Autosomal dominant Emery-Dreifuss muscular dystrophy 4 LMNA
Autosomal dominant limb-girdle muscular dystrophy type IB 1 LMNA
Autosomal recessive Emery-Dreifuss muscular dystrophy 1 LMNA
Charcot-Marie-Tooth disease type 2B1 1 LMNA
Congenital muscular dystrophy due to LMNA mutation 1 LMNA
Dilated cardiomyopathy - hypergonadotropic hypogonadism 1 LMNA
Familial dilated cardiomyopathy with conduction defect due to LMNA 1 LMNA
121
Table IX-21781338.1 mutation
Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 LMNA
Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 LMNA
Familial isolated arrhythmogenic ventricular dysplasia, right dominant
form 10 LMNA
Familial partial lipodystrophy, Dunnigan type 1 LMNA
Familial partial lipodystrophy, Kobberling type 1 LMNA
Heart-hand syndrome, Slovenian type 1 LMNA
Hutchinson-Gilford progeria syndrome 2 LMNA
Laminopathy type Decaudain-Vigouroux 1 LMNA
Left ventricular noncompaction 11 LMNA
Lethal restrictive dermopathy 2 LMNA
Mandibuloacral dysplasia with type A lipodystrophy 1 LMNA
Progeria-associated arthropathy 1 LMNA
Adult-onset autosomal dominant leukodystrophy 1 LMNB1
Partial acquired lipodystrophy 1 LMNB2
Nail-patella syndrome 1 LMX1B
Keratoderma hereditarium mutilans with ichthyosis 1 LOR
Progressive symmetric erythrokeratodermia 1 LOR
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 LOXHD1
Hypotrichosis simplex . 6 LPAR6
Woolly hair 4 LPAR6
Genetic recurrent myoglobinuria 3 LPIN1
Majeed syndrome 1 LPIN2
Familial lipoprotein lipase deficiency 1 LPL
Hyperlipoproteinemia type 5 4 LPL
Leber congenital amaurosis 18 LRAT
Retinitis pigmentosa 61 LRAT
Common variable immunodeficiency 10 LRBA
Ochoa syndrome 2 LRIG2
Congenital stationary night blindness 12 LRIT3
Donnai-Barrow syndrome 1 LRP2
Cenani-Lenz syndrome 1 LRP4
Sclerosteosis, 2 LRP4
Autosomal dominant osteopetrosis type 1 1 LRP5
Autosomal dominant osteosclerosis, Worth type 1 LRP5
Familial exudative vitreoretinopathy 5 LRP5
Hyperostosis corticalis generalisata 2 LRP5
Idiopathic juvenile osteoporosis 4 LRP5
Osteoporosis - pseudoglioma 1 LRP5
Osteosclerosis - developmental delay - craniosynostosis 1 LRP5
122
Table IX-21781338.1 Retinopathy of prematurity 3 LRP5
Coronary artery disease - hyperlipidemia - hypertension - diabetes - osteoporosis 1 LRP6
Congenital lactic acidosis, Saguenay-Lac-St. Jean type 1 LRPPRC
Primary ciliary dyskinesia 21 LRRC50
Primary ciliary dyskinesia 21 LRRC6
Autosomal agammaglobulinemia 7 LRRC8A
Hereditary nonpolyposis colon cancer 11 LRRFIP2
Young adult-onset Parkinsonism 13 LRRK2
Autosomal dominant Charcot-Marie-Tooth disease type 2P 1 LRSAM1
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 LRTOMT
Congenital glaucoma 3 LTBP2
Glaucoma secondary to spherophakia/ectopia lentis and megalocornea 1 LTBP2
Weill-Marchesani syndrome 4 LTBP2
Oligodontia 10 LTBP3
Cutis laxa with severe pulmonary, gastrointestinal and urinary anomalies 1 LTBP4
Duchenne muscular dystrophy 2 LTBP4
Hypotonia - failure to thrive - microcephaly 1 LTC4S
Attenuated Chediak-Higashi syndrome 1 LYST
Chediak-Higashi syndrome 1 LYST
Familial renal amyloidosis due to lysozyme variant 1 LYZ
Bardet-Biedl syndrome 17 LZTFL1
Cataract-microcornea syndrome 8 MAF
Cerulean cataract 4 MAF
Pulverulent cataract 7 MAF
Multicentric carpo-tarsal osteolysis with or without nephropathy 1 MAFB
Prader-Willi syndrome 6 MAGEL2
West syndrome 4 MAGI2
X-linked immunodeficiency with magnesium defect, Epstein-Barr virus
infection and neoplasia 1 MAGT1
X-linked nonsyndromic intellectual deficit 24 MAGT1
Retinitis pigmentosa 61 MAK
MALT lymphoma 4 MALT1
Familial hypospadias 2 MAMLD1
X-linked centronuclear myopathy 2 MAMLD1
Autosomal recessive nonsyndromic intellectual deficit 15 MAN1B1
Alpha-mannosidosis, adult form 1 MAN2B1
Alpha-mannosidosis, infantile form 1 MAN2B1
Beta-mannosidosis 1 MANBA
Familial pancreatic carcinoma 8 MANF
Monoamine oxidase-A deficiency 1 MAOA
123
Table IX- 21781338.1 Cardiofaciocutaneous syndrome 4 MAP2K1
Cardiofaciocutaneous syndrome 4 MAP2K2
46,XY complete gonadal dysgenesis 8 MAP3K1
46,XY partial gonadal dysgenesis 7 MAP3K1
Distal 22qll.2 microdeletion syndrome 3 MAPK1
Lennox-Gastaut syndrome 2 MAPK10
Behavioral variant of frontotemporal dementia 3 MAPT
Classical progressive supranuclear palsy 1 MAPT
Progressive non-fluent aphasia 3 MAPT
Progressive supranuclear palsy - corticobasal syndrome 1 MAPT
Progressive supranuclear palsy - parkinsonism 1 MAPT
Progressive supranuclear palsy - progressive non fluent aphasia 1 MAPT
Progressive supranuclear palsy - pure akinesia with gait freezing 1 MAPT
Semantic dementia 2 MAPT
Autosomal recessive spastic ataxia with leukoencephalopathy 1 MARS2
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 MARVELD2
Craniofacial-ulnar-renal syndrome 2 MASP1
Immunodeficiency due to MASP-2 deficiency 1 MASP2
Autosomal thrombocytopenia with normal platelets 3 MASTL
Brain demyelination due to methionine adenosyltransferase deficiency 1 MAT1A
Multiple epiphyseal dysplasia type 5 1 MATN3
Spondyloepimetaphyseal dysplasia, matrilin-3 type 1 MATN3
Distal myopathy with vocal cord weakness 1 MATR3
Hereditary pheochromocytoma-paraganglioma 7 MAX
2q23.1 microdeletion syndrome 1 MBD5
Autosomal dominant nonsyndromic intellectual deficit 15 MBD5
BRESEK syndrome 1 MBTPS2
Ichthyosis follicularis - alopecia - photophobia 1 MBTPS2
Keratosis follicularis spinulosa decalvans 2 MBTPS2
Mutilating palmoplantar keratoderma with periorificial keratotic plaques 2 MBTPS2
Familial melanoma 6 MC1R
Large congenital melanocytic nevus 1 MC1R
Oculocutaneous albinism type 2 2 MC1R
Familial glucocorticoid deficiency 4 MC2R
Obesity due MC3 deficiency 1 MC3R
Obesity due to melanocortin-4 receptor deficiency 1 MC4R
Isolated 3-methylcrotonyl-CoA carboxylase deficiency 2 MCCC1
Isolated 3-methylcrotonyl-CoA carboxylase deficiency 2 MCCC2
Methylmalonic acidemia due to methylmalonyl-CoA epimerase deficiency 1 MCEE
Combined deficiency of factor V and factor VIII 2 MCFD2
Immunodeficiency with natural-killer cell deficiency 1 MCM4
124
Table IX-21781338.1 Mucolipidosis type 4 1 MCOLN1
Autosomal recessive primary microcephaly 11 MCPH1
Premature chromosome condensation with microcephaly and intellectual
deficit 1 MCPH1
Dedifferentiated liposarcoma 3 MDM2
Well-differentiated liposarcoma 3 MDM2
Myelodysplastic syndromes 2 MECOM
Atypical Rett syndrome 4 MECP2
Intellectual deficit, X-linked - psychosis - macroorchidism 1 MECP2
Rett syndrome 1 MECP2
Severe neonatal-onset encephalopathy with microcephaly 1 MECP2
^Trisomy Xq28 1 MECP2
X-linked nonsyndromic intellectual deficit 24 MECP2
Blepharophimosis-intellectual deficit syndrome, MKB type 1 MED12
FG syndrome type 1 1 MED12
X-linked intellectual deficit with marfanoid habitus 3 MED12
X-linked nonsyndromic intellectual deficit 24 MED12
Autosomal recessive nonsyndromic intellectual deficit 15 MED23
Charcot-Marie-Tooth disease type 2B2 1 MED25
5ql4.3 microdeletion syndrome 1 MEF2C
Behcet disease 9 MEFV
Familial mediterranean fever 1 MEFV
Intermittent hydrarthrosis 2 MEFV
Maternal uniparental disomy of chromosome 14 3 MEG3 ·
Paternal uniparental disomy of chromosome 14 3 MEG3
Spinal muscular atrophy with respiratory distress 2 MEGF10
Carpenter syndrome 2 MEGF8
Familial isolated hyperparathyroidism 3 MEN1
Familial parathyroid/adenoma 2 MEN1
Multiple endocrine neoplasia type 1 5 MEN1
Zollinger-Ellison syndrome 1 MEN1
Zollinger-Ellison syndrome 1 MEN1
Isolated Klippel-Feil syndrome 3 MEOX1
Retinitis pigmentosa 61 MERTK
Autosomal recessive spondylocostal dysostosis 4 MESP2
Familial papillary renal cell carcinoma 1 MET
Hepatocellular carcinoma, childhood-onset 2 MET
Autosomal dominant Charcot-Marie-Tooth disease type 2A2 1 MFN2
Autosomal dominant optic atrophy plus syndrome 2 MFN2
Hereditary motor and sensory neuropathy type 5 1 MFN2
Hereditary motor and sensory neuropathy type 6 1 MFN2
125
Table IX-21781338.1 Severe early-onset axonal neuropathy due to MFN2 deficiency 1 MFN2
Microphthalmia - retinitis pigmentosa - foveoschisis - optic disc drusen 1 MF P
Nanophthalmia 1 MFRP
CLN7 disease 1 MFSD8
MGAT2-CDG syndrome 1 MGAT2
Progressive external ophthalmoplegia - myopathy - emaciation 1 MGME1
Giant cell glioblastoma 10 MGMT
Gliosarcoma 10 MGMT
Keutel syndrome 1 MGP
Left ventricular noncompaction 11 MIB1
X-linked Opitz G/BBB syndrome 1 MIDI
Systemic-onset juvenile idiopathic arthritis 2 MIF
Cataract with Y-shaped suture opacities 3 MIP
Cerulean cataract 4 MIP
Nuclear cataract 9 MIP
Total congenital cataract 5 MIP
Zonular cataract 9 MIP
Laurin-Sandrow syndrome 1 MIPOL1
Feingold syndrome 3 MIR17HG
EDICT syndrome 1 MIR184
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 MIR96
Clear cell renal carcinoma 1 MITF
MITF-related melanoma and renal cell carcinoma predisposition syndrome 1 MITF
Ocular albinism with congenital sensorineural deafness 2 MITF
Papillary renal cell carcinoma 1 MITF
Tietz syndrome 1 MITF
Waardenburg syndrome type 2 4 MITF
Bardet-Biedl syndrome 17 MKKS
Laurence-Moon syndrome 1 MKKS
McKusick-Kaufman syndrome 1 MKKS
Bardet-Biedl syndrome 17 MKS1
Meckel syndrome 13 MKS1
Megalencephalic leukoencephalopathy with subcortical cysts 2 MLC1
Constitutional mismatch repair deficiency syndrome 4 MLH1
Hereditary nonpolyposis colon cancer 11 MLH1
Muir-Torre syndrome 3 MLH1
Nonpolyposis Turcot syndrome 4 MLH1
Hereditary nonpolyposis colon cancer 11 MLH3
Acute biphenotypic leukemia 2 MLL
Acute myeloid leukemia with llq23 abnormalities 1 MLL
Acute undifferentiated leukemia 1 MLL
126
Table IX-21781338.1 Bilineal acute leukemia 1 MLL
Precursor B-cell acute lymphoblastic leukemia 13 MLL
Wiedemann-Steiner syndrome 1 MLL
Kabuki syndrome 2 MLL2
Precursor T-cell acute lymphoblastic leukemia 19 MLLT10
Griscelli disease type 3 2 MLPH
Williams syndrome 17 MLXIPL
Malonic aciduria 1 MLYCD
Vitamin B12-responsive methylmalonic acidemia type cbIA 1 MMAA
Methylmalonic acidemia with homocystinuria, type cbIC 1 MMACHC
Methylcobalamin deficiency type cblDvl 1 MMADHC
Vitamin B12-responsive methylmalonic acidemia, type cblDv2 1 MMADHC
Primary biliary cirrhosis 8 MMEL1
Severe generalized recessive dystrophic epidermolysis bullosa 2 MMP1
Metaphyseal anadysplasia 2 MMP13
Spondyloepimetaphyseal dysplasia, Missouri type 1 MMP13
Torg-Winchester syndrome 2 MMP14
Nodulosis-arthropathy-osteolysis syndrome 1 MMP2
Torg-Winchester syndrome 2 MMP2
Hypomaturation amelogenesis imperfecta 5 MMP20
Metaphyseal anadysplasia 2 MMP9
Familial multiple meningioma 5 MN1
Currarino triad 1 MNX1
Xanthinuria type II 1 MOCOS
Sulfite oxidase deficiency due to molybdenum cofactor deficiency type A 1 MOCS1
Sulfite oxidase deficiency due to molybdenum cofactor deficiency type B 1 MOCS2
Sulfite oxidase deficiency due to molybdenum cofactor deficiency 1 MOCS3
Narcolepsy-cataplexy 4 MOG
GCS1-CDG syndrome 1 MOGS
MPDU1-CDG syndrome 1 MPDU1
Congenital communicating hydrocephalus 1 MPDZ
MPI-CDG syndrome 1 MPI
Congenital amegakaryocytic thrombocytopenia 1 MPL
Essential thrombocythemia 4 MPL
Familial thrombocytosis 3 MPL
Myelofibrosis with myeloid metaplasia 3 MPL
Polycythemia vera 3 MPL
BIDS syndrome 1 MPLKIP
Myeloperoxidase deficiency 1 MPO
Encephalopathy due to beta-mercaptolactate-cysteine disulfiduria 1 MPST
Navajo neurohepatopathy 1 MPV17 '
127
Table IX-21781338.1 Autosomal dominant Charcot-Marie-Tooth disease type 21 1 MPZ
Autosomal dominant Charcot-Marie-Tooth disease type 2J 1 MPZ
Autosomal dominant intermediate Charcot-Marie-Tooth disease type D 1 MPZ
Autosomal dominant intermediate Charcot-Marie-Tooth disease with
neuropathic pain 1 MPZ
Charcot-Marie-Tooth disease type IB 1 MPZ
Dejerine-Sottas syndrome 4 MPZ
Roussy-Levy syndrome 2 MPZ
Familial glucocorticoid deficiency 4 MRAP
Ataxia-telangiectasia-like disorder 1 MRE11A
Hereditary breast and ovarian cancer syndrome 13 MRE11A
Combined oxidative phosphorylation defect type 9 1 MRPL3
Infantile hypertrophic cardiomyopathy due to MRPL44 deficiency 1 MRPL44
Combined oxidative phosphorylation defect type 2 1 MRPS16
Hypotonia with lactic acidemia and hyperammonemia 1 MRPS22
Common variable immunodeficiency 10 MS4A1
Constitutional mismatch repair deficiency syndrome 4 MSH2
Hereditary nonpolyposis colon cancer 11 MSH2
Muir-Torre syndrome 3 MSH2
Nonpolyposis Turcot syndrome 4 MSH2
Constitutional mismatch repair deficiency syndrome 4 MSH6
Hereditary nonpolyposis colon_cancer 11 MSH6
Muir-Torre syndrome 3 MSH6
Nonpolyposis Turcot syndrome 4 MSH6
Familial prostate cancer 14 M5MB
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 MSRB3
Myostatin-related muscle hypertrophy 1 MSTN
Hypodontia - dysplasia of nails 1 MSX1
Oligodontia 10 MSX1
Craniosynostosis, Boston type 1 M5X2
Parietal foramina 2 MSX2
Parietal foramina with cleidocranial dysplasia 1 MSX2
Diaphyseal medullary stenosis - bone malignancy 1 MTAP
Familial infantile bilateral striatal necrosis 2 MT-ATP6
Leber hereditary optic neuropathy 11 MT-ATP6
Maternally-inherited Leigh syndrome 14 MT-ATP6
Maternally-inherited spastic paraplegia 1 MT-ATP6
NARP syndrome 1 MT-ATP6
Kearns-Sayre syndrome 3 MT-ATP8
Genetic recurrent myoglobinuria 3 MT-COl
Isolated cytochrome C oxidase deficiency 9 MT-COl
128
Table IX-21781338.1 Leber hereditary optic neuropathy 11 MT-COl
Maternally-inherited Leigh syndrome 14 MT-COl
MELAS syndrome 13 MT-COl
Mitochondrial nonsyndromic sensorineural deafness 6 MT-COl
Mitochondrial nonsyndromic sensorineural deafness with susceptibility to
aminoglycoside exposure 4 MT-COl
Isolated cytochrome C oxidase deficiency 9 MT-C02
Maternally-inherited Leigh syndrome 14 MT-C02
MELAS syndrome 13 MT-C02
Genetic recurrent myoglobinuria 3 MT-C03
Isolated cytochrome C oxidase deficiency 9 MT-C03
Leber hereditary optic neuropathy 11 MT-C03
Maternally-inherited Leigh syndrome 14 MT-C03
MELAS syndrome 13 MT-C03
Histiocytoid cardiomyopathy 1 MT-CYB
Isolated CoQ-cytochrome C reductase deficiency 6 MT-CYB
Leber hereditary optic neuropathy 11 MT-CYB
Combined oxidative phosphorylation defect type 15 1 MTFMT
Isolated NADH-CoQ reductase deficiency 25 MTFMT
Cervical spina bifida aperta 6 MTHFD1
Cervical spina bifida cystica 6 MTHFD1
Cervicothoracic spina bifida aperta 6 MTHFD1
Cervicothoracic spina bifida cystica 6 MTHFD1
Lumbosacral spina bifida aperta 6 MTHFD1
Lumbosacral spina bifida cystica 6 MTHFD1
Thoracolumbosacral spina bifida aperta 6 MTHFD1
Thoracolumbosacral spina bifida cystica 6 MTHFD1
Total spina bifida aperta 6 MTHFD1
Total spina bifida cystica 6 MTHFD1
Upper thoracic spina bifida aperta 6 MTHFD1 .
Upper thoracic spina bifida cystica 6 MTHFD1
Cervical spina bifida aperta 6 MTHF
Cervical spina bifida cystica 6 MTHFR
Cervicothoracic spina bifida aperta 6 MTHF
Cervicothoracic spina bifida cystica 6 MTHFR
Homocystinuria due to methylenetetrahydrofolate reductase deficiency 1 MTHFR
Isolated anencephaly/exencephaly 2 MTHFR
Lumbosacral spina bifida aperta 6 MTHFR
Lumbosacral spina bifida cystica 6 MTHFR
Methotrexate poisoning 1 MTHFR
Thoracolumbosacral spina bifida aperta 6 MTHFR
129
Table IX-21781338.1 Thoracolumbosacral spina bifida cystica 6 MTHFR
Total spina bifida aperta 6 MTHFR
Total spina bifida cystica 6 MTHFR
Upper thoracic spina bifida aperta 6 MTHFR
Upper thoracic spina bifida cystica 6 MTHFR
X-linked centronuclear myopathy 2 MTM1
Autosomal dominant centronuclear myopathy 4 MTMR14 ·
Leber hereditary optic neuropathy 11 MT-ND1
Maternally-inherited Leigh syndrome 14 MT-ND1
MELAS syndrome 13 MT-ND1
Isolated NADH-CoQ reductase deficiency 25 MT-ND2
Leber hereditary optic neuropathy 11 MT-ND2
Maternally-inherited Leigh syndrome 14 MT-ND2
Leber hereditary optic neuropathy . 11 MT-ND3
Leber 'plus' disease 3 MT-ND3
Maternally-inherited Leigh syndrome 14 MT- D3
Sporadic Leigh syndrome 3 MT-ND3
Leber hereditary optic neuropathy 11 MT-ND4
Leber 'plus' disease 3 MT-ND4
Maternally-inherited Leigh syndrome 14 MT- D4
MELAS syndrome 13 MT-ND4
Mitochondrial nonsyndromic sensorineural deafness with susceptibility to
aminoglycoside exposure 4 MT-ND4
Leber hereditary optic neuropathy 11 MT-ND4L
Leber hereditary optic neuropathy 11 MT-ND5
Maternally-inherited Leigh syndrome 14 MT-ND5
MELAS syndrome 13 MT-ND5
ME RF syndrome 10 MT-ND5
Sporadic Leigh syndrome 3 MT-ND5
Leber hereditary optic neuropathy 11 MT-ND6
Leber 'plus' disease 3 MT-ND6
Maternally-inherited Leigh syndrome 14 MT-ND6
MELAS syndrome 13 MT-ND6
Sporadic Leigh syndrome 3 MT-ND6
Mitochondrial hypertrophic cardiomyopathy with lactic acidosis due to
MTOl deficiency 1 MTOl
Autosomal recessive spastic ataxia - optic atrophy - dysarthria 1 MTPAP
Methylcobalamin deficiency type cbIG 1 MTR
MERRF syndrome 10 MT-RNR1
Mitochondrial nonsyndromic sensorineural deafness 6 MT-RNR1
Mitochondrial nonsyndromic sensorineural deafness with susceptibility to
aminoglycoside exposure 4 MT-RNR1
130 '
Table IX-21781338.1 Methylcpbalamin deficiency type cblE 1 MTRR
Maternally inherited diabetes and deafness 3 MT-TE
Mitochondrial myopathy with reversible cytochrome C oxidase deficiency 2 MT-TE
Myopathy and diabetes mellitus 1 MT-TE
MELAS syndrome 13 MT-TF
ME RF syndrome 10 MT-TF
Maternally-inherited mitochondrial hypertrophic cardiomyopathy 2 MT-TG
MELAS syndrome 13 MT-TH
MERRF syndrome 10 MT-TH
Mitochondrial nonsyndromic sensorineural deafness 6 MT-TH
Maternally-inherited mitochondrial hypertrophic cardiomyopathy 2 MT-TI
Maternally inherited diabetes and deafness 3 MT-TK
Maternally-inherited cardiomyopathy and hearing loss 1 MT-TK
Maternally-inherited Leigh syndrome 14 MT-TK
MERRF syndrome 10 MT-TK
Hypertrophic cardiomyopathy and renal tubular disease due to
mitochondrial DNA mutation 1 MT-TL1
Kearns-Sayre syndrome 3 MT-TL1
Maternally inherited diabetes and deafness 3 MT-TL1
Maternally-inherited Leigh syndrome 14 MT-TL1
Maternally-inherited progressive external ophthalmoplegia 1 MT-TL1
MELAS syndrome 13 MT-TL1
MERRF syndrome 10 MT-TL1
Endomyocardial fibroelastosis 1 MT-TL2
Abetalipoproteinemia 2 MTTP
MERRF syndrome 10 MT-TP
MELAS syndrome 13 MT-TQ
MERRF syndrome 10 MT-TQ
MELAS syndrome 13 MT-TS1
MERRF syndrome 10 MT-TS1
Mitochondrial nonsyndromic sensorineural deafness 6 MT-TS1
Mitochondrial nonsyndromic sensorineural deafness with susceptibility to
aminoglycoside exposure 4 MT-TS1
Palmoplantar keratoderma-deafness syndrome 2 MT-TS1
MELAS syndrome 13 MT-TS2
MERRF syndrome 10 MT-TS2
Usher syndrome type 3 3 MT-TS2
Lethal infantile mitochondrial myopathy 1 MT-TT
Maternally-inherited Leigh syndrome 14 MT-TV
Maternally-inherited Leigh syndrome 14 MT-TW
Autosomal dominant medullary cystic kidney disease with or without
hyperuricemia 2 MUC1
13:
Table IX-21781338.1 Diffuse panbronchiolitis 2 MUC5B
Idiopathic pulmonary fibrosis 11 MUC5B
Postsynaptic congenital myasthenic syndromes 9 MUSK
Vitamin B12-unresponsive methylmalonic acidemia type mut- 1 MUT
Vitamin B12-unresponsive methylmalonic acidemia type mutO 1 MUT
Familial gastric cancer 2 MUTYH
MUTYH-related attenuated familial adenomatous polyposis 1 MUTYH
Disseminated superficial actinic porokeratosis 1 MVK
Hyperimmunoglobulinemia D with periodic fever 1 MVK
Mevalonic aciduria 1 MVK
Familial prostate cancer 14 MXI1
Acute basophilic leukemia 2 MYB
Precursor T-cell acute lymphoblastic leukemia 19 MYB "
Digitotalar dysmorphism 5 MYBPC1
Lethal congenital contracture syndrome type 3 2 MYBPC1
Familial isolated dilated cardiomyopathy 38 MYBPC3
Left ventricular noncompaction 11 MYBPC3
Burkitt lymphoma 1 MYC
Precursor T-cell acute lymphoblastic leukemia 19 MYC
Feingold syndrome 3 MYCN
Neuroblastoma 7 MYCN
Pyogenic bacterial infections due to MyD88 deficiency 1 MYD88
Waldenstrom macroglobulinemia 1 MYD88
Autosomal dominant centronuclear myopathy 4 MYF6
Acute myeloid leukemia with abnormal bone marrow eosinophils
inv(16)(pl3q22) or t(16;16)(pl3;q22) 2 MYH11
Familial aortic dissection 1 MYH11
Familial thoracic aortic aneurysm and aortic dissection 8 MYH11
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 MYH14
Hereditary inclusion body myopathy - joint contractures - ophthalmoplegia 1 MYH2
Digitotalar dysmorphism 5 MYH3
Freeman-Sheldon syndrome 1 MYH3
Sheldon-Hall syndrome 4 MYH3
Atrial septal defect, ostium secundum type 8 MYH6
Familial isolated dilated cardiomyopathy 38 MYH6
Sick sinus syndrome 3 MYH6
Classic multiminicore myopathy 2 MYH7
Familial isolated dilated cardiomyopathy 38 MYH7
Hyaline body myopathy 1 MYH7
Laing distal myopathy 1 MYH7
Left ventricular noncompaction 11 MYH7
132
Table IX-21781338.1 Scapuloperoneal amyotrophy 4 MYH7
Carney complex-trismus-pseudocamptodactyly syndrome 1 MYH8
Trismus - pseudocamptodactyly 2 MYH8
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 MYH9
Congenital fiber-type disproportion myopathy 5 MYL2
Familial thoracic aortic aneurysm and aortic dissection 8 MYLK
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 MY015A
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 MYOIA
Familial idiopathic steroid-resistant nephrotic syndrome with focal
segmental hyalinosis 11 MYOIE
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 MY03A
Griscelli disease type 1 1 MY05A
Griscelli disease type 3 2 MY05A
Neuroectodermal melanolysosomal disease 1 MY05A
Microvillous inclusion disease 1 MY05B
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 MY06
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 MY06
Progressive sensorineural hearing loss - hypertrophic cardiomyopathy 1 MY06
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 MY07A
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 MY07A
Usher syndrome type 1 9 MY07A
Congenital glaucoma 3 MYOC
Juvenile glaucoma 2 MYOC
Autosomal dominant limb-girdle muscular dystrophy type 1A 1 MYOT
Myotilin-related myofibrillar myopathy without spheroid body 1 MYOT
Spheroid body myopathy 1 MYOT
Familial isolated dilated cardiomyopathy 38 MYPN
Familial isolated restrictive cardiomyopathy 3 MYPN
Premature ageing appearance-developmental delay-cardiac arrhythmia
syndrome 1 NAA10
Solitary fibrous tumor 2 NAB2 .
Acute promyelocytic leukemia 4 NABP1
Alpha-N-acetylgalactosaminidase deficiency type 1 1 NAGA
Sanfilippo syndrome type B • 1 NAGLU
Hyperammonemia due to N-acetylglutamate synthetase deficiency 1 NAGS
Proximal spinal muscular atrophy type 1 3 NAIP
Proximal spinal muscular atrophy type 2 3 NAIP
Proximal spinal muscular atrophy type 3 3 NAIP
Male infertility due to NANOS1 mutation 1 NANOS1
Gray platelet syndrome 1 NBEAL2
Familial prostate cancer 14 NBN
133
Table IX-21781338.1 Hereditary breast and ovarian cancer syndrome 13 NBN
Nijmegen breakage syndrome 1 NBN
Papillary or follicular thyroid carcinoma 14 NCOA4
Hydranencephaly 1 NDE1
Microlissencephaly 1 NDE1
Prader-Willi syndrome 6 NDN
Coats disease 1 NDP
Familial exudative vitreoretinopathy 5 NOP
Norrie disease 2 NDP
Persistent hyperplastic primary vitreous 3 NDP
Retinopathy of prematurity 3 NDP
Charcot-Marie-Tooth disease type 4D 1 ND G1
Isolated NADH-CoQ reductase deficiency 25 NDUFA1
Leigh syndrome with leukodystrophy 13 NDUFA10
Isolated NADH-CoQ reductase deficiency 25 NDUFA11
Isolated NADH-CoQ reductase deficiency 25 NDUFA2
Leigh syndrome with leukodystrophy 13 NDUFA2
Isolated NADH-CoQ reductase deficiency 25 NDUFA9
Fatal infantile hypertrophic cardiomyopathy due to mitochondrial complex
1 deficiency 1 NDUFAF1
Isolated NADH-CoQ reductase deficiency 25 NDUFAF1
Isolated NADH-CoQ reductase deficiency 25 NDUFAF2
Isolated NADH-CoQ reductase deficiency 25 NDUFAF3
Isolated NADH-CoQ reductase deficiency 25 NDUFAF4
Isolated NADH-CoQ reductase deficiency 25 NDUFAF5
Isolated NADH-CoQ reductase deficiency . 25 NDUFB3
Isolated NADH-CoQ reductase deficiency 25 NDUFB9
Isolated NADH-CoQ reductase deficiency 25 NDUFS1
Leigh syndrome with leukodystrophy 13 NDUFS1
Isolated NADH-CoQ reductase deficiency 25 NDUFS2
Isolated NADH-CoQ reductase deficiency 25 NDUFS3
Leigh syndrome with leukodystrophy 13 NDUFS3
Isolated NADH-CoQ reductase deficiency 25 NDUFS4
Leigh syndrome with leukodystrophy 13 NDUFS4
Isolated NADH-CoQ reductase deficiency 25 NDUFS6
Isolated NADH-CoQ reductase deficiency 25 NDUFS7
Leigh syndrome with leukodystrophy 13 NDUFS7
Isolated NADH-CoQ reductase deficiency 25 NDUFS8
Leigh syndrome with leukodystrophy . 13 NDUFS8
Isolated NADH-CoQ reductase deficiency 25 NDUFV1
Leigh syndrome with leukodystrophy 13 NDUFV1
134
Table IX-21781338.1 Isolated NADH-CoQ reductase deficiency 25 NDUFV2
Childhood-onset nemaline myopathy 5 NEB
Intermediate nemaline myopathy 3 NEB
Severe congenital nemaline myopathy 3 NEB
Typical nemaline myopathy 4 NEB
Amyotrophic lateral sclerosis 23 NEFH
Autosomal dominant Charcot-Marie-Tooth disease type 2E 1 NEFL
Charcot-Marie-Tooth disease type IF 1 NEFL
Severe early-onset axonal neuropathy due to NEFL deficiency 1 NEFL
Severe early-onset axonal neuropathy due to NEFL deficiency 1 NEFL
Short rib-polydactyly syndrome, Majewski type 2 NEK1
Infantile autosomal recessive medullary cystic kidney disease 3 NEK8
Renal-hepatic-pancreatic dysplasia 2 NEK8
Congenital sialidosis type 2 1 NEU1
Sialidosis type 1 1 NEU1
MODY syndrome 12 NEUROD1
Congenital malabsorptive diarrhea due to paucity of enteroendocrine cells 1 NEUROG3
Familial isolated dilated cardiomyopathy 38 NEXN
17qll microdeletion syndrome 3 NF1
17qll.2 microduplication syndrome 1 NF1
Familial segmental neurofibromatosis 1 NF1
Familial spinal neurofibromatosis 1 NF1
Juvenile myelomonocytic leukemia 3 NF1
Neurofibromatosis type 1 1 NF1
Noonan syndrome 10 NF1
Watson syndrome 1 NF1
Neurofibromatosis type 2 1 NF2
Neurofibromatosis type 3 2 NF2
Marshall-Smith syndrome 1 NFIX
Sotos syndrome 2 NFIX
Giant cell glioblastoma 10 NFKBIA
Gliosarcoma 10 NFKBIA
Hypohidrotic ectodermal dysplasia with immunodeficiency 2 NFKBIA
Fatal infantile encephalopathy-pulmonary hypertension syndrome 1 NFU1
Fatal multiple mitochondrial dysfunction syndrome 2 NFU1
Hereditary sensory and autonomic neuropathy type 5 2 NGF
Bilateral generalized polymicrogyria 2 NHEJ1
Cernunnos-XLF deficiency 1 NHEJ1
Lafora disease 2 NHLRC1
Dyskeratosis congenita 10 NHP2
Nance-Horan syndrome 1 NHS
135
Table IX-21781338.1 Nuclear cataract 9 NHS
Total congenital cataract 5 NHS
Microcephalic primordial dwarfism, Dauber type 1 NIN
Autosomal dominant spastic paraplegia type 6 1 NIPA1
Congenital nonbullous ichthyosiform erythroderma 6 NIPAL4
Lamellar ichthyosis 6 NIPAL4
5pl3 microduplication syndrome 1 NIPBL
Cornelia de Lange syndrome 5 NIPBL
Athyreosis 5 NKX2-1
Benign familial chorea 1 NKX2-1
Brain-lung-thyroid syndrome 1 NKX2-1
Papillary or follicular thyroid carcinoma 14 NKX2-1
Thyroid hypoplasia 5 NKX2-1
Athyreosis 5 NKX2-5
Atrial septal defect - atrioventricular conduction defects 1 NKX2-5
Atrial septal defect, ostium secundum type 8 NKX2-5
Familial atrial fibrillation 14 NKX2-5
Familial isolated congenital asplenia 2 NKX2-5
Familial progressive cardiac conduction defect 3 NKX2-5
Hypoplastic left heart syndrome 2 NKX2-5
Tetralogy of Fallot 8 NKX2-5
Thyroid hypoplasia 5 NKX2-5
Ventricular septal defect 4 NKX2-5
Truncus arteriosus 1 NKX2-6
Spondylo-megaepiphyseal-metaphyseal dysplasia 1 NKX3-2
Corneal intraepithelial dyskeratosis with palmoplantar hyperkeratosis and
laryngeal dyskeratosis 1 NLRP1
Vitiligo-associated autoimmune disease 2 NLRP1
NLRP12-associated hereditary periodic fever syndrome 1 NLRP12
CINCA syndrome with CIAS1 mutations 1 NLRP3
Familial cold urticaria 1 NLRP3
Muckle-Wells syndrome 1 NLRP3
Complete hydatidiform mole 2 NLRP7
Partial hydatidiform mole 1 NLRP7
Primary ciliary dyskinesia 21 NME8
Leber congenital amaurosis 18 NMNAT1
Familial glucocorticoid deficiency 4 NNT
Behcet disease 9 NOD2
Blau syndrome 1 NOD2
Alobar holoprosencephaly 14 NODAL
Lobar holoprosencephaly 14 NODAL
136
Table IX-21781338.1 Microform holoprosencephaly 14 NODAL
Midline interhemispheric variant of holoprosencephaly 14 NODAL
Semilobar holoprosencephaly 14 NODAL
Septopreoptic holoprosencephaly 14 NODAL
Situs ambiguus 6 NODAL
Situs inversus totalis 3 NODAL
Brachydactyly type B2 1 NOG
Multiple synostoses syndrome 3 NOG
Proximal symphalangism 2 NOG
Stapes ankylosis with broad thumbs and toes 1 NOG
Tarsal-carpal coalition syndrome 1 NOG
Familial cortical myoclonus 1 NOL3
Translocation renal cell carcinoma 7 NONO
Dyskeratosis congenita 10 NOP10
Spinocerebellar ataxia type 36 1 NOP56
Romano-Ward syndrome 13 NOS1AP
Acroosteolysis dominant type 1 NOTCH2
Alagille syndrome due to a NOTCH2 point mutation 1 NOTCH2
CADASIL 1 NOTCH3
Niemann-Pick disease type C, adult neurologic onset 2 NPC1
Niemann-Pick disease type C, juvenile neurologic onset 2 NPC1
Niemann-Pick disease type C, late infantile neurologic onset 2 NPC1
Niemann-Pick disease type C, severe early infantile neurologic onset 2 NPC1
Niemann-Pick disease type C, severe perinatal 2 NPC1
Niemann-Pick disease type C, adult neurologic onset 2 NPC2
Niemann-Pick disease type C, juvenile neurologic onset 2 NPC2
Niemann-Pick disease type C, late infantile neurologic onset 2 NPC2
Niemann-Pick disease type C, severe early infantile neurologic onset 2 NPC2
Niemann-Pick disease type C, severe perinatal 2 NPC2
Joubert syndrome with renal defect 5 NPHP1
Juvenile autosomal recessive medullary cystic kidney disease 4 NPHP1
Senior-Loken syndrome 8 NPHP1
Late-onset autosomal recessive medullary cystic kidney disease 3 NPHP3
NPHP3-related Meckel-like syndrome 1 NPHP3
Renal-hepatic-pancreatic dysplasia 2 NPHP3
Senior-Loken syndrome 8 NPHP3
Juvenile autosomal recessive medullary cystic kidney disease 4 NPHP4
Senior-Loken syndrome 8 NPHP4
Congenital nephrotic syndrome, Finnish type 1 NPHS1
Familial idiopathic steroid-resistant nephrotic syndrome with diffuse
mesangial proliferation 2 NPHS1
137
Table IX-21781338.1 Familial idiopathic steroid-resistant nephrotic syndrome with focal
segmental hyalinosis 11 NPHS1
Familial idiopathic steroid-resistant nephrotic syndrome with minimal
changes . 3 NPHS1
Familial idiopathic steroid-resistant nephrotic syndrome with focal
segmental hyalinosis 11 NPHS2
Familial idiopathic steroid-resistant nephrotic syndrome with minimal
changes 3 NPHS2
Sporadic idiopathic steroid-resistant nephrotic syndrome with focal
segmental hyalinosis 2 NPHS2
Sporadic idiopathic steroid-resistant nephrotic syndrome with minimal
changes 1 NPHS2
Acute promyelocytic leukemia 4 NPM1
Familial atrial fibrillation 14 NPPA
Acromesomelic dysplasia, Maroteaux type 1 NPR2
Tall stature - scoliosis - macrodactyly of the great toes 1 NPR2
46,XY complete gonadal dysgenesis 8 NR0B1
46,XY partial gonadal dysgenesis 7 NR0B1
Cytomegalic congenital adrenal hypoplasia 1 NR0B1
Amyotrophic lateral sclerosis 23 NR1H3
Intrahepatic cholestasis of pregnancy 4 NR1H4
Goldmann-Favre syndrome 1 NR2E3
Retinitis pigmentosa 61 NR2E3 '
Glucocorticoid resistance 1 NR3C1
Glucocorticoid resistance 1 NR3C1
Pseudohyperaldosteronism type 2 1 NR3C2
Renal pseudohypoaldosteronism type 1 1 NR3C2
Young adult-onset Parkinsonism 13 NR4A2
46,XX gonadal dysgenesis 4 NR5A1
46(XY complete gonadal dysgenesis 8 NR5A1
46,XY disorder of sex development - adrenal insufficiency due to CYP11A1
deficiency 2 NR5A1
46,XY partial gonadal dysgenesis 7 NR5A1
Autoimmune lymphoproliferative syndrome 6 NRAS
Noonan syndrome 10 NRAS
Retinitis pigmentosa 61 NRL
Hirschsprung disease 7 NRTN
Pitt-Hopkins-like syndrome 2 NRXN1
5q35 microduplication syndrome 1 NSD1
5q35 microduplication syndrome 1 NSD1
Beckwith-Wiedemann syndrome due to NSD1 mutation 1 NSD1 .
Sotos syndrome 2 NSD1
Weaver syndrome 2 NSD1
138
Table IX-21781338.1 CHILD syndrome 1 NSDHL
CK syndrome 1 NSDHL
Kallmann syndrome 19 NSMF
Normosmic congenital hypogonadotropic hypogonadism 18 NSMF
Autosomal recessive nonsyndromic intellectual deficit 15 NSUN2
Dubowitz syndrome 1 NSUN2
Hemolytic anemia due to pyrimidine 5' nucleotidase deficiency 1 NT5C3
Hereditary arterial and articular multiple calcification syndrome 1 NT5E
Hereditary arterial and articular multiple calcification syndrome 1 NT5E
Atypical Rett syndrome 4 NTNG1
Familial medullary thyroid carcinoma 2 NTRK1
Hereditary sensory and autonomic neuropathy type 4 1 NTRK1
Hereditary sensory and autonomic neuropathy type 5 2 NTRK1
Papillary or follicular thyroid carcinoma 14 NTRK1
Congenital mesoblastic nephroma 2 NTRK3
Fibrosarcoma 2 NTRK3
Isolated NADH-CoQ reductase deficiency 25 NUBPL
Familial atrial fibrillation 14 NUP155
Familial infantile bilateral striatal necrosis 2 NUP62
Familial infantile bilateral striatal necrosis 2 NUP62
Congenital stationary night blindness 12 NYX
Gyrate atrophy of choroid and retina 1 OAT
Idiopathic pulmonary fibrosis 11 OBFC1
3M syndrome 3 OBSL1
Angelman syndrome 6 OCA2
Oculocutaneous albinism type 2 2 OCA2
Prader-Willi syndrome 6 OCA2
Congenital intrauterine infection-like syndrome 1 OCLN
Dent disease type 2 1 OCRL
Oculocerebrorenal syndrome 1 OCRL
Colobomatous microphthalmia 8 ODZ3
Joubert syndrome with orofaciodigital defect 4 OFD1
Oral-facial-digital syndrome type 1 1 OFD1
Primary ciliary dyskinesia 21 OFD1
Retinitis pigmentosa 61 OFD1
Simpson-Golabi-Behmel syndrome type 2 1 O.FD1
Oxoglutaricaciduria 1 OGDH
Autosomal dominant optic atrophy and congenital deafness 1 OPA1
Autosomal dominant optic atrophy plus syndrome 2 OPA1
Autosomal dominant optic atrophy, classic type 1 OPA1
Early-onset X-linked optic atrophy 1 OPA2
139
Table IX-21781338.1 3-methylglutaconic aciduria type 3 2 OPA3
Autosomal dominant optic atrophy and cataract 1 OPA3
X-linked intellectual deficit - cerebellar hypoplasia 1 OPHN1
5-oxoprolinase deficiency 1 OPLAH
Cone rod dystrophy 22 OPN1LW
Blue cone monochromatism 2 OPN1MW
Cone rod dystrophy 22 OPN1MW
Tritanopia 1 OPN1SW
Amyotrophic lateral sclerosis 23 OPTN
Combined immunodeficiency due to O AI1 deficiency 1 ORAI1
Combined immunodeficiency due to ORAI1 deficiency 1 ORAI1
Ear-patella-short stature syndrome 5 ORC1
Ear-patella-short stature syndrome 5 ORC4
Ear-patella-short stature syndrome 5 ORC6
Familial primary localized cutaneous amyloidosis 2 OSMR
Infantile osteopetrosis with neuroaxonal dysplasia 1 OSTM1
Ornithine transcarbamylase deficiency 1 OTC
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 OTOA
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 OTOF
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 OTOG
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 OTOGL
Agnathia - holoprosencephaly - situs inversus 2 OTX2
Combined pituitary hormone deficiencies, genetic forms 5 OTX2
Septo-optic dysplasia 6 OTX2
Syndromic microphthalmia type 5 - 1 OTX2
Succinyl-CoA acetoacetate transferase deficiency 1 OXCT1
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 P2RX2
P2Y12 deficiency 1 P2RY12
Oculopharyngeal muscular dystrophy 1 PABPN1
Young adult-onset Parkinsonism 13 PACRG
Intellectual deficit - craniofacial dysmorphism - cryptorchidism 1 PACS1
17pl3.3 microduplication syndrome 2 PAFAH1B1
Lissencephaly due to LIS1 mutation 1 PAFAH1B1
Miller-Dieker syndrome 3 PAFAH1B1
Subcortical band heterotopia 2 PAFAH1B1
Maternal hyperphenylalaninemia 1 PAH
Mild hyperphenylalaninemia 1 PAH
Mild phenylketonuria 1 PAH
Tetrahydrobiopterin-responsive hyperphenylalaninemia/phenylketonuria 1 PAH
Tetrahydrobiopterin-responsive hyperphenylalaninemia/phenylketonuria 1 PAH
X-linked nonsyndromic intellectual deficit 24 PAK3
140
Table IX-21781338.1 Familial pancreatic carcinoma 8 PALB2
Fanconi anemia 16 PALB2
Hereditary breast and ovarian cancer syndrome 13 PALB2
Atypical pantothenate kinase associated neurodegeneration 1 PANK2
Classic pantothenate kinase associated neurodegeneration 1 PANK2
Brachyolmia type 1, Toledo type 1 PAPSS2
Spondyloepimetaphyseal dysplasia, Pakistani type 1 PAPSS2
Young adult-onset Parkinsonism 13 PARK2
Amyotrophic lateral sclerosis-parkinsonism-dementia complex 2 PARK7
Young adult-onset Parkinsonism 13 PARK7
Renal coloboma syndrome 1 PAX 2
Alveolar rhabdomyosarcoma 4 PAX3
Craniofacial-deafness-hand syndrome 1 PAX3
Waardenburg syndrome type 1 1 PAX3
Waardenburg syndrome type 3 1 PAX3
MODY syndrome 12 PAX4
Aniridia - cerebellar ataxia - intellectual deficit 1 PAX6
Autosomal dominant keratitis 1 PAX6
Foveal hypoplasia - presenile cataract 1 PAX6
Isolated aniridia 1 PAX6
Isolated aniridia 1 PAX6
Isolated optic nerve hypoplasia 1 PAX6
Morning glory syndrome 1 PAX6
Peters anomaly 6 PAX6
WAG syndrome 3 PAX6
Alveolar rhabdomyosarcoma 4 PAX7
Athyreosis 5 PAX8
Thyroid hypoplasia 5 PAX8
Oligodontia 10 PAX9
Precursor B-cell acute lymphoblastic leukemia 13 PBX1
Pyruvate carboxylase deficiency, benign type 1 PC
Pyruvate carboxylase deficiency, infantile type 1 PC
Pyruvate carboxylase deficiency, severe neonatal type 1 PC
Dehydratase deficiency 1 PCBD1
Propionic acidemia 2 PCCA
Propionic acidemia 2 PCCB
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 PCDH15
Usher syndrome type 1 9 PCDH15
Female restricted epilepsy with intellectual deficit 1 PCDH19
Phosphoenolpyruvate carboxykinase 1 deficiency 1 PCK1
Papillary or follicular thyroid carcinoma 14 PCM1
14J
Table IX-21781338.1 Microcephalic osteodysplastic primordial dwarfism type 2 1 PCNT
Seckel syndrome 6 PCNT
Obesity due to prohormone convertase-l deficiency 1 PCSK1
Hereditary cerebral cavernous malformation 3 PDCD10
Primary pigmented nodular adrenocortical disease 3 PDE11A
Acrodysostosis 2 PDE4D
Acrodysostosis with multiple hormone resistance 2 PDE4D
Retinitis pigmentosa 61 PDE6A
Congenital stationary night blindness 12 PDE6B
Retinitis pigmentosa 61 PDE6B
Achromatopsia 6 PDE6C
Progressive cone dystrophy 4 PDE6C
Retinitis pigmentosa 61 PDE6G
Achromatopsia 6 PDE6H
Autosomal dominant striatal neurodegeneration 1 PDE8B
Primary pigmented nodular adrenocortical disease 3 PDE8B
Dermatofibrosarcoma protuberans 2 PDGFB
Familial multiple meningioma 5 PDGFB
Gastrointestinal stromal tumor 4 PDGFRA
Idiopathic hypereosinophilic syndrome 3 PDGFRA
Myeloid neoplasm associated with PDGFRA rearrangement 1 PDGFRA
Precursor B-cell acute lymphoblastic leukemia 13 PDGFRA
Bilateral striopallidodentate calcinosis 2 PDGFRB
Chronic myelomonocytic leukemia 2 PDGFRB
Idiopathic hypereosinophilic syndrome 3 PDGFRB
Infantile myofibromatosis 1 PDGFRB
Myeloid neoplasm associated with PDGFRB rearrangement 1 PDGFRB
Unclassified chronic myeloproliferative disease 1 PDGFRB
Leigh syndrome with cardiomyopathy 8 PDHA1
Pyruvate dehydrogenase El-alpha deficiency 1 PDHA1
Pyruvate dehydrogenase El-beta deficiency 1 PDHB
Pyruvate dehydrogenase E3-binding protein deficiency 1 PDHX
X-linked Charcot-Marie-Tooth disease type 6 1 PDK3
Pyruvate dehydrogenase phosphatase deficiency 1 PDP1
Deafness - encephaloneuropathy - obesity - valvulopathy 1 PDSS1
Leigh syndrome with nephrotic syndrome 4 PDSS2
MODY syndrome 12 PDX1
Partial pancreatic agenesis 1 PDX1
Permanent neonatal diabetes mellitus 5 PDX1
Spinocerebellar ataxia type 23 1 PDYN
Usher syndrome type 2 4 PDZD7
142
Table IX-21781338.1 Prolidase deficiency 1 PEPD
Familial advanced sleep-phase syndrome 2 PER2
Infantile efsum disease 13 PEX1
Neonatal adrenoleukodystrophy 13 PEX1
Zellweger syndrome 13 PEX1
Autosomal recessive ataxia due to PEXIO deficiency 1 PEXIO
Infantile Refsum disease 13 PEXIO
Neonatal adrenoleukodystrophy 13 PEXIO
Zellweger syndrome 13 PEXIO
Infantile Refsum disease 13 PEX11B
Neonatal adrenoleukodystrophy 13 PEX11B
Zellweger syndrome 13 PEX11B
Infantile Refsum disease 13 PEX12
Neonatal adrenoleukodystrophy 13 PEX12
Zellweger syndrome 13 PEX12
Infantile Refsum disease 13 PEX13
Neonatal adrenoleukodystrophy 13 PEX13
Zellweger syndrome 13 PEX13
Infantile Refsum disease 13 PEX14
Neonatal adrenoleukodystrophy 13 PEX14
Zellweger syndrome 13 PEX14
Infantile Refsum disease 13 PEX16
Neonatal adrenoleukodystrophy 13 PEX16
Zellweger syndrome 13 PEX16
Infantile Refsum disease 13 PEX19
Neonatal adrenoleukodystrophy 13 PEX19
Zellweger syndrome 13 PEX19
Infantile Refsum disease 13 PEX2
Neonatal adrenoleukodystrophy 13 PEX2
Zellweger syndrome 13 PEX2
Infantile Refsum disease 13 PEX26
Neonatal adrenoleukodystrophy 13 PEX26
Zellweger syndrome 13 PEX26
Infantile Refsum disease 13 PEX3
Neonatal adrenoleukodystrophy 13 PEX3
Zellweger syndrome 13 PEX3
Infantile Refsum disease 13 PEX5
Neonatal adrenoleukodystrophy 13 PEX5
Zellweger syndrome 13 PEX5
Infantile Refsum disease 13 PEX6
Neonatal adrenoleukodystrophy 13 PEX6
143
Table IX-21781338.1 Zellweger syndrome 13 PEX6
Refsum disease 2 PEX7
Rhizomelic chondrodysplasia punctata type 1 1 PEX7
Glycogen storage disease due to muscle phosphofructokinase deficiency 1 PFKM
Amyotrophic lateral sclerosis 23 PFN1
Glycogen storage disease due to phosphoglycerate mutase deficiency 1 PGAM2
Hyperphosphatasia-intellectual deficiency syndrome 3 PGAP2
Glycogen storage disease due to phosphoglycerate kinase 1 deficiency 1 PGK1
Glycogen storage disease due to phosphoglucomutase deficiency 1 PGM1
PGM-CDG syndrome 1 PGM1
Autosomal recessive primary microcephaly 11 PHC1
X-linked hypophosphatemia 1 PHEX
Potocki-Shaffer syndrome 3 PHF21A
Borjeson-Forssman-Lehmann syndrome 1 PHF6
Intellectual deficit, X-linked, Siderius type 1 PHF8
3-Phosphoglycerate dehydrogenase deficiency 1 PHGDH
Glycogen storage disease due to muscle phosphorylase kinase deficiency 2 PHKA1
Glycogen storage disease due to liver phosphorylase kinase deficiency 2 PHKA2
Glycogen storage disease due to liver and muscle phosphorylase kinase
deficiency 1 PHKB
Glycogen storage disease due to muscle phosphorylase kinase deficiency 2 PHKG1
Glycogen storage disease due to liver phosphorylase kinase deficiency 2 PHKG2
Congenital fibrosis of extraocular muscles 4 PHOX2A
Haddad syndrome 3 PHOX2B
Neuroblastoma 7 PHOX2B
Ondine syndrome 5 PHOX2B
Refsum disease 2 PHYH
Dehydrated hereditary stomatocytosis 1 PIEZOl
Arthrogryposis with oculomotor limitation and electroretinal anomalies 1 PIEZ02
Multiple congenital anomalies-hypotonia-seizures syndrome type 2 1 PIGA
Paroxysmal nocturnal hemoglobinuria 1 PIGA
Zunich-Kaye syndrome 1 PIGL
Hypercoagulability syndrome due to glycosylphosphatidylinositol
deficiency ' 1 PIGM
Multiple congenital anomalies - hypotonia - seizures syndrome 1 PIGN
Hyperphosphatasia-intellectual deficiency syndrome 3 PIGO
Hyperphosphatasia-intellectual deficiency syndrome 3 PIGV
CLOVE syndrome 1 PIK3CA
Cowden syndrome 7 PIK3CA
Hemimegalencephaly 2 PIK3CA
Hereditary nonpolyposis colon cancer 11 PIK3CA
144
Table IX-21781338.1 Macrocephaly-capillary malformation syndrome 1 PIK3CA acrodactyly of fingers, unilateral 1 PIK3CA
Segmental progressive overgrowth syndrome with fibroadipose
hyperplasia 1 PIK3CA
Autosomal agammaglobulinemia 7 PIK3R1
Megalencephaly - polymicrogyria - post-axial Polydactyly - hydrocephalus 2 PIK3R2
Spinocerebellar ataxia with axonal neuropathy type 2 2 PIK3R5
Fleck corneal dystrophy 1 PIKFYVE
Young adult-onset Parkinsonism 13 PINK1
Lethal congenital contracture syndrome type 3 2 PIP5K1C
Cone rod dystrophy 22 PITPNM3
Brachydactyly - elbow wrist dysplasia 1 PITX1
Familial clubfoot due to 5q31 microdeletion 1 PITX1
Familial clubfoot due to PITX1 point mutation 1 PITX1
Axenfeld-Rieger syndrome 2 PITX2
Axenfeld's anomaly 2 PITX2
Peters anomaly 6 PITX2
Rieger's anomaly 2 PITX2
Ring dermoid of cornea l' PITX2
Cataract-glaucoma 1 PITX3
Familial ocular anterior segment mesenchymal dysgenesis 2 PITX3
Posterior polar cataract 5 PITX3
Autosomal dominant polycystic kidney disease type 1 with tuberous
sclerosis 2 PKD1
Autosomal recessive polycystic kidney disease 1 PKHD1
Hemolytic anemia due to red cell pyruvate kinase deficiency 1 PKLR
Epidermolysis bullosa simplex due to plakophilin deficiency 1 PKP1
Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 PKP2
Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 PKP2
Familial isolated arrhythmogenic ventricular dysplasia, right dominant
form 10 PKP2
Adult-onset dystonia-parkinsonism 1 PLA2G6
Infantile neuroaxonal dystrophy 1 PLA2G6
Paternal uniparental disomy of chromosome 6 2 PLAGL1
Transient neonatal diabetes mellitus 5 PLAGLl
Quebec platelet disorder 1 PLAU
Early infantile epileptic encephalopathy 9 PLCB1
Malignant migrating partial seizures of infancy 4 PLCB1
Auriculo-condylar syndrome 2 PLCB4
Leukonychia totalis 1 PLCD1
Familial idiopathic steroid-resistant nephrotic syndrome with diffuse
mesangial sclerosis 4 PLCE1
145
Table IX-21781338.1 Familial idiopathic steroid-resistant nephrotic syndrome with focal
segmental hyalinosis 11 PLCE1
Autoinflammation and PLCG2-associated antibody deficiency and immune
dysregulation 1 PLCG2
PLCG2-associated antibody deficiency and immune dysregulation 1 PLCG2
Hermansky-Pudlak syndrome type 9 1 PLDN
Autosomal recessive limb-girdle muscular dystrophy type 2Q 1 PLEC
Epidermolysis bullosa simplex with muscular dystrophy 1 PLEC
Epidermolysis bullosa simplex with pyloric atresia 2 PLEC
Epidermolysis bullosa simplex, Ogna type 1 PLEC
Spinocerebellar ataxia type 4 1 PLEKHG4
Autosomal recessive lower motor neuron disease with childhood onset 1 PLEKHG5
Intermediate osteopetrosis 2 PLEKHM1
Hypoplasminogenemia 1 PLG
Ligneous conjunctivitis 1 PLG
Familial partial lipodystrophy associated with PLIN1 mutations 1 PLINl
Familial isolated dilated cardiomyopathy 38 PLN
Ehlers-Danlos syndrome, kyphoscoliotic type , 1 PLOD1
Nevo syndrome 1 PLOD1
Bruck syndrome 2 PLOD2
Connective tissue disorder due to lysyl hydroxylase-3 deficiency 1 PLOD3
Null syndrome 1 PLP1
Pelizaeus-Merzbacher disease in female carriers 1 PLP1
Pelizaeus-Merzbacher disease, classic form 1 PLP1
Pelizaeus-Merzbacher disease, connatal form 1 PLP1
Pelizaeus-Merzbacher disease, transitional form 1 PLP1
Spastic paraplegia type 2 1 PLP1
Acute promyelocytic leukemia 4 PML
PMM2-CDG syndrome 1 PMM2 .
Acute inflammatory demyelinating polyradiculoneuropathy 1 PMP22
Charcot-Marie-Tooth disease type 1A 1 PMP22
Charcot-Marie-Tooth disease type IE 1 PMP22
Dejerine-Sottas syndrome 4 PMP22
Hereditary neuropathy with liability to pressure palsies 1 PMP22
Roussy-Levy syndrome 2 PMP22
Hereditary nonpolyposis colon cancer 11 PMS1
Constitutional mismatch repair deficiency syndrome 4 PMS2
Hereditary nonpolyposis colon cancer 11 PMS2
Nonpolyposis Turcot syndrome 4 PMS2
Paroxysmal non-kinesigenic dyskinesia 1 PNKD
Microcephaly - seizures - developmental delay 1 PNKP
146
Table IX-21781338.1 Purine nucleoside phosphorylase deficiency 1 PNP
Congenital nonbullous ichthyosiform erythroderma 6 PNPLA1
Neutral lipid storage myopathy 1 PNPLA2
Autosomal recessive spastic paraplegia type 39 1 PNPLA6
Pyridoxal phosphate-responsive seizures 1 PNPO
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 PNPT1
Combined oxidative phosphorylation defect type 13 1 PNPT1
Short stature-onychodysplasia-facial dysmorphism-hypotrichosis
syndrome 1 POC1A
Dowling-Degos disease 2 POFUT1
Facial dysmorphism - immunodeficiency - livedo - short stature 1 POLE
Alpers syndrome 1 POLG
Autosomal dominant progressive external ophthalmoplegia 5 POLG
Autosomal recessive progressive external ophthalmoplegia 2 POLG
Mitochondrial neurogastrointestinal encephalomyopathy 3 POLG
Recessive mitochondrial ataxic syndrome 1 POLG
Sensory ataxic neuropathy - dysarthria - ophthalmoparesis 2 POLG
Spinocerebellar ataxia with epilepsy 1 POLG
Autosomal dominant progressive external ophthalmoplegia 5 POLG 2
Xeroderma pigmentosum variant 1 POLH
Treacher-Collins syndrome 3 POLR1C
Treacher-Collins syndrome 3 POLR1D
Hypomyelination - hypogonadotropic hypogonadism - hypodontia 2 POLR3A
Odontoleukodystrophy 1 POLR3A
Hypomyelination - hypogonadotropic hypogonadism - hypodontia 2 POLR3B
Obesity due to pro-opiomelanocortin deficiency 1 POMC
Autosomal recessive limb-girdle muscular dystrophy type 20 1 POMGNT1
Muscle eye brain disease 7 POMGNT1
Walker-Warburg syndrome · 13 POMGNT1
Keratosis linearis-ichthyosis congenita-sclerosing keratoderma syndrome 1 POMP
Autosomal recessive limb-girdle muscular dystrophy type 2K 1 POMT1
Congenital muscular dystrophy - muscle hypertrophy - severe intellectual
deficit 1 POMT1
Muscle eye brain disease 7 POMT1
Walker-Warburg syndrome 13 POMT1
Autosomal recessive limb-girdle muscular dystrophy type 2N 1 POMT2
Muscle eye brain disease 7 POMT2
Walker-Warburg syndrome 13 POMT2
Amyotrophic lateral sclerosis 23 PON1
Amyotrophic lateral sclerosis 23 PON2
Amyotrophic lateral sclerosis 23 PON3
147
Table IX-21781338.1 Anauxetic dysplasia 2 POP1
Congenital adrenal hyperplasia due to cytochrome P450 oxidoreductase
deficiency 1 POR
Focal dermal hypoplasia 1 PORCN
B-cell chronic lymphocytic leukemia 7 POT1
Combined pituitary hormone deficiencies, genetic forms 5 POU1F1
Hypothyroidism due to deficient transcription factors involved in pituitary
development or function 5 POU1F1
Primary biliary cirrhosis 8 POU2AF1
Developmental delay - deafness, Hildebrand type 1 POU3F4
Gusher syndrome 1 POU3F4
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 POU4F3
Nephroblastoma 5 POU6F2
Familial partial lipodystrophy associated with PPARG mutations 1 PPARG
Giant cell glioblastoma 10 PPARG
Gliosarcoma 10 PPARG
Osteogenesis imperfecta type 2 5 PPIB
Osteogenesis imperfecta type 3 9 PPIB
Osteogenesis imperfecta type 4 7 PPIB
2p21 microdeletion syndrome 4 PPM1B
Intermediate maple syrup urine disease 5 PPM1K
Porphyria variegata 2 PPOX
Spinocerebellar ataxia type 12 1 PPP2R2B
CLN1 disease 1 PPT1
Hamel cerebro-palato-cardiac syndrome 1 PQBP1
X-linked intellectual deficit, Porteous type 1 PQBP1
X-linked intellectual deficit, Sutherland-Haan type 1 PQBP1
Translocation renal cell carcinoma 7 PRCC
Retinitis pigmentosa 61 PRCD lp36 deletion syndrome 4 PRDM16
Familial isolated dilated cardiomyopathy 38 PRDM16
Left ventricular noncompaction 11 PRDM16
Brittle cornea syndrome 2 PRDM5
Early-onset Lafora body disease 1 PRDM8
2p21 microdeletion syndrome 4 PREPL
Atypical hypotonia - cystinuria syndrome 3 PREPL
Hypotonia - cystinuria syndrome 2 PREPL
Familial hemophagocytic lymphdhistiocytosis 4 PRF1
Idiopathic aplastic anemia 5 PRF1
Camptodactyly-arthropathy-coxa-vara-pericarditis syndrome 1 PRG4
Unverricht-Lundborg disease 3 PRICKLEl
118
Table IX-21781338.1 Acrodysostosis 2 PRKAR1A
Acrodysostosis with multiple hormone resistance 2 PRKAR1A
Carney complex 1 PRKAR1A
Familial atrial myxoma 1 PRKAR1A
Primary pigmented nodular adrenocortical disease 3 PRKAR1A
Autoimmune lymphoproliferative syndrome 6 PRKCD
Common variable immunodeficiency 10 PRKCD
Spinocerebellar ataxia type 14 1 PRKCG
Isolated polycystic liver disease 2 PRKCSH
Severe combined immunodeficiency due to DNA-PKcs deficiency 1 PRKDC
Dystonia 16 1 PRKRA
Familial Alzheimer-like prion disease 1 PRNP
Fatal familial insomnia 1 PRNP
Gerstmann-Straussler-Scheinker syndrome 1 PRNP
Gerstmann-Straussler-Scheinker syndrome 1 PRNP
Huntington disease-like 1 1 PRNP
Inherited Creutzfeldt-Jakob disease 1 PRNP
Hereditary thrombophilia due to congenital protein C deficiency 1 PROC
Hyperprolinemia type 1 1 PRODH
Kallmann syndrome 19 PROK2
Normosmic congenital hypogonadotropic hypogonadism 18 PROK2
Kallmann syndrome 19 PROKR2
Normosmic congenital hypogonadotropic hypogonadism 18 PROKR2
Septo-optic dysplasia 6 PROKR2
Cone rod dystrophy 22 PROM1
Retinal macular dystrophy type 2 1 PROM1
Retinitis pigmentosa 61 PROM1
Stargardt disease 3 PROM1
Combined pituitary hormone deficiencies, genetic forms 5 PROP1
Hypothyroidism due to deficient transcription factors involved in pituitary
development or function 5 PROP1
Panhypopituitarism 2 PROP1
Hereditary thrombophilia due to congenital protein S deficiency 1 PROS1
Retinitis pigmentosa 61 PRPF3
Retinitis pigmentosa 61 PRPF31
Retinitis pigmentosa 61 PRPF6
Retinitis pigmentosa 61 PRPF8
Amyotrophic lateral sclerosis 23 PRPH
Adult-onset foveomacular vitelliform dystrophy 2 PRPH2
Butterfly-shaped pigment dystrophy 1 PRPH2
Central areolar choroidal dystrophy 2 PRPH2
149
Table IX-21781338.1 Cone rod dystrophy 22 PRPH2
Fundus albipunctatus 3 PRPH2
Retinitis pigmentosa 61 PRPH2
Retinitis punctata albescens 4 PRPH2
Lethal ataxia with deafness and optic atrophy 1 PRPS1
Phosphoribosylpyrophosphate synthetase superactivity 1 PRPS1
X-linked Charcot-Marie-Tooth disease type 5 1 PRPS1
X-linked nonsyndromic sensorineural deafness type DFN 2 PRPS1
Benign familial infantile seizures 4 PRRT2
Familial or sporadic hemiplegic migraine 4 PRRT2
ICCA syndrome 1 PRRT2
Paroxysmal kinesigenic dyskinesia 1 PRRT2
Agnathia - holoprosencephaly - situs inversus 2 PRRX1
Hereditary chronic pancreatitis 6 PRSS1
Autosomal recessive nonsyndromic intellectual deficit 15 PRSS12
Hereditary chronic pancreatitis 6 PRSS2
Isolated anophthalmia - microphthalmia 5 PRSS56
Wegener granulomatosis 3 PRTN3
Charcot-Marie-Tooth disease type 4F 1 PRX
Dejerine-Sottas syndrome 4 PRX
Atypical Gaucher disease due to saposin C deficiency 1 PSAP
Encephalopathy due to prosaposin deficiency 1 PSAP
Infantile Krabbe disease 2 PSAP
Metachromatic leukodystrophy, adult form 2 PSAP
Metachromatic leukodystrophy, juvenile form 2 PSAP
Metachromatic leukodystrophy, late infantile form 2 PSAP
Phosphoserine aminotransferase deficiency 1 PSAT1
Behavioral variant of frontotemporal dementia 3 PSEN1
Early-onset autosomal dominant Alzheimer disease 4 PSEN1
Familial isolated dilated cardiomyopathy 38 PSEN1
Progressive non-fluent aphasia 3 PSEN1
Semantic dementia 2 PSEN1
Early-onset autosomal dominant Alzheimer disease 4 PSEN2
Familial isolated dilated cardiomyopathy 38 PSEN2
CANDLE syndrome 1 PSMB8
JMP syndrome 1 PSMB8
Nakajo-Nishimura syndrome 1 •PSMB8
46,XX gonadal dysgenesis 4 PSMC3IP
3-Phosphoserine phosphatase deficiency 1 PSPH
Pyogenic arthritis - pyoderma gangrenosum - acne 1 PSTPIP1
Alobar holoprosencephaly 14 PTCH1
150
Table IX-2178 J 338.1 Gorlin syndrome 1 PTCH1
Lobar holoprosencephaly 14 PTCH1
Microform holoprosencephaly 14 PTCH1
Midline interhemispheric variant of holoprosencephaly 14 PTCH1
Semilobar holoprosencephaly 14 PTCH1
Septopreoptic holoprosencephaly 14 PTCH1
Commissural facial cleft 2 PTCH2
Bannayan-Riley-Ruvalcaba syndrome 1 PTEN
Cowden syndrome 7 PTEN
Hereditary breast and ovarian cancer syndrome 13 PTEN
Juvenile polyposis of infancy 2 PTEN
Lhermitte-Duclos disease 1 PTEN
Macrocephaly-autism syndrome 1 PTEN
Proteus syndrome 2 PTEN
Proteus-like syndrome 1 PTEN
Segmental outgrowth - lipomatosis - arteriovenous malformation - epidermal nevus 1 PTEN
Squamous cell carcinoma of head and neck 4 PTEN
Permanent neonatal diabetes mellitus - pancreatic and cerebellar agenesis 1 PTF1A
Familial isolated hypoparathyroidism due to impaired PTH secretion 2 PTH
Chondrodysplasia, Blomstrand type 1 PTH1R
Dental ankylosis 1 PTH1R
Eiken syndrome 1 PTH1R
Enchondromatosis 3 PTH1R
Metaphyseal chondrodysplasia, Jansen type 1 PTH1R
Brachydactyly type E 2 PTHLH
Juvenile myelomonocytic leukemia 3 PTPN11
LEOPARD syndrome 3 PTPN11
Metachondromatosis 1 PTPN11
Noonan syndrome 10 PTPN11
Juvenile rheumatoid factor-negative polyarthritis 9 PTPN2
Oligoarticular juvenile arthritis 9 PTPN2
Juvenile rheumatoid factor-negative polyarthritis 9 PTPN22
Oligoarticular juvenile arthritis 9 PTPN22
Wegener granulomatosis 3 PTPN22
T-B+ severe combined immunodeficiency due to CD45 deficiency 1 PTPRC
Familial idiopathic steroid-resistant nephrotic syndrome with diffuse
mesangial proliferation 2 PTPRO
Familial idiopathic steroid-resistant nephrotic syndrome with diffuse
mesangial sclerosis 4 PTPRO
Familial idiopathic steroid-resistant nephrotic syndrome with focal
segmental hyalinosis 11 PTPRO
15]
Table IX-21781338.1 Familial idiopathic steroid-resistant nephrotic syndrome with minimal
changes 3 PTPRO
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 PTPRQ
Generalized congenital lipodystrophy with myopathy 1 PTRF
6-pyruvoyl-tetrahydropterin synthase deficiency 1 PTS
Mitochondrial myopathy and sideroblastic anemia 2 PUS1
Zlotogora-Ogur syndrome 1 PVRL1
Ectodermal dysplasia - syndactyly syndrome 1 PVRL4
Congenital cataract microcornea with corneal opacity 2 PXDN
Autosomal recessive cutis laxa type 2B 1 PYCR1
Geroderma osteodysplastica 2 PYCR1
PYC l-related DeBarsy syndrome 1 PYCR1
Glycogen storage disease due to liver glycogen phosphorylase deficiency 1 PYGL
Glycogen storage disease due to liver glycogen phosphorylase deficiency 1 PYGL
Glycogen storage disease due to muscle glycogen phosphorylase
deficiency 1 PYGM
Glycogen storage disease due to muscle glycogen phosphorylase
deficiency 1 PYGM
Dihydropteridine reductase deficiency 1 QDPR
Micro syndrome 3 RAB18
Carpenter syndrome 2 RAB23
Griscelli disease type 2 1 RAB27A
Cone rod dystrophy 22 RAB28
Smith-McCort dysplasia 2 RAB33B
X-linked nonsyndromic intellectual deficit 2 RAB39B
Cataract - intellectual deficit - hypogonadism 2 RAB3GAP1
Micro syndrome 3 RAB3GAP1
Cataract - intellectual deficit - hypogonadism 2 RAB3GAP2
Micro syndrome 3 RAB3GAP2
Deafness - intellectual deficit, Martin-Probst type 1 RAB40AL
Autosomal dominant Charcot-Marie-Tooth disease type 2B 1 RAB7A
Neutrophil immunodeficiency syndrome 1 RAC2
Cornelia de Lange syndrome 5 RAD21
Hereditary breast and ovarian cancer syndrome 13 RAD50
Nijmegen breakage syndrome-like disorder 1 RAD50
Familial congenital mirror movements 2 RAD51
Hereditary breast and ovarian cancer syndrome 13 RAD51
Fanconi anemia 16 RAD51C
Hereditary breast and ovarian cancer syndrome 13 RAD51C
Hereditary breast and ovarian cancer syndrome 13 RAD51D
LEOPARD syndrome 3 RAF1
Noonan syndrome 10 RAF1
152
Table IX-21781338.1 Combined immunodeficiency T+ B+ due to partial RAG1 deficiency 1 RAG1
Combined immunodeficiency with skin granulomas 2 RAG1
Omenn syndrome - 9 RAG1
Severe combined immunodeficiency due to complete RAGl/2 deficiency 2 RAG1
Combined immunodeficiency with skin granulomas 2 RAG 2
Omenn syndrome 9 RAG2
Severe combined immunodeficiency due to complete RAGl/2 deficiency 2 RAG 2
17pll.2 microduplication syndrome 1 RAI1
Smith-Magenis syndrome 3 RAI1
Acute necrotizing encephalopathy of childhood 2 RANBP2
Familial acute necrotizing encephalopathy 1 RANBP2
Inflammatory myofibroblastic tumor 6 RANBP2
Fetal akinesia deformation sequence 2 RAPSN
Lethal multiple pterygium syndrome 4 RAPSN
Postsynaptic congenital myasthenic syndromes 9 RAPSN
Acute promye!ocytic leukemia 4 RARA
Pontocerebellar hypoplasia type 1 4 RARS2
Pontocerebellar hypoplasia type 6 1 RARS2
Parkes Weber syndrome 1 RASA1
Isolated anophthalmia - microphthalmia 5 RAX
Cone rod dystrophy 22 RAX 2
Familial retinoblastoma 1 RBI
Unilateral retinoblastoma 1 RBI
Jawad syndrome 1 RBBP8
Seckel syndrome 6 RBBP8
Autoinflammatory syndrome with pyogenic bacterial infection and
amylopectinosis * 1 RBCK1
TARP syndrome 1 RBM10
Familial isolated dilated cardiomyopathy 38 RBM20
ANE syndrome 1 RBM28
Thrombocytopenia - absent radius 1 RBM8A
Partial chromosome Y deletion 8 RBMY1A1
Retinitis pigmentosa 61 RBP3
Progressive retinal dystrophy due to retinol transport defect 1 RBP4
Adams-Oliver syndrome 4 RBPJ
Leber congenital amaurosis 18 RD3
Leber congenital amaurosis 18 RDH12
Retinitis pigmentosa 61 RDH12
Fundus albipunctatus 3 RDH5
Retinitis punctata albescens 4 RDH5
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 RDX
153
Table IX-21781338.1 Baller-Gerold syndrome 1 RECQL4
RAPADILINO syndrome 1 RECQL4
Rothmund-Thomson syndrome type 2 1 RECQL4
Autosomal dominant spastic paraplegia type 31 1 REEP1
Distal hereditary motor neuropathy type 5 3 REEP1
Hereditary chronic pancreatitis 6 REG1A
Lissencephaly syndrome, Norman-Roberts type 1 RELN
Hyperuricemia - anemia - renal failure 1 REN
Renal tubular dysgenesis of genetic origin 4 REN
Bilateral renal agenesis 2 RET
Bilateral renal dysplasia 1 RET
Familial medullary thyroid carcinoma 2 RET
Haddad syndrome 3 RET
Hirschsprung disease 7 RET
Multiple endocrine neoplasia type 2A 1 RET
Multiple endocrine neoplasia type 2B 1 RET
Papillary or follicular thyroid carcinoma 14 RET
Unilateral renal dysplasia 1 RET
Williams syndrome 17 RFC2
RFT1-CDG syndrome 1 RFT1
Immunodeficiency by defective expression of HLA class 2 5 RFX5
Hypoplastic pancreas-intestinal atresia-hypoplastic gallbalder syndrome 1 RFX6
Immunodeficiency by defective expression of HLA class 2 5 RFXANK
Immunodeficiency by defective expression of HLA class 2 5 RFXAP
Retinitis pigmentosa 61 RGR
Bradyopsia 2 RGS9
Bradyopsia 2 RGS9BP
Overhydrated hereditary stomatocytosis 1 RHAG
Rh deficiency syndrome 3 RHAG
Paimoplantar keratoderma-esophageal carcinoma syndrome 1 RHBDF2
Rh deficiency syndrome 3 RHCE
Rh deficiency syndrome 3 RHD
Congenital stationary night blindness 12 RHO
Retinitis pigmentosa 61 RHO
Retinitis punctata albescens 4 RHO
T-cell immunodeficiency with epidermodysplasia verruciformis 1 RHOH
Cone rod dystrophy 22 RIMS1
MACS syndrome 1 RIN2
Bartsocas-Papas syndrome 1 RIPK4
Noonan syndrome 10 RIT1
Bothnia retinal dystrophy 1 RLBP1
154
Table IX-21781338.1 Fundus albipunctatus 3 RLBP1
Retinitis pigmentosa 61 RLBP1
Retinitis punctata albescens 4 RLBP1
Combined oxidative phosphorylation defect type 11 1 RMND1
Anauxetic dysplasia 2 RMRP
Cartilage-hair hypoplasia 1 RMRP
Omenn syndrome 9 RMRP
Alveolar rhabdomyosarcoma 4 RMST
Aicardi-Goutieres syndrome 6 RNASEH2A
Aicardi-Goutieres syndrome 6 RNASEH2B
Aicardi-Goutieres syndrome 6 RNASEH2C
Familial prostate cancer 14 RNASEL
Cystic leukoencephalopathy without megalencephaly 1 R ASET2
17qll microdeletion syndrome 3 RNF135
Overgrowth - macrocephaly - facial dysmorphism 1 RNF135 Familial renal cell carcinoma 6 RNF139
Moyamoya disease 2 RNF213
Cerebellar ataxia - hypogonadism 1 RNF216
Microcephalic osteodysplastic primordial dwarfism types 1 and 3 1 RNU4ATAC
Familial vesicoureteral reflux 2 ROB02
Horizontal gaze palsy with progressive scoliosis 1 R0B03
Amelo-cerebro-hypohidrotic syndrome 1 ROGDI
Retinitis pigmentosa 61 ROMl
Autosomal recessive Robinow syndrome 1 ROR2
Brachydactyly type B 1 ROR2
Retinitis pigmentosa 61 RP1
Occult macular dystrophy 1 RP1L1
Retinitis pigmentosa 61 RP1L1
Retinitis pigmentosa 61 RP2
Retinitis pigmentosa 61 RP9
Choroideremia 2 RPE65
Leber congenital amaurosis 18 RPE65
Retinitis pigmentosa 61 RPE65
Achromatopsia 6 RPGR
Cone rod dystrophy 22 RPGR
Primary ciliary dyskinesia 21 RPGR
Primary ciliary dyskinesia - retinitis pigmentosa 1 RPGR
Retinitis pigmentosa 61 RPGR
Cone rod dystrophy 22 RPGRIP1
Leber congenital amaurosis 18 RPGRIP1
Meckel syndrome 13 RPGRIP1
155
Table IX-21781338.1 Joubert syndrome with hepatic defect 3 RPGRIP1L
Joubert syndrome with renal defect 5 RPGRIP1L
Meckel syndrome 13 RPGRIP1L
Blackfan-Diamond anemia 11 RPL11
Hypotrichosis simplex 6 RPL21
Blackfan-Diamond anemia 11 RPL26
Blackfan-Diamond anemia 11 RPL35A
Blackfan-Diamond anemia 11 RPL5
Blackfan-Diamond anemia 11 RPS10
Myelodysplastic syndrome associated with isolated del(5q) chromosome
abnormality 1 RPS14
Blackfan-Diamond anemia 11 RPS17
Blackfan-Diamond anemia 11 RPS19
Blackfan-Diamond anemia 11 RPS24
Blackfan-Diamond anemia 11 RPS26
Coffin-Lowry syndrome 1 RPS6KA3
X-linked nonsyndromic intellectual deficit 24 RPS6KA3
Blackfan-Diamond anemia 11 RPS7
Familial isolated congenital asplenia 2 RPSA
Adult-onset chronic progressive external ophthalmoplegia with
mitochondrial myopathy 1 RRM2B
Autosomal dominant progressive external ophthalmoplegia 5 RRM2B
Kearns-Sayre syndrome 3 RRM2B
Mitochondrial DNA depletion syndrome, encephalomyopathic form with
renal tubulopathy 1 RRM2B
Mitochondrial neurogastrointestinal encephalomyopathy 3 RRM2B
X-linked retinoschisis 1 RSI
Primary ciliary dyskinesia 21 RSPH4A
Primary ciliary dyskinesia 21 RSPH9
Palmoplantar keratoderma - XX sex reversal - predisposition to squamous .
cell carcinoma 1 RSPOl
Congenital anonychia 1 RSP04
Dyskeratosis congenita 10 RTEL1
Hoyeraal-Hreidarsson syndrome 4 RTEL1
Maternal uniparental disomy of chromosome 14 3 RTL1
Paternal uniparental disomy of chromosome 14 3 RTL1
Autosomal dominant spastic paraplegia type 12 1 RTN2
Bilateral generalized polymicrogyria 2 RTTN
Acute myeloid leukemia with t(8;21)(q22;q22) translocation 3 RUNX1
Chronic myeloid leukemia 3 RUNX1
Familial platelet syndrome with predisposition to acute myelogenous
leukemia 1 RUNX1
15G
Table IX-21781338.1 Precursor B-cell acute lymphoblastic leukemia 13 RUNX1
Acute myeloid leukemia with t(8;21)(q22;q22) translocation 3 RUNX1T1
Cleidocranial dysplasia 1 RUNX2
Metaphyseal dysplasia - maxillary hypoplasia - brachydacty 1 RUNX2
Autosomal dominant centronuclear myopathy 4 RYR1
Benign Samaritan congenital myopathy 1 RYR1
Central core disease 1 RYR1
Congenital multicore myopathy with external ophthalmoplegia 1 RYR1
King-Denborough syndrome 1 RYR1
Malignant hyperthermia 2 RYR1
Moderate multiminicore disease with hand involvement 1 RYR1
Catecholaminergic polymorphic ventricular tachycardia 4 RYR2
Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 RYR2
Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 RYR2
Familial isolated arrhythmogenic ventricular dysplasia, right dominant
form 10 RYR2
Secondary amyloidosis 1 SAA1
Autosomal recessive spastic ataxia of Charlevoix-Saguenay 1 SACS
Oguchi disease 2 SAG
Retinitis pigmentosa 61 SAG
Townes-Brocks syndrome 1 SALL1
Acro-renal-ocular syndrome 1 SALL4
IVIC syndrome 1 SALL4
Okihiro syndrome due to 20ql3 microdeletion 1 SALL4
Okihiro syndrome due to a point mutation 1 SALL4
Normocalcemic tumoral calcinosis 1 SAMD9
Aicardi-Goutieres syndrome 6 SAMHD1
Chilblain lupus 2 SAMHD1
Chylomicron retention disease 1 SAR1B
Sarcosinemia 1 SARDH
Keratosis follicularis spinulosa decalvans 2 SAT1
2q32q33 microdeletion syndrome 1 SATB2
2q33.1 microdeletion syndrome 1 SATB2
Idiopathic aplastic anemia * 5 SBDS
Shwachman-Diamond syndrome 1 SBDS
Charcot-Marie-Tooth disease type 4B2 1 SBF2
Lathosterolosis 1 SC5DL
Spinocerebellar ataxia type 20 1 SCA20
Spinocerebellar ataxia type 21 1 SCA21
Spinocerebellar ataxia type 25 1 SCA25
Spinocerebellar ataxia type 26 1 SCA26
157
Table IX-21781338.1 Spinocerebellar ataxia type 30 1 SCA30
Action myoclonus - renal failure syndrome 1 SCARB2
Action myoclonus - renal failure syndrome 1 SCARB2
Gaucher disease type 1 2 SCARB2
Unverricht-Lundborg disease 3 SCARB2
Van den Ende-Gupta syndrome 1 SCARF2
Channelopathy-associated congenital insensitivity to pain 3 SCN10A
Paroxysmal extreme pain disorder 3 SCN10A
Primary erythermalgia 3 SCN10A
Sodium channelopathy-related small fiber neuropathy 2 SCN10A
Channelopathy-associated congenital insensitivity to pain 3 SCN11A
Paroxysmal extreme pain disorder 3 SCN11A
Primary erythermalgia 3 SCN11A
Dravet syndrome 4 SCN1A
Epilepsy with myoclonic-astatic seizures 2 SCN1A
Familial or sporadic hemiplegic migraine 4 SCN1A
Generalized epilepsy with febrile seizures-plus context 6 SCN1A
Lennox-Gastaut syndrome 2 SCN1A
Malignant migrating partial seizures of infancy 4 SCN1A
Brugada syndrome 12 SCN1B
Dravet syndrome 4 SCN1B
Generalized epilepsy with febrile seizures-plus context 6 SCN1B
Benign familial infantile seizures 4 SCN2A
Benign familial neonatal-infantile seizures 2 SCN2A
Early infantile epileptic encephalopathy 9 SCN2A
Generalized epilepsy with febrile seizures-plus context 6 SCN2A
Brugada syndrome 12 SCN3B
Acetazolamide-responsive myotonia 1 SCN4A
Hyperkalemic periodic paralysis 1 SCN4A
Hypokalemic periodic paralysis 3 SCN4A
Myotonia fluctuans 1 SCN4A
Myotonia permanens 1 SCN4A
Paramyotonia congenita of Von Eulenburg 1 SCN4A
Postsynaptic congenital myasthenic syndromes 9 SCN4A
Romano-Ward syndrome 13 SCN4B
Atrial stand still 2 SCN5A
Brugada syndrome 12 SCN5A
Familial atrial fibrillation 14 SCN5A
Familial isolated dilated cardiomyopathy 38 SCN5A
Familial progressive cardiac conduction defect f 3 SCN5A
Idiopathic ventricular fibrillation, not Brugada type 2 SCN5A
158
Table IX-21781338.1 Romano-Ward syndrome 13 SCN5A
Sick sinus syndrome 3 SCN5A
Early infantile epileptic encephalopathy 9 SCN8A -
Channelopathy-associated congenital insensitivity to pain 3 SCN9A
Dravet syndrome 4 SCN9A
Erythromelalgia 1 SCN9A
Generalized epilepsy with febrile seizures-plus context 6 SCN9A
Hereditary sensory and autonomic neuropathy type 2 4 SCN9A
Paroxysmal extreme pain disorder 3 SCN9A
Primary erythermalgia 3 SCN9A
Sodium channelopathy-related small fiber neuropathy 2 SCN9A
Generalized pseudohypoaldosteronism type 1 3 SCNN1A
Idiopathic bronchiectasis 4 SCNN1A
Generalized pseudohypoaldosteronism type 1 3 SCNN1B
Idiopathic bronchiectasis 4 SCNN1B
Liddle syndrome 2 SCNN1B
Generalized pseudohypoaldosteronism type 1 3 SCNN1G
Idiopathic bronchiectasis 4 SCNN1G
Liddle syndrome 2 SCNN1G
Fatal infantile cytochrome C oxidase deficiency 5 SCOl
Fatal infantile cytochrome C oxidase deficiency 5 SC02
Leigh syndrome with cardiomyopathy 8 SC02
Leukoencephalopathy - dystonia - motor neuropathy 1 SCP2
Leukoencephalopathy - dystonia - motor neuropathy 1 SCP2
Bardet-Biedl syndrome 17 SDCCAG8
Senior-Loken syndrome 8 SDCCAG8
Familial isolated dilated cardiomyopathy 38 SDHA
Hereditary pheochromocytoma-paraganglioma 7 SDHA
Isolated succinate-CoQ reductase deficiency 3 SDHA
Leigh syndrome with leukodystrophy 13 SDHA
Isolated succinate-CoQ reductase deficiency 3 SDHAF1
Hereditary pheochromocytoma-paraganglioma 7 SDHAF2
Carney-Stratakis syndrome 3 SDHB
Cowden syndrome 7 SDHB
Gastrointestinal stromal tumor 4 SDHB
Hereditary pheochromocytoma-paraganglioma 7 SDHB
Isolated succinate-CoQ reductase deficiency 3 SDHB
Carney-Stratakis syndrome 3 SDHC
Cowden syndrome 7 SDHC
Gastrointestinal stromal tumor 4 SDHC
Hereditary pheochromocytoma-paraganglioma . 7 SDHC
159
Table IX-21781338.1 Carcinoid tumor and carcinoid syndrome 1 SDHD
Carney-Stratakis syndrome 3 SDHD
Cowden syndrome 7 SDHD
Hereditary pheochromocytoma-paraganglioma 7 SDHD
Craniolenticulosutural dysplasia 1 SEC23A
Congenital dyserythropoietic anemia type 2 1 SEC23B
Isolated polycystic liver disease 2 SEC63
Short stature-delayed bone age due to thyroid hormone metabolism
deficiency 1 SECISBP2
Kallmann syndrome 19 SEMA3A
CHARGE syndrome 2 SEMA3E
Cone rod dystrophy 22 SEMA4A
Retinitis pigmentosa 61 SEMA4A
Monosomy 5p 2 SEMA5A
Classic multiminicore myopathy 2 SEPN1
Congenital fiber-type disproportion myopathy 5 SEPN1
Desmin-related myopathy with Mallory body-like inclusions 1 SEPN1
Rigid spine syndrome 1 SEPN1
Pontocerebellar hypoplasia type 2 4 SEPSECS
Progressive cerebello-cerebral atrophy 1 SEPSECS
Neuralgic amyotrophy 1 SEPT9
MEGDEL syndrome 1 SERAC1
Alpha-1 antitrypsin deficiency 1 SERPINA1
Hemorrhagic disease due to alpha-1 antitrypsin Pittsburgh mutation 1 SERPINA1
Alpha-l-antichymotrypsin deficiency 1 SERPINA3
Corticosteroid-binding globulin deficiency 1 SERPINA6
Congenital isolated thyroxine-binding globulin deficiency 1 SERPINA7
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 SERPINB6
Hereditary thrombophilia due to congenital antithrombin deficiency 1 SERPINC1
Congenital plasminogen activator inhibitor type 1 deficiency 1 SERPINE1
Osteogenesis imperfecta type 3 9 SERPINF1
Congenital alpha2 antiplasmin deficiency 1 SERPINF2
Hereditary angioedema type 1 1 SERPING1
Immunodeficiency due to an early component of complement deficiency 8 SERPING1
Osteogenesis imperfecta type 3 9 SERPINH1
Familial encephalopathy with neuroserpin inclusion bodies 1 SERPINI1
Schinzel-Giedion syndrome 1 SETBP1
Amyotrophic lateral sclerosis type 4 1 SETX
Spinocerebellar ataxia with axonal neuropathy type 2 2 SETX
Nager syndrome 1 SF3B4
Translocation renal cell carcinoma 7 SFPQ
160
Table IX-21781338.1 Idiopathic pulmonary fibrosis 11 SFTPA1
Idiopathic pulmonary fibrosis 11 SFTPA2
Congenital pulmonary alveolar proteinosis 5 SFTPB
Infant acute respiratory distress syndrome 2 SFTPB
Neonatal acute respiratory distress with surfactant metabolism deficiency 2 SFTPB
Chronic respiratory distress with surfactant metabolism deficiency 1 SFTPC
Congenital pulmonary alveolar proteinosis 5 SFTPC
Desquamative interstitial pneumonia 1 SFTPC
Idiopathic pulmonary fibrosis 11 SFTPC
Infant acute respiratory distress syndrome 2 SFTPC
Autosomal recessive limb-girdle muscular dystrophy type 2D 1 SGCA
Autosomal recessive limb-girdle muscular dystrophy type 2E 1 SGCB
Autosomal recessive limb-girdle muscular dystrophy type 2F 1 SGCD
Familial isolated dilated cardiomyopathy 38 SGCD
Myoclonic dystonia 11 3 SGCE
Autosomal recessive limb-girdle muscular dystrophy type 2C 1 SGCG
Autosomal recessive limb-girdle muscular dystrophy type 2C 1 SGCG
Walker-Warburg syndrome 13 SGK196
Sanfilippo syndrome type A 1 SGSH
Distal 16pll.2 microdeletion syndrome 1 SH2B1
Proximal 16pll.2 microdeletion syndrome 1 SH2B1
Severe early-onset obesity-insulin resistance syndrome due to SH2B1
deficiency 1 SH2B1
X-linked lymphoproliferative disease 2 SH2D1A
Cherubism 1 SH3BP2
Frank-Ter Haar syndrome 1 SH3PXD2B
Charcot-Marie-Tooth disease type 4C 1 SH3TC2
Monosomy 22ql3 1 SHANK3
Split hand-split foot malformation 4 SHFM1
Alobar holoprosencephaly 14 SHH
Colobomatous microphthalmia 8 SHH
Hypoplastic tibiae - postaxial Polydactyly 2 SHH
Lobar holoprosencephaly 14 SHH
Microform holoprosencephaly 14 SHH
Midline interhemispheric variant of holoprosencephaly 14 SHH
Polydactyly of a triphalangeal thumb, bilateral 2 SHH
Polydactyly of a triphalangeal thumb, unilateral · 2 SHH
Radial hemimelia, bilateral 2 SHH
Radial hemimelia, unilateral 2 SHH
Schizencephaly 4 SHH
Semilobar holoprosencephaly 14 SHH
161
Table IX-21781338.1 Septopreoptic holoprosencephaly 14 SHH
Solitary median maxillary central incisor syndrome 1 SHH
Syndactyly type 4 2 SHH
Triphalangeal thumb - polysyndactyly syndrome 2 SHH
Noonan-like syndrome with loose anagen hair 1 SHOC2
Langer mesomelic dysplasia 2 SHOX
Leri-Weill dyschondrosteosis 1 SHOX
Shox-related short stature 1 SHOX
Intellectual deficit, X-linked, Stocco Dos Santos type 1 SHROOM4
Congenital sucrase-isomaltase deficiency with minimal starch tolerance 1 SI
Congenital sucrase-isomaltase deficiency with starch and lactose
intolerance 1 SI
Congenital sucrase-isomaltase deficiency with starch intolerance 1 SI
Congenital sucrase-isomaltase deficiency without starch intolerance 1 SI
Congenital sucrase-isomaltase deficiency without sucrose intolerance 1 SI
Juvenile amyotrophic lateral sclerosis 3 SIGMAR1
Marinesco-Sjogren syndrome 1 SI LI
6ql6 deletion syndrome 1 SIM1
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 SIX1
BOR syndrome 3 SIX1
Branchio-otic syndrome 2 SIX1
Alobar holoprosencephaly 14 SIX3
Lobar holoprosencephaly 14 SIX3
Microform holoprosencephaly 14 SIX3
Midline interhemispheric variant of holoprosencephaly 14 SIX3
Schizencephaly 4 SIX3
Semilobar holoprosencephaly 14 SIX3
Septopreoptic holoprosencephaly 14 SIX3
BOR syndrome 3 SIX5
14q22q23 microdeletion syndrome 2 SIX6
Microphthalmia - cataract 4 SIX6 lp36 deletion syndrome 4 SKI
Shprintzen-Goldberg syndrome 2 SKI
Syndromic diarrhea 2 SKIV2L
Tuberculosis 1 SLC11A1
Microcytic anemia with liver iron overload 1 SLC11A2
Antenatal Bartter syndrome 2 SLC12A1
Gitelman syndrome 2 SLC12A3
Corpus callosum agenesis - neuronopathy 1 SLC12A6
Exercise-induced hyperinsulinism 1 SLC16A1
Exercise-induced hyperinsulinism 1 SLC16A1
162
Table IX-21781338.1 Metabolic myopathy due to lactate transporter defect 1 SLC16A1
Juvenile cataract - microcornea - renal glucosuria 1 SLC16A12
Allan-Hemdon-Dudley syndrome 1 SLC16A2
Free sialic acid storage disease, infantile form 1 SLC17A5
Intermediate severe Salla disease 1 SLC17A5
Salla disease 1 SLC17A5
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 SLC17A8
Thiamine-responsive megaloblastic anemia syndrome 1 SLC19A2
Biotin-responsive basal ganglia disease 1 SLC19A3
Infantile spams - psychomotor retardation - progressive brain atrophy - basal ganglia disease 1 SLC19A3
Leigh syndrome with leukodystrophy 13 SLC19A3
Thiamine-responsive encephalopathy 1 SLC19A3
Alternating hemiplegia of childhood 4 SLC1A3
Episodic ataxia type 6 1 SLC1A3
Bilateral striopallidodentate calcinosis 2 SLC20A2
Hereditary renal hypouricemia 2 SLC22A12
Carnitine uptake deficiency 1 SLC22A5
Congenital stationary night blindness 12 SLC24A1
Hypocalcified amelogenesis imperfecta 2 SLC24A4
D,L-2-hydroxyglutaric aciduria 1 SLC25A1
Epileptic encephalopathy with global cerebral demyelination 1 SLC25A12
Citrullinemia type II 1 SLC25A13
Neonatal intrahepatic cholestasis due to citrin deficiency 1 SLC25A13
Hyperornithinemia-hyperammonemia-homocitrullinuria 1 SLC25A15
Amish lethal microcephaly 1 SLC25A19
Progressive demyelinating neuropathy with bilateral striatal necrosis 1 SLC25A19
Carnitine-acylcarnitine translocase deficiency 1 SLC25A20
Early infantile epileptic encephalopathy 9 SLC25A22
Early myoclonic encephalopathy 1 SLC25A22
Cardiomyopathy - hypotonia - lactic acidosis 1 SLC25A3
Autosomal recessive pyridoxine-refractory sideroblastic anemia 2 SLC25A38
Autosomal dominant progressive external ophthalmoplegia 5 SLC25A4
Congenital cataract - hypertrophic cardiomyopathy - mitochondrial
myopathy 2 SLC25A4
Facioscapulohumeral dystrophy 4 SLC25A4
Achondrogenesis type IB 1 SLC26A2
Atelosteogenesis type II 1 SLC26A2
Diastrophic dwarfism 1 SLC26A2
Multiple epiphyseal dysplasia type 4 1 SLC26A2
Congenital chloride diarrhea 1 SLC26A3
163
Table IX-21781338.1 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 SLC26A4
Pendred syndrome 3 SLC26A4
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 SLC26A5
Ichthyosis prematurity syndrome 1 SLC27A4
Dysosteosclerosis 1 SLC29A3
Faisalabad histiocytosis 1 SLC29A3
Familial sinus histiocytosis with massive lymphadenopathy 1 SLC29A3
H syndrome 1 SLC29A3
Pigmented hypertrichosis with insulin dependent diabetes mellitus
syndrome 1 SLC29A3
Childhood absence epilepsy 6 SLC2A1
Encephalopathy due to GLUT1 deficiency 1 SLC2A1
Epilepsy with myoclonic-astatic seizures 2 SLC2A1
Hereditary cryohydrocytosis with reduced stomatin 1 SLC2A1
Paroxysmal dystonic choreathetosis with episodic ataxia and spasticity 1 SLC2A1
Paroxysmal exertion-induced dyskinesia 1 SLC2A1
Arterial tortuosity syndrome 1 SLC2A10
Glycogen storage disease due to GLUT2 deficiency 1 SLC2A2
Hereditary renal hypouricemia 2 SLC2A9
Cirrhosis-dystonia-polycythemia-hypermanganesemia syndrome 1 SLC30A10 .
Autosomal dominant spastic paraplegia type 42 1 SLC33A1
Congenital cataract-hearing loss-severe developmental delay syndrome 1 SLC33A1
Dominant hypophosphatemia with nephrolithiasis or osteoporosis 2 SLC34A1
Primary Fanconi syndrome 1 SLC34A1
Pulmonary alveolar microlithiasis 1 SLC34A2
Pulmonary alveolar microlithiasis 1 SLC34A2
Hereditary hypophosphatemic rickets with hypercalciuria 1 SLC34A3
SLC35A1-CDG syndrome 1 SLC35A1
SLC35A2-CDG syndrome 1 SLC35A2
Leukocyte adhesion deficiency type II 1 SLC35C1
Schneckenbecken dysplasia •1 SLC35D1
Iminoglycinuria 4 SLC36A2
Glycogen storage disease due to glucose-6-phosphatase deficiency type b 1 SLC37A4
Ehlers-Danlos syndrome, spondylocheiro dysplastic type 1 SLC39A13
Acrodermatitis enteropathica, zinc deficiency type 1 SLC39A4
2p21 microdeletion syndrome 4 SLC3A1
Atypical hypotonia - cystinuria syndrome 3 SLC3A1
Cystinuria type A 1 SLC3A1
Hypotonia - cystinuria syndrome 2 SLC3A1
Hemochromatosis type 4 1 SLC40A1
Oculocutaneous albinism type 4 1 SLC45A2
164
Table IX-21781338.1 Hereditary folate malabsorption 1 SLC46A1
Autosomal dominant distal renal tubular acidosis 1 SLC4A1
Distal renal tubular acidosis with anemia 1 SLC4A1
Hereditary elliptocytosis 3 SLC4A1
Hereditary spherocytosis 5 SLC4A1
Congenital hereditary endothelial dystrophy II 1 SLC4A11
Corneal dystrophy - perceptive deafness 1 SLC4A11
Fuchs endothelial corneal dystrophy 4 SLC4A11
Transient neonatal multiple acyl-CoA dehydrogenase deficiency 1 SLC52A1
Riboflavin transporter deficiency 2 SLC52A2
Riboflavin transporter deficiency 2 SLC52A3
Glucose-galactose malabsorption 1 SLC5A1
Renal glucosuria 1 SLC5A2
Familial thyroid dyshormonogenesis 6 SLC5A5
Distal hereditary motor neuropathy type 7 2 SLC5A7
Iminoglycinuria 4 SLC6A18
Hartnup syndrome 1 SLC6A19
Iminoglycinuria 4 SLC6A19
Iminoglycinuria 4 SLC6A20
Infantile dystonia-parkinsonism 1 SLC6A3
Hereditary hyperekplexia 4 SLC6A5
X-linked creatine transporter deficiency 1 SLC6A8
Lysinuric protein intolerance 1 SLC7A7
Cystinuria type B 1 SLC7A9
Dominant hypophosphatemia with nephrolithiasis or osteoporosis 2 SLC9A3R1
Christianson syndrome 1 SLC9A6
Rotor syndrome 2 SLC01B1
Rotor syndrome 2 SLC01B3
Pachydermoperiostosis 2 SLC02A1
Mesomelia-synostoses syndrome 2 SLC05A1
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 SLITRK6
Mai de Meleda 1 SLURP1
Fanconi anemia 16 SLX4
Aneurysm - osteoarthritis syndrome 1 SMAD3
Familial thoracic aortic aneurysm and aortic dissection 8 SMAD3
Familial pancreatic carcinoma 8 SMAD4
Generalized juvenile polyposis/juvenile polyposis coli 3 SMAD4
Myhre syndrome 1 SMAD4
Rendu-Osler-Weber disease 3 SMAD4
Heritable pulmonary arterial hypertension 5 SMAD9
Intellectual deficit - sparse hair - brachydactyly 1 SMARCA2
165
Table IX-21781338.1 Coffin-Siris syndrome 4 SMARCA4
Familial rhabdoid tumor 2 SMARCA4
Isolated adermatoglyphia 1 SMARCAD1
Schimke immuno-osseous dysplasia 1 SMARCAL1
Atypical teratoid tumor 1 SMARCB1
Coffin-Siris syndrome 4 SMARCB1
Familial multiple meningioma 5 SMARCB1
Familial rhabdoid tumor 2 SMARCB1
Neurofibromatosis type 3 2 SMARCB1
Familial multiple meningioma 5 SMARCE1
Cornelia de Lange syndrome 5 SMC1A
Cornelia de Lange syndrome 5 SMC3
Facioscapulohumeral dystrophy 4 SMCHD1
Proximal spinal muscular atrophy type 1 3 SMN1
Proximal spinal muscular atrophy type 2 3 SMN1
Proximal spinal muscular atrophy type 3 3 SMN1
Proximal spinal muscular atrophy type 4 2 SMN1
Proximal spinal muscular atrophy type 1 3 SMN2
Proximal spinal muscular atrophy type 2 3 SMN2
Proximal spinal muscular atrophy type 3 3 SMN2
Proximal spinal muscular atrophy type 4 2 SMN2
Microphthalmia with limb anomalies 1 SMOC1
Atypical dentin dysplasia due to SMOC2 deficiency 1 SMOC2
Niemann-Pick disease type A 1 SMPD1
Niemann-Pick disease type B 1 SMPD1
X-linked nonsyndromic sensorineural deafness type DFN 2 SMPX
Intellectual deficit, X-linked, Snyder type 1 SMS
Piebaldism 2 SNAI2
Waardenburg syndrome type 2 4 SNAI2
CEDNIK syndrome 1 SNAP29
Parkinsonian-pyramidal syndrome 2 SNCA
Young adult-onset Parkinsonism 13 SNCA
SNORD11S
Prader-Willi syndrome 6 @
SNORD116
Prader-Willi syndrome 6 @
Retinitis pigmentosa 61 SNRNP200
Hypotrichosis simplex 6 SNRPE
Angelman syndrome 6 SNRPN
Prader-Willi syndrome 6 SNRPN
Romano-Ward syndrome 13 SNTA1
166
Table IX-21781338.1 Autosomal recessive malignant osteopetrosis 5 SNX10
MMEP syndrome 1 SNX3
Amyotrophic lateral sclerosis 23 SOD1
Male infertility with normal virilization due to meiosis defect 2 SOHLH1
Early-onset autosomal dominant Alzheimer disease 4 SORL1
Hereditary gingival fibromatosis 1 SOS1
Noonan syndrome 10 SOS1
Craniodiaphyseal dysplasia 1 SOST
Hyperostosis corticalis generalisata 2 SOST
Sclerosteosis 2 SOST
Kallmann syndrome 19 SOX10
Neurologic Waardenburg-Shah syndrome 1. SOX10
Waardenburg syndrome type 2 4 SOX10
Waardenburg-Shah syndrome 3 SOX10
Familial vesicoureteral reflux 2 SOX17
Hypotrichosis - lymphedema - telangiectasia 1 . SOX18
Anophthalmia/microphthalmia - esophageal atresia 1 SOX2
Colobomatous microphthalmia 8 SOX2
Septo-optic dysplasia 6 SOX2
Panhypopituitarism 2 SOX3
Septo-optic dysplasia 6 SOX3
X-linked congenital generalized hypertrichosis 1 SOX3
X-linked intellectual deficit with isolated growth hormone deficiency 1 SOX3
12pl2.1 microdeletion syndrome 1 SOX5
Developmental and speech delay due to SOX5 deficiency 1 SOX5
46,XX ovotesticular disorder of sex development 1 SOX9
Campomelic dysplasia 1 SOX9
Hepatic veno-occlusive disease - immunodeficiency 1 SP110
Osteogenesis imperfecta type 4 7 SP7
Autosomal dominant spastic paraplegia type 4 1 SPAST
Globozoospermia 2 SPATA16
Leber congenital amaurosis 18 SPATA7
Retinitis pigmentosa 61 SPAT A 7
Commissural facial cleft 2 SPECC1L
Tessier number 4 facial cleft 1 SPECC1L
Autosomal recessive spastic paraplegia type 11 1 SPG11
Autosomal recessive spastic paraplegia type 14 1 SPG14
X-linked spastic paraplegia type 16 1 SPG16
Autosomal dominant spastic paraplegia type 19 1 SPG19
Autosomal recessive spastic paraplegia type 20 1 SPG20
Autosomal recessive spastic paraplegia type 21 1 SPG21
167
Table IX-21781338.1 Autosomal recessive spastic paraplegia type 21 1 SPG21
Autosomal recessive spastic paraplegia type 23 1 SPG23
Autosomal recessive spastic paraplegia type 23 1 SPG23
Autosomal recessive spastic paraplegia type 24 1 SPG 24
Autosomal recessive spastic paraplegia type 24 1 SPG24
Autosomal recessive spastic paraplegia type 25 1 SPG 25
Autosomal recessive spastic paraplegia type 27 1 SPG27
Autosomal dominant spastic paraplegia type 29 1 SPG29
Autosomal recessive spastic paraplegia type 32 1 SPG32
X-linked spastic paraplegia type 34 1 SPG34
Autosomal dominant spastic paraplegia type 36 1 SPG36
Autosomal dominant spastic paraplegia type 37 1 SPG37
Autosomal dominant spastic paraplegia type 38 1 SPG38
Autosomal dominant spastic paraplegia type 41 1 SPG41
Autosomal recessive spastic paraplegia type 43 1 SPG43
Autosomal recessive spastic paraplegia type 45 1 SPG45
Autosomal dominant optic atrophy and peripheral neuropathy 1 SPG7
Autosomal recessive spastic paraplegia type 7 1 SPG7
Autosomal dominant spastic paraplegia type 9 1 SPG9
Primary biliary cirrhosis 8 SPIB
Hereditary chronic pancreatitis 6 SPINK1
Netherton syndrome 1 SPINK5
Congenital sodium diarrhea 1 SPINT2
Dopa responsive dystonia due to sepiapterin reductase deficiency 1 SPR
Legius syndrome 1 SPRED1
Kallmann syndrome 19 SPRY4
Normosmic congenital hypogonadotropic hypogonadism 18 SPRY4
Common hereditary elliptocytosis 1 SPTA1
Hereditary elliptocytosis 3 SPTA1
Hereditary pyropoikilocytosis 1 SPTA1
Hereditary spherocytosis 5 SPTA1
Early infantile epileptic encephalopathy 9 SPTAN1
Hereditary spherocytosis 5 SPTB
Spectrin-associated autosomal recessive cerebellar ataxia 1 SPTBN2
Spinocerebellar ataxia type 5 1 SPTBN2
Hereditary sensory and autonomic neuropathy type 1 4 SPTLC1
Hereditary sensory and autonomic neuropathy type 1 4 SPTLC2
Floating-Harbor syndrome 1 SRCAP
46,XY disorder of sex development due to 5-alpha-reductase 2 deficiency 1 SRD5A2
Familial prostate cancer 14 SRD5A2
5RD5A3-CDG syndrome 1 SRD5A3
168
Table IX-21781338.1 Autosomal dominant aplasia and myelodysplasia 1 SRP72
Benign familial epilepsy of childhood with rolandic spikes 1 SRPX2
Bilateral perisylvian polymicrogyria 1 SRPX2
Rolandic epilepsy - speech dyspraxia 1 SRPX2
45,X/46,XY mixed gonadal dysgenesis 2 SRY
46,XX testicular disorder of sex development 1 SRY
46,XY complete gonadal dysgenesis 8 SRY
46,XY partial gonadal dysgenesis 7 SRY
Testicular regression syndrome 1 SRY
Synovial sarcoma 3 SS18
Acromegaly 2 SSTR5
Synovial sarcoma 3 SSX1
Synovial sarcoma 3 SSX2
Ichthyosis-hypotrichosis syndrome 1 ST14
Autosomal recessive nonsyndromic intellectual deficit 15 ST3GAL3
West syndrome 4 ST3GAL3
Amish infantile epilepsy syndrome 1 ST3GAL5
Native American myopathy 1 STAC3
Microcephaly-capillary malformation syndrome 1 STAMBP
Classic congenital lipoid adrenal hyperplasia due to STAR deficency 1 STAR
Familial glucocorticoid deficiency 4 STAR
Non-classic congenital lipoid adrenal hyperplasia due to STAR deficency 1 STAR
Chronic mucocutaneous candidiasis 6 STATl
Mendelian susceptibility to mycobacterial diseases due to partial STATl
deficiency 1 STATl
Autosomal dominant hyper IgE syndrome 1 STAT3
Behcet disease 9 STAT4
Juvenile rheumatoid factor-negative polyarthritis 9 STAT4
Oligoarticular juvenile arthritis 9 STAT4
Laron syndrome with immunodeficiency 1 STAT5B
Solitary fibrous tumor 2 STAT6
Severe congenital hypochromic anemia with ringed sideroblasts 1 STEAP3
Autosomal recessive primary microcephaly 11 STIL
Precursor T-cell acute lymphoblastic leukemia 19 STIL
Combined immunodeficiency due to STIM1 deficiency 1 STIM1
Combined immunodeficiency due to STIM1 deficiency 1 STIM1
Tubular aggregate myopathy 1 STIM1
Peutz-Jeghers syndrome 1 STK11
Combined immunodeficiency due to STK4 deficiency 1 STK4
Colobomatous microphthalmia 8 STRA6
Matthew-Wood syndrome 1 STRA6
169
Table IX-21781338.1 Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 ST C
Deafness-infertility syndrome 2 STRC
Recessive X-linked ichthyosis 1 STS
Syndromic X-linked ichthyosis 1 STS
Familial hemophagocytic lymphohistiocytosis 4 STX11
Pseudohypoparathyroidism type IB 2 STX16
Early infantile epileptic encephalopathy 9 STXBP1
Familial hemophagocytic lymphohistiocytosis 4 STXBP2
Mitochondrial DNA depletion syndrome, encephalomyopathic form with
methylmalonic aciduria 1 SUCLA2
Fatal infantile lactic acidosis with methylmalonic aciduria 1 SUCLG1
Desmoplastic/nodular medulloblastoma 1 SUFU
Familial multiple meningioma 5 SUFU
Medulloblastoma with extensive nodularity 1 SUFU
Mesomelia-synostoses syndrome 2 SULF1
Multiple sulfatase deficiency 1 SUMF1
Isolated sulfite oxidase deficiency 1 SUOX
Fatal infantile cytochrome C oxidase deficiency 5 SURF1
Leigh syndrome with cardiomyopathy 8 SURF1
Leigh syndrome with leukodystrophy 13 SURF1
17qll microdeletion syndrome 3 SUZ12
Male infertility with normal virilization due to meiosis defect 2 SYCP3
X-linked epilepsy - learning disabilities - behavior disorders 1 SYN1
Autosomal dominant Emery-Dreifuss muscular dystrophy 4 SYNE1
Autosomal recessive ataxia, Beauce type 1 SYNE1
Autosomal recessive myogenic arthrogryposis multiplex congenita 1 SYNE1
Autosomal dominant Emery-Dreifuss muscular dystrophy 4 SYNE2
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 SYNE4
Autosomal dominant nonsyndromic intellectual deficit 15 SYNGAP1
X-linked nonsyndromic intellectual deficit 24 SYP
Autosomal recessive cerebellar ataxia - psychomotor retardation 1 SYT14
Cervical spina bifida aperta 6 T
Cervical spina bifida cystica 6 T
Cervicothoracic spina bifida aperta 6 T
Cervicothoracic spina bifida cystica 6 T
Chordoma 1 T
Lumbosacral spina bifida aperta 6 T
Lumbosacral spina bifida cystica 6 T
Thoracolumbosacral spina bifida aperta 6 T
Thoracolumbosacral spina bifida cystica 6 T
Total spina bifida aperta 6 T
170
Table IX-21781338.1 Total spina bifida cystica 6 T
Upper thoracic spina bifida aperta 6 T
Upper thoracic spina bifida cystica 6 T
Normosmic congenital hypogonadotropic hypogonadism 18 TAC3
Giant cell glioblastoma 10 TACC1
Gliosarcoma 10 TACC1
Giant cell glioblastoma 10 TACC3
Gliosarcoma 10 TACC3
Leigh syndrome with cardiomyopathy 8 TACOl
Kallmann syndrome 19 TAC 3
Normosmic congenital hypogonadotropic hypogonadism 18 TACR3
Gelatinous drop-like corneal dystrophy 1 TACSTD2
X-linked dystonia-parkinsonism 1 TAF1
Precursor T-cell acute lymphoblastic leukemia 19 TALI
Transaldolase deficiency 1 TALDOl
Immunodeficiency by defective expression of HLA class 1 3 TAP1
Immunodeficiency by defective expression of HLA class 1 3 TAP2
Immunodeficiency by defective expression of HLA class 2 5 TAP2
Immunodeficiency by defective expression of HLA class 1 3 TAPBP
Amyotrophic lateral sclerosis 23 TARDBP
Frontotemporal dementia with motor neuron disease 3 TARDBP
Tyrosinemia type 2 1 TAT
Barth syndrome 1 TAZ
Familial isolated dilated cardiomyopathy 38 TAZ
Left ventricular noncompaction 11 TAZ
Familial infantile myoclonic epilepsy 1 TBC1D24
Focal epilepsy - intellectual deficit - cerebro-cerebellar malformation 1 TBC1D24
Malignant migrating partial seizures of infancy 4 TBC1D24
Progressive myoclonic epilepsy with dystonia 1 TBC1D24
Autosomal recessive Kenny-Caffey syndrome 1 TBCE
Sanjad-Sakati syndrome 1 TBCE
Herpetic encephalitis 5 TBK1
Williams syndrome 17 TBL2
Spinocerebellar ataxia type 17 1 TBP
22qll.2 deletion syndrome 7 TBX1
22qll.2 microduplication syndrome 1 TBX1
Pelviscapular dysplasia 1 TBX15
Congenital isolated ACTH deficiency 1 TBX19
17q23.1q23.2 microdeletion syndrome 2 TBX2
Atrial septal defect, ostium secundum type 8 TBX20
Abruzzo-Erickson syndrome 1 TBX22
171
Table IX-21781338.1 X-linked cleft palate and ankyloglossia 1 TBX22
Ulnar-mammary syndrome 1 TBX3
17q23.1q23.2 microdeletion syndrome 2 TBX4
Coxo-podo-patellar syndrome 1 TBX4
Familial clubfoot due to 17q23.1q23.2 microduplication 1 TBX4
Familial clubfoot due to 17q23.1q23.2 microduplication 1 TBX4
Holt-Oram syndrome 1 TBX5
Autosomal dominant spondylocostal dysostosis 1 TBX6
Bleeding diathesis due to thromboxane synthesis deficiency 1 TBXA2
Ghosal hematodiaphyseal dysplasia 1 TBXAS1
Autosomal recessive limb-girdle muscular dystrophy type 2G 1 TCAP
Familial isolated dilated cardiomyopathy 38 TCAP
Isolated brachycephaly 3 TCF12
Isolated plagiocephaly 3 TCF12
Saethre-Chotzen syndrome 4 TCF12
Precursor B-cell acute lymphoblastic leukemia 13 TCF3
Autosomal dominant nonsyndromic intellectual deficit 15 TCF4
Fuchs endothelial corneal dystrophy 4 TCF4
Pallister-Hall syndrome 2 TCF4
Pitt-Hopkins syndrome 1 TCF4
Autosomal recessive malignant osteopetrosis 5 TCIRG1
Precursor T-cell acute lymphoblastic leukemia 19 TCL1A
Transcobalamin II deficiency 1 TCN2
Treacher-Collins syndrome 3 TCOF1
Joubert syndrome 9 TCTN1
Joubert syndrome with renal defect 5 TCTN2
Meckel syndrome 13 TCTN2
Joubert syndrome with orofaciodigital defect 4 TCTN3
Oral-facial-digital syndrome type 4 1 TCTN3
Alobar holoprosencephaly 14 TDGF1
Lobar holoprosencephaly 14 TDGF1
Microform holoprosencephaly 14 TDGF1
Midline interhemispheric variant of holoprosencephaly 14 TDGF1
Semilobar holoprosencephaly 14 TDGF1
Septopreoptic holoprosencephaly 14 TDGF1
Spinocerebellar ataxia type 1 with axonal neuropathy 1 TDP1
Helicoid peripapillary chorioretinal degeneration 1 TEADl
Autosomal recessive spastic paraplegia type 49 1 TECPR2
Autosomal recessive nonsyndromic intellectual deficit 15 TECR
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 TECTA
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 TECTA
172
Table IX-21781338.1 Mucocutaneous venous malformations 1 TEK
Dyskeratosis congenita 10 TERC
Idiopathic aplastic anemia 5 TERC
Idiopathic pulmonary fibrosis 11 TERC
Dyskeratosis congenita 10 TERT
Familial melanoma 6 TERT
Hoyeraal-Hreidarsson syndrome 4 TERT
Idiopathic aplastic anemia 5 TERT
Idiopathic pulmonary fibrosis 11 TERT
Acquired idiopathic sideroblastic anemia 1 TET2
Acute myeloid leukemia with multilineage dysplasia 1 TET2
Essential thrombocythemia 4 TET2
Myelofibrosis with myeloid metaplasia 3 TET2
Polycythemia vera 3 TET2
Refractory anemia 1 TET2
Refractory anemia with excess blasts 1 TET2
Congenital atransferrinemia 1 TF
Branchio-oculo-facial syndrome 1 TFAP2A
Char syndrome 1 TFAP2B
Mitochondrial nonsyndromic sensorineural deafness 6 TFB1M
Alveolar soft-part sarcoma 2 TFE3
Translocation renal cell carcinoma 7 TFE3
Translocation renal cell carcinoma 7 TFEB
Hereditary motor and sensory neuropathy, Okinawa type 1 TFG
Papillary or follicular thyroid carcinoma 14 TFG
Spastic paraplegia-optic atrophy-neuropathy syndrome 1 TFG
Hemochromatosis type 3 1 TFR2
Familial thyroid dyshormonogenesis 6 TG
Oligodontia 10 TGFA
Camurati-Engelmann disease 1 TGFB1
Cystic fibrosis 4 TGFB1
Familial thoracic aortic aneurysm and aortic dissection > 8 TGFB2
Peters anomaly 6 TGFB2
Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 TGFB3
Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 TGFB3
Familial isolated arrhythmogenic ventricular dysplasia, right dominant
form IP TGFB3
Granular corneal dystrophy type 1 1 TGFBI
Granular corneal dystrophy type II 1 TGFBI
Microcystic corneal dystrophy 1 TGFBI
Reis-Bucklers corneal dystrophy 1 TGFBI
173
Table IX-21781338.1 Familial thoracic aortic aneurysm and aortic dissection 8 TGFBR1
Loeys-Dietz syndrome type 1 2 TGFBR1
Multiple keratoacanthoma, Ferguson-Smith type 1 TGFBR1
Aortic dilatation - joint hypermobility - arterial tortuosity 1 TGFBR2
Familial thoracic aortic aneurysm and aortic dissection 8 TGFBR2
Hereditary nonpolyposis colon cancer 11 TGFBR2
Loeys-Dietz syndrome type 1 2 TGFBR2
Marfan syndrome type 2 1 TGFBR2
Alobar holoprosencephaly 14 TGIF1
Lobar holoprosencephaly 14 TGIF1
Microform holoprosencephaly 14 TGIF1
Midline interhemispheric variant of holoprosencephaly 14 TGIF1
Semilobar holoprosencephaly 14 TGIF1
Septopreoptic holoprosencephaly 14 TGIF1
Acral self-healing collodion baby 1 TGM1
Bathing suit ichthyosis 1 TGM1
Congenital nonbullous ichthyosiform erythroderma 6 TGM1
Lamellar ichthyosis 6 TGM1
Self-healing collodion baby 3 TGM1
Localized peeling skin syndrome 1 TGM5
Spinocerebellar ataxia type 35 1 TGM6
Autosomal recessive dopa-responsive dystonia 1 TH
Primary dystonia, DYT6 type 1 THAP1
Atypical hemolytic uremic syndrome with thrombomodulin anomaly 1 THBD
Familial thrombomodulin anomalies 1 THBD
Familial thrombocytosis 3 THPO
Hereditary thrombocytosis with transverse limb defect 1 THPO
Peripheral resistance to thyroid hormones 2 THRA
Generalized resistance to thyroid hormone 1 THRB
Peripheral resistance to thyroid hormones 2 THRB
Selective pituitary resistance to thyroid hormone 1 THRB
Distal myopathy, Welander type 1 TIA1
Distal myopathy, Welander type 1 TIA1
Herpetic encephalitis 5 TICAM1
Mohr-Tranebjaerg syndrome 1 TIMM8A
Sorsby's fundus dystrophy 1 TIMP3
Dyskeratosis congenita 10 TINF2
Hoyeraal-Hreidarsson syndrome 4 TINF2
Retinopathy - anemia- central nervous system anomalies 1 TINF2
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 TJP2
Familial hypercholanemia 3 TJP2
174
Table IX-21781338.1 Autosomal recessive progressive external ophthalmoplegia 2 TK2
Mitochondrial DNA depletion syndrome, myopathic form 1 TK2
Atrial septal defect, ostium primum type 1 TLL1
Atrial septal defect, ostium secundum type 8 TLL1
Herpetic encephalitis 5 TLR3
Behcet disease 9 TLR4
Precursor T-cell acute lymphoblastic leukemia 19 TLX1
Precursor T-cell acute lymphoblastic leukemia 19 TLX3
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 TMC1
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 TMC1
Epidermodysplasia verruciformis 2 TMC6
Epidermodysplasia verruciformis 2 TMC8
TMCOl defect syndrome 1 TMCOl
Microphthalmia - cataract 4 TMEM114
Autosomal recessive optic atrophy, OPA7 type 1 TMEM126A
Hereditary pheochromocytoma-paraganglioma 7 TMEM127
Joubert syndrome with oculorenal defect 6 TMEM138
TMEM165-CDG syndrome 1 TMEM165
FRAXF syndrome 1 TMEM185A
Joubert syndrome with oculorenal defect 6 TMEM216
Joubert syndrome with orofaciodigital defect 4 TMEM216
Meckel syndrome 13 TMEM216
Joubert syndrome 9 TMEM231
Joubert syndrome with ocular defect 5 TMEM231
Meckel syndrome 13 TMEM231
Joubert syndrome 9 TMEM237
Joubert syndrome with ocular defect 5 TMEM237
Joubert syndrome with oculorenal defect 6 TMEM237
Joubert syndrome with renal defect 5 TMEM237
Osteogenesis imperfecta type 4 7 TMEM38B
Autosomal dominant Emery-Dreifuss muscular dystrophy 4 TMEM43
Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 TMEM43
Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 TMEM43
Familial isolated arrhythmogenic ventricular dysplasia, right dominant
form 10 TMEM43
Walker-Warburg syndrome 13 TMEM5
Joubert syndrome 9 TMEM67
Joubert syndrome with hepatic defect 3 TMEM67
Meckel syndrome 13 TMEM67 '
Senior-Boichis syndrome 1 TMEM67
Isolated ATP synthase deficiency 4 TMEM70
175
Table IX-21781338.1 Mitochondrial encephalo-cardio-myopathy due to TMEM70 deficiency 1 TMEM70
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 TMIE
Familial isolated dilated cardiomyopathy 38 TMPO
Congenital enteropathy due to enteropeptidase deficiency 1 TMPRSS15
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 TMPRSS3
IRIDA syndrome 1 TMPRSS6
Squamous cell carcinoma of head and neck 4 TNFRSF10B
Autosomal recessive malignant osteopetrosis 5 TNFRSF11A
Familial expansile osteolysis 1 TNFRSF11A
Osteopetrosis - hypogammaglobulinemia 1 TNFRSF11A
Juvenile Paget's disease 1 TNFRSF11B
Common variable immunodeficiency 10 TNFRSF13B
Common variable immunodeficiency 10 TNFRSF13C
Intermittent hydrarthrosis 2 TNFRSF1A
TRAPS syndrome 1 TNFRSF1A
Autosomal recessive malignant osteopetrosis 5 TNFSF11
Common variable immunodeficiency 10 TNFSF12
Primary biliary cirrhosis 8 TNFSF15
Familial isolated dilated cardiomyopathy 38 TNNC1
Digitotalar dysmorphism 5 TNNI2
Sheldon-Hall syndrome 4 TNNI2
Familial isolated dilated cardiomyopathy 38 TNNI3
Familial isolated restrictive cardiomyopathy 3 TNNI3
Amish nemaline myopathy 1 TNNT1
Familial isolated dilated cardiomyopathy 38 TNNT2
Familial isolated restrictive cardiomyopathy 3 TNNT2
Left ventricular noncompaction 11 TN T2
Digitotalar dysmorphism 5 TNNT3
Sheldon-Hall syndrome 4 TNNT3
Autosomal dominant limb-girdle muscular dystrophy type IF 1 TNP03
Primary biliary cirrhosis 8 TNP03
Ehlers-Danlos syndrome due to tenascin-X deficiency 1 TNXB
Ehlers-Danlos syndrome, hypermobility type 1 TNXB
Neuroblastoma 7 TOP2A
Retinitis pigmentosa 61 TOPORS
Early onset torsion dystonia 1 TOR1A
Myoclonic dystonia 11 3 TOR1A
Adrenocortical carcinoma 1 TP53
B-cell chronic lymphocytic leukemia 7 TP53
Essential thrombocythemia 4 TP53
Familial pancreatic carcinoma 8 TP53
176
Table IX-21781338.1 Giant cell glioblastoma 10 TP53
Gliosarcoma 10 TP53
Li-Fraumeni syndrome 2 TP53
Papilloma of choroid plexus 1 TP53
ADULT syndrome 1 TP63
Ankyloblepharon - ectodermal defects - cleft lip/palate 1 TP63
Bladder exstrophy 1 TP63
EEC syndrome 1 TP63
Limb-mammary syndrome 1 TP63
Split hand-split foot malformation 4 TP63
Triose phosphate-isomerase deficiency 1 TPI1
Childhood encephalopathy due to thiamine pyrophosphokinase deficiency 1 TPK1
Familial isolated dilated cardiomyopathy 38 TPM1
Left ventricular noncompaction 11 TPM1
Cap myopathy 2 TPM2
Childhood-onset nemaline myopathy 5 TPM2
Congenital fiber-type disproportion myopathy 5 TPM2
Digitotalar dysmorphism 5 TPM2
Sheldon-Hall syndrome 4 TPM2
Trismus - pseudocamptodactyly 2 TPM2
Typical nemaline myopathy 4 TPM2
Childhood-onset nemaline myopathy 5 TPM3
Congenital fiber-type disproportion myopathy 5 TPM3
Inflammatory myofibroblastic tumor 6 TPM3
Intermediate nemaline myopathy 3 TPM3
Inflammatory myofibroblastic tumor 6 TPM4
Thiopurine S-methyltransferase deficiency 1 TPMT
Familial thyroid dyshormonogenesis 6 TPO
Childhood-onset autosomal recessive slowly progressive spinocerebellar
ataxia 1 TPP1
CLN2 disease 1 TPP1
CLN2 disease 1 TPP1
Papillary or follicular thyroid carcinoma 14 TPR
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 TPRN
Precursor T-cell acute lymphoblastic leukemia 19 TRA
Herpetic encephalitis 5 TRAF3
Autosomal dominant hypohidrotic ectodermal dysplasia 3 TRAF6
Spondyloepiphyseal dysplasia tarda 1 TRAPPC2
Autosomal recessive nonsyndromic intellectual deficit 15 TRAPPC9
Intellectual deficit - obesity - brain malformations - facial dysmorphism 1 TRAPPC9
Precursor T-cell acute lymphoblastic leukemia 19 TRB
177
Table IX-21781338.1 Precursor T-cell acute lymphoblastic leukemia 19 TRD
Catecholaminergic polymorphic ventricular tachycardia 4 TRDN
Diarrhea-vomiting due to trehalase deficiency 1 TREH
Nasu-Hakola disease 2 TREM2
Aicardi-Goutieres syndrome 6 TREX1
Cerebroretinal vasculopathy 1 TREX1
Chilblain lupus 2 TREX1
Hereditary vascular retinopathy 1 TREX1
HERNS syndrome 1 TREX1
Precursor T-cell acute lymphoblastic leukemia 19 TRG
Precursor T-cell acute lymphoblastic leukemia 19 TRG
Isolated thyrotropin-releasing hormone deficiency 1 TRH
Resistance to thyrotropin-releasing hormone syndrome 1 TRHR
Papillary or follicular thyroid carcinoma 14 TRIM24
Papillary or follicular thyroid carcinoma 14 TRIM27
Autosomal recessive limb-girdle muscular dystrophy type 2H 1 TRIM32
Bardet-Biedl syndrome 17 TRIM32
Papillary or follicular thyroid carcinoma 14 TRIM33
MULIBREY nanism 1 TRIM37
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 TRIOBP
Achondrogenesis type 1A 1 TRIP11
Acute infantile liver failure due to mtDNA-encoded proteins synthesis
defect 1 TRMU
Mitochondrial myopathy with reversible cytochrome C oxidase deficiency 2 TRMU
Mitochondrial nonsyndromic sensorineural deafness 6 TRMU
Familial idiopathic steroid-resistant nephrotic syndrome with focal
segmental hyalinosis 11 TRPC6
Congenital stationary night blindness 12 TRPM1
Brugada syndrome 12 TRPM4
Familial progressive cardiac conduction defect 3 TRPM4
Autosomal recessive primary hypomagnesemia with normocalcuria and
hypocalcemia 1 TRPM6
Amyotrophic lateral sclerosis-parkinsonism-dementia complex 2 TRPM7
Langer-Giedion syndrome 2 TRPS1
Trichorhinophalangeal syndrome type 1 and 3 1 TRPS1
Mutilating palmoplantar keratoderma with periorificial keratotic plaques 2 TRPV3
Autosomal dominant Charcot-Marie-Tooth disease type 2C 1 TRPV4
Autosomal dominant congenital benign spinal muscular atrophy 1 TRPV4
Brachyolmia type 3 1 TRPV4
Familial digital arthropathy-brachydactyly 1 TRPV4
Metatropic dysplasia type 1 1 TRPV4
Parastremmatic dwarfism 1 TRPV4
178
Table IX-21781338.1 Scapuloperoneal amyotrophy ' 4 TRPV4
Spondyloepiphyseal dysplasia, Maroteaux type 1 TRPV4
Spondylometaphyseal dysplasia, Kozlowski type 1 TRPV4
Isolated focal cortical dysplasia 1 TSC1
Isolated focal cortical dysplasia 1 TSC1
Lymphangioleiomyomatosis 2 TSC1
Tuberous sclerosis 2 TSC1
Autosomal dominant polycystic kidney disease type 1 with tuberous
sclerosis 2 TSC2
Lymphangioleiomyomatosis 2 TSC2
Tuberous sclerosis 2 TSC2
Pontocerebellar hypoplasia type 2 4 TSEN2
Pontocerebellar hypoplasia type 2 4 TSEN34
Pontocerebellar hypoplasia type 1 4 TSEN54
Pontocerebellar hypoplasia type 2 4 TSEN54
Pontocerebellar hypoplasia type 4 1 TSEN54
Pontocerebellar hypoplasia type 5 1 TSEN54
Fatal mitochondrial disease due to combined oxidative phosphorylation
deficiency 3 1 TSFM
Isolated thyroid-stimulating hormone deficiency 1 TSHB
Athyreosis 5 TSHR-
Familial gestational hyperthyroidism 1 TSHR
Familial hyperthyroidism due to mutations in TSH receptor 1 TSHR
Hypothyroidism due to TSH receptor mutations 1 TSHR
Thyroid hypoplasia 5 TSHR
Microtia 1 TSHZ1
Familial exudative vitreoretinopathy 5 TSPAN12
X-linked nonsyndromic intellectual deficit 24 TSPAN7
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 TSPEAR
45,X/46,XY mixed gonadal dysgenesis 2 TSPY1
Partial chromosome Y deletion 8 TSPY1
Sudden infant death - dysgenesis of the testes 1 TSPYL1
Spinocerebellar ataxia type 11 1 TTBK2
Isolated CoQ-cytochrome C reductase deficiency 6 TTC19
Infantile autosomal recessive medullary cystic kidney disease 3 TTC21B
Jeune syndrome 6 TTC21B
Syndromic diarrhea 2 TTC37
Multiple intestinal atresia 1 TTC7A
Bardet-Biedl syndrome 17 TTC8
Retinitis pigmentosa 61 TTC8
Autosomal recessive limb-girdle muscular dystrophy type 2J 1 TTN
179
Table IX-21781338.1 Early-onset myopathy with fatal cardiomyopathy 1 TTN
Familial isolated arrhythmogenic ventricular dysplasia, biventricular form 10 TTN
Familial isolated arrhythmogenic ventricular dysplasia, left dominant form 10 TTN
Familial isolated arrhythmogenic ventricular dysplasia, right dominant
form 10 TTN
Familial isolated dilated cardiomyopath 38 TTN
Hereditary proximal myopathy with early respiratory failure 1 TTN
Tibial muscular dystrophy 2 TTN
Ataxia with vitamin E deficiency 1 TTPA
Transthyretin-related familial amyloid cardiomyopathy 1 TTR
Lissencephaly due to TUBA1A mutation 1 TUBA1A
Polymicrogyria with optic nerve hypoplasia 1 TUBA8
Autosomal dominant macrothrombocytopenia 3 TUBB1
Congenital fibrosis of extraocular muscles 4 TUBB2B
Polymicrogyria due to TUBB2B mutation 1 TUBB2B
Congenital fibrosis of extraocular muscles 4 TUBB3
Cortical dysgenesis with pontocerebellar hypoplasia due to TUBB3
mutation 1 TUBB3
Hypomyelination with atrophy of basal ganglia and cerebellum 1 TUBB4A
Primary dystonia, DYT4 type 1 TUBB4A
Autosomal recessive chorioretinopathy - microcephaly 1 TUBGCP6
Combined oxidative phosphorylation defect type 4 1 TUFM
Leber congenital amaurosis 18 TULP1
Retinitis pigmentosa 61 TULP1
Autosomal recessive npnsyndromic intellectual deficit 15 TUSC3
Isolated brachycephaly 3 TWIST1
Isolated scaphocephaly 3 TWISTl
Saethre-Chotzen syndrome 4 TWIST1
Focal facial dermal dysplasia 2 TWIST2
Familial isolated dilated cardiomyopathy 38 TXNRD2
Autosomal recessive hyper IgE syndrome due to TYK2 deficiency 1 TYK2
Juvenile rheumatoid factor-negative polyarthritis 9 TYK2
Oligoarticular juvenile arthritis 9 TYK2
Mitochondrial neurogastrointestinal encephalomyopathy 3 TYMP
Ocular albinism with congenital sensorineural deafness 2 TYR
Oculocutaneous albinism type 1A 1 TYR
Oculocutaneous albinism type IB 1 TYR
Type 1 minimal pigment oculocutaneous albinism 1 TYR
Type 1 temperature-sensitive oculocutaneous albinism 1 TYR
Waardenburg syndrome type 2 4 TYR
Nasu-Hakola disease 2 TYROBP
180
Table IX-21781338.1 Oculocutaneous albinism type 3 1 TYRP1
X-linked distal arthrogryposis multiplex congenita 1 UBA1
Behcet disease 9 UBAC2
Intellectual deficit, X-linked, Nascimento type 1 UBE2A
Angelman syndrome 6 UBE3A
Blepharophimosis-intellectual deficit syndrome due to UBE3B deficiency 1 UBE3B
Schnyder corneal dystrophy 1 UBIAD1
Amyotrophic lateral sclerosis 23 UBQLN2
Johanson-Blizzard syndrome 1 UBR1
Early-onset progressive neurodegeneration - blindness - ataxia - spasticity 1 UCHL1
Early-onset progressive neurodegeneration - blindness - ataxia - spasticity 1 UCHL1
Young adult-onset Parkinsonism 13 UCHL1
Hyperinsulinism due to UCP2 deficiency 1 UCP2
22qll.2 deletion syndrome 7 UFD1L
Crigler-Najjar syndrome type 1 1 UGT1A1
Transient familial neonatal hyperbilirubinemia 1 UGT1A1
Autosomal dominant medullary cystic kidney disease with or without
hyperuricemia 2 UMOD
Familial juvenile hyperuricemic nephropathy type 1 1 UMOD
Hereditary orotic aciduria 1 UMPS
Cone rod dystrophy 22 UNC119
Cone rod dystrophy 22 UNC119
Idiopathic CD4 lymphocytopenia 1 UNC119
Familial hemophagocytic lymphohistiocytosis 4 UNC13D
Herpetic encephalitis 5 UNC93B1
Hyper-lgM syndrome type 5 1 UNG
Beta-ureidopropionase deficiency 1 UPB1
X-linked intellectual deficit with marfanoid habitus 3 UPF3B
X-linked nonsyndromic intellectual deficit 24 UPF3B
Bilateral renal agenesis 2 UPK3A
Isolated CoQ-cytochrome C reductase deficiency 6 UQCRB
Isolated CoQ-cytochrome C reductase deficiency 6 UQCRC2
Isolated CoQ-cytochrome C reductase deficiency 6 UQCRQ
Urocanic aciduria ' 1 UROC1
Hepatoerythropoietic porphyria 1 UROD
Porphyria cutanea tarda 2 UROD
Congenital erythropoietic porphyria 2 UROS
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 USH1C
Autosomal recessive nonsyndromic sensorineural deafness type DFNB 52 USH1C
Usher syndrome type 1 9 USH1C
Usher syndrome type 1 9 USH1E
181
Table IX-21781338.1 Usher syndrome type 1 9 USH1G
Usher syndrome type 1 9 USH1H
Usher syndrome type 1 9 USH1K
Retinitis pigmentosa 61 USH2A
Usher syndrome type 2 4 USH2A
Partial chromosome Y deletion 8 USP9Y
UV-sensitive syndrome 3 UVSSA
Caudal regression sequence 2 VANGL1
Cervical spina bifida aperta 6 VANGL1
Cervical spina bifida cystica 6 VANGL1
Cervicothoracic spina bifida aperta 6 VANGL1
Cervicothoracic spina bifida cystica 6 VANGL1
Familial caudal dysgenesis 1 VANGL1
Lumbosacral spina bifida aperta 6 VANGL1
Lumbosacral spina bifida cystica 6 VANGL1
Thoracolumbosacral spina bifida aperta 6 VANGL1
Thoracolumbosacral spina bifida cystica 6 VANGL1
Total spina bifida aperta 6 VANGL1
Total spina bifida cystica 6 VANGL1
Upper thoracic spina bifida aperta 6 VANGL1
Upper thoracic spina bifida cystica 6 VANGL1
Cervical spina bifida aperta 6 VANGL2
Cervical spina bifida cystica 6 VANGL2
Cervicothoracic spina bifida aperta 6 VANGL2
Cervicothoracic spina bifida cystica 6 VANGL2
Isolated anencephaly/exencephaly . 2 VANGL2
Lumbosacral spina bifida aperta 6 VANGL2
Lumbosacral spina bifida cystica 6 VANGL2
Thoracolumbosacral spina bifida aperta 6 VANGL2
Thoracolumbosacral spina bifida cystica 6 VANGL2
Total spina bifida aperta 6 VANGL2
Total spina bifida cystica 6 VANGL2
Upper thoracic spina bifida aperta 6 VANGL2
Upper thoracic spina bifida cystica 6 VANGL2
Adult-onset proximal spinal muscular atrophy, autosomal dominant 1 VAPB
Amyotrophic lateral sclerosis 23 VAPB
Wagner disease 1 VCAN
Familial isolated dilated cardiomyopathy 38 VCL
Adult-onset distal myopathy due to VCP mutation 1 CP
Amyotrophic lateral sclerosis 23 VCP
Inclusion body myopathy with Paget disease of bone and frontotemporal 2 VCP
182
Table IX-21781338.1 dementia
Spastic paraplegia - Paget disease of bone 1 VCP
Hypocalcemic vitamin D resistant rickets 1 VDR
Chuvash erythrocytosis 1 VHL
Von Hippel-Lindau disease 1 VHL
Pulverulent cataract 7 VIM
Arthrogryposis - renal dysfunction - cholestasis 2 VIPAR
Hereditary combined deficiency of vitamin K-dependent clotting factors 2 VKORC1
Dysequilibrium syndrome 4 VLDLR
X-linked myopathy with excessive autophagy 1 VMA21
Choreoacanthocytosis 1 VPS13A
Cohen syndrome 1 VPS13B
Arthrogryposis - renal dysfunction - cholestasis 2 VPS33B
Young adult-onset Parkinsonism 13 VPS35
Autosomal recessive spastic paraplegia type 53 1 VPS37A
Pontocerebellar hypoplasia type 1 4 VRK1
Posterior polymorphous corneal dystrophy 3 VSX1
Colobomatous microphthalmia 8 VSX2
Colobomatous microphthalmia 8 VSX2
Isolated anophthalmia - microphthalmia 5 VSX2
Microphthalmia - cataract 4 VSX2
Von Willebrand disease type 1 1 VWF
Von Willebrand disease type 2A 1 VWF
Von Willebrand disease type 2B 1 VWF
Von. Willebrand disease type 2M 1 VWF
Von Willebrand disease type 2N 1 VWF
Von Willebrand disease type 3 1 VWF
Wiskott-Aldrich syndrome 2 WAS
X-linked severe congenital neutropenia 1 WAS
X-linked thrombocytopenia with normal platelets 1 WAS
Williams syndrome 17 WBSCR16
Williams syndrome 17 WBSCR22
Williams syndrome 17 WBSCR27
Bardet-Biedl syndrome 17 WDPCP
Meckel syndrome 13 WDPCP
Kallmann syndrome 19 WDR11
Normosmic congenital hypogonadotropic hypogonadism 18 WDR11
Cranioectodermal dysplasia 4 WDR19
Jeune syndrome 6 WDR19
Juvenile autosomal recessive medullary cystic kidney disease 4 WDR19
Cranioectodermal dysplasia 4 WDR35
183
Table IX-21781338.1 Short rib-polydactyly syndrome, Verma-Naumoff type 3 WDR35
Beta-propeller protein-associated neurodegeneration 1 WDR45
Autosomal recessive primary microcephaly 11 WDR62
Autosomal recessive primary microcephaly 11 WDR62
Hypomaturation amelogenesis imperfecta 5 WDR72
Dysequilibrium syndrome 4 WDR81~
Autosomal dominant nonsyndromic sensorineural deafness type DFNA 30 WFS1
Wolfram syndrome 2 WFS1
Wolf-Hirschhorn syndrome 3 WHSC1
Wolf-Hirschhorn syndrome 3 WHSC2
Wiskott-Aldrich syndrome 2 WIPF1
Progressive pseudorheumatoid arthropathy of childhood 1 WISP3
Hereditary sensory and autonomic neuropathy type 2 4 WNK1
Pseudohypoaldosteronism type 2C 1 WNK1
Pseudohypoaldosteronism type 2B 1 WNK4
Idiopathic juvenile osteoporosis 4 WNT1
Osteogenesis imperfecta type 3 9 WNT1
Osteogenesis imperfecta type 4 7 WNT1
Autosomal recessive hypohidrotic ectodermal dysplasia 3 WNT10A
Odonto-onycho-dermal dysplasia 1 WNT10A
Oligodontia 10 WNT10A
Schopf-Schulz-Passarge syndrome 1 WNT10A
Split hand-split foot malformation 4 WNT10B
Tetraamelia - multiple malformations 1 WNT3
Idiopathic juvenile osteoporosis 4 WNT3A
Atypical Mayer-Rokitansky-Kuster-Hauser syndrome 1 WNT4
SERKAL syndrome 1 WNT4
Autosomal dominant Robinow syndrome 1 WNT5A
Fuhrmann syndrome 1 WNT7A
Phocomelia, Schinzel type 1 WNT7A
Dyskeratosis congenita 10 WRAP53
Werner syndrome 1 WRN
46,XY partial gonadal dysgenesis 7 WT1
Denys-Drash syndrome 1 WT1
Denys-Drash syndrome 1 WT1
Desmoplastic small round cell tumor 2 WT1
Familial idiopathic steroid-resistant nephrotic syndrome with diffuse
mesangial sclerosis 4 WT1
Familial idiopathic steroid-resistant nephrotic syndrome with focal
segmental hyalinosis 11 WT1
Frasier syndrome 1 WT1
184
Table IX-21781338.1 Meacham syndrome 1 WT1
Nephroblastoma 5 WT1
Sporadic idiopathic steroid-resistant nephrotic syndrome with diffuse
mesangial sclerosis 1 WT1
WAGR syndrome 3 WT1
46,XY partial gonadal dysgenesis 7 WWOX
Wieacker-Wolff syndrome 2 WWS
Xanthinuria type 1 1 XDH
X-linked lymphoproliferative disease 2 XIAP cLeod neuroacanthocytosis syndrome 1 XK
Xeroderma pigmentosum complementation group A 1 XPA
Xeroderma pigmentosum complementation group C 1 XPC
Renin-angiotensin-aldosterone system-blocker-induced angioedema 1 XPNPEP2
Late-onset autosomal recessive medullary cystic kidney disease 3 XPNPEP3
Hereditary breast cancer 4 XRCC2
Autosomal dominant intermediate Charcot-Marie-Tooth disease type C 1 YARS
Mitochondrial myopathy and sideroblastic anemia 2 YARS2
17pl3.3 microduplication syndrome 2 YWHAE
Distal 17pl3.3 microdeletion syndrome 1 YWHAE
Miller-Dieker syndrome 3 YWHAE
Combined immunodeficiency due to ZAP70 deficiency 1 ZAP70
ICF syndrome 2 ZBTB24
Autosomal recessive nonsyndromic intellectual deficit 15 ZC3H14
Wieacker-Wolff syndrome 2 ZC4H2
Intellectual deficit, X-linked, Raymond type 1 ZDHHC9
X-linked intellectual deficit with marfanoid habitus 3 ZDHHC9
Fuchs endothelial corneal dystrophy 4 ZEB1
Posterior polymorphous corneal dystrophy 3 ZEB1
Mowat-Wilson syndrome due to a point mutation 1 ZEB2
Mowat-Wilson syndrome due to monosomy 2q22 1 ZEB2
Transient neonatal diabetes mellitus 5 ZFP57
3-methylglutaconic aciduria type 3 2 ZFP90
Congenital diaphragmatic hernia 2 ZFPM2
Tetralogy of Fallot 8 ZFPM2
Autosomal recessive spastic paraplegia type 15 1 ZFYVE26
Isolated Dandy-Walker malformation 2 ZIC1
Alobar holoprosencephaly 14 ZIC2
Lobar holoprosencephaly 14 ZIC2
Microform holoprosencephaly 14 ZIC2
Midline interhemispheric variant of holoprosencephaly 14 ZIC2
Semilobar holoprosencephaly 14 ZIC2
185
Table IX-21781338.1 Septopreoptic holoprosencephaly 14 ZIC2
Double outlet right ventricle 3 ZIC3
Isolated congenitally uncorrected transposition of the great arteries 1 ZIC3
Situs ambiguus 6 ZIC3
Isolated Dandy-Walker malformation 2 ZIC4
Hutchinson-Gilford progeria syndrome 2 ZMPSTE24
Lethal restrictive dermopathy 2 ZMPSTE24
Mandibuloacral dysplasia with type B lipodystrophy 1 ZMPSTE24
Postaxial Polydactyly type A, bilateral 2 ZNF141
Microcephalic primordial dwarfism due to ZNF335 deficiency 1 ZNF335
Familial exudative vitreoretinopathy 5 ZNF408
X-linked nonsyndromic intellectual deficit 24 ZNF41
Joubert syndrome with oculorenal defect 6 ZNF423
Brittle cornea syndrome 2 ZNF469
Retinitis pigmentosa 61 ZNF513
CAMOS syndrome 1 ZNF592
X-linked nonsyndromic intellectual deficit 24 ZNF674
X-linked nonsyndromic intellectual deficit 24 ZNF711
Seborrhea-like dermatitis with psoriasiform elements 1 ZNF750
X-linked nonsyndromic intellectual deficit 24 ZNF81
Myxofibrosarcoma 3
Ondine syndrome 5
186
Table IX-21781338.1

Claims

CLAIMS What is claimed is:
1. A gRNA molecule comprising a targeting domain which is complementary with a target sequence from a target nucleic acid disclosed herein, e.g., a sequence from:
a gene or pathway described herein, e.g., in Section VIIB, e.g., in Table VIT13, VIT14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX-1, IX- 1 A, LX-2, LX-3, XIV- 1, or Section VIII.
2. A composition, e.g., pharmaceutical composition, comprising a gRNA molecule of claim 1.
3. The composition of claim 2, further comprising a Cas9 molecule, e.g., an eaCas9 or an eiCas9 molecule.
4. The composition of claim 2, wherein said Cas9 molecule is an eaCas9 molecule.
5. The composition of claim 2, wherein said Cas9 molecule is an eiCas9 molecule.
6. The composition of claim 2, wherein said composition comprises a payload, e.g., a payload described herein, e.g., in Section VI, e.g., in Table VI-1, VI-2, VI-3, VI-4, VI-5, VI-6, or VI-7.
7. The composition of claim 6, wherein the payload comprises:
an epigenetic modifier, e.g., a molecule that modifies DNA or chromatin component, e.g., a molecule that modifies a histone, e.g., an epigenetic modifier described herein, e.g., in Section VI;
a transcription factor, e.g., a transcription factor described herein, e.g., in Section VI;
a transcriptional activator domain;
298 an inhibitor of a transcription factor, e.g., an anti-transcription factor antibody, or other inhibitor;
a small molecule;
an antibody;
an enzyme;
an enzyme that interacts with DNA, e.g., a helicase, restriction enzyme, ligase, or polymerase;
an a nucleic acid, e.g., an enzymatically active nucleic acid, e.g., a ribozyme, or an mRNA, siRNA, of antisense oligonucleotide.
8. The composition of claim 2, further comprising a Cas9 molecule, e.g., an eiCas9, molecule.
9. The composition of claim 7, wherein said payload is coupled, e.g., covalently or noncovalently, to a Cas9 molecule, e.g., an eiCas9 molecule.
10. The composition of claim 9, wherein said payload is coupled to said Cas9 molecule by a linker.
11. The composition of claim 10, wherein said linker is or comprises a bond that is cleavable under physiological, e.g., nuclear, conditions.
12. The composition of claim 11, wherein said linker is, or comprises, a bond described herein, e.g., in Section XL
13. The composition of claim 10, wherein said linker is, or comprises, an ester bond.
14. The composition of claim 9, wherein said payload comprises a fusion partner fused to a Cas9 molecule, e.g., an eaCas9 molecule or an eiCas9 molecule.
299
15. The composition of claim 7, wherein said payload is coupled, e.g., covalently or noncovalently, to the gRNA molecule.
16. The composition of claim 15, wherein said payload is coupled to said gRNA molecule by a linker.
17. The composition of claim 16, wherein said linker is or comprises a bond that is cleavable under physiological, e.g., nuclear, conditions.
18. The composition of claim 16, wherein said linker is, or comprises, a bond described herein, e.g., in Section XL
19. The composition of claim 16, wherein said linker is, or comprises, an ester bond.
20. The composition of claim 2, comprising an eaCas9 molecule.
21. The composition of claim 2, comprising an eaCas9 molecule which forms a double stranded break in the target nucleic acid.
22. The composition of claim 2, comprising an eaCas9 molecule which forms a single stranded break in the target nucleic acid.
23. The composition of claim 22, wherein said single stranded break is formed in the complementary strand of the target nucleic acid.
24. The composition of claim 22, wherein said single stranded break is formed in the strand which is not the complementary strand of the target nucleic acid.
25. The composition of claim 22, comprising HNH-like domain cleavage activity but having no, or no significant, N-terminal RuvC-like domain cleavage activity.
300
26. The composition of claim 22, comprising N-terminal RuvC-like domain cleavage activity but having no, or no significant, HNH-like domain cleavage activity.
27. The composition of claim 21, wherein said double stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
28. The composition of claim 22, wherein said single stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
29. The composition of claim 20, further comprising a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.
30. The composition of claim 29, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
31. The composition of claim 29, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ,-2, ΓΧ-3, XIV- 1, or Section VIII.
32. The composition of claim 29, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ,-2, ΓΧ-3, XIV- 1, or Section VIII.
33. The composition of claim 29, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:
301 a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ,-2, ΓΧ-3, XIV- 1, or Section VIII.
34. The composition of claim 3, further comprising a second gRNA molecule, e.g., a second gRNA molecule from claim 100.
35. The composition of claim 34, wherein said gRNA molecule and said second gRNA molecule mediate breaks at different sites in the target nucleic acid, e.g., flanking a target position.
36. The composition of claim 34, wherein said gRNA molecule and said second gRNA molecule are complementary to the same strand of the target.
37. The composition of claim 34, wherein said gRNA molecule and said second gRNA molecule are complementary to the different strands of the target.
38. The composition of claim 34, wherein said Cas9 molecule mediates a double stranded break.
39. The composition of claim 34, wherein said gRNA molecule and said second gRNA molecule are configured such that first and second break made by the Cas9 molecule flank a target position.
40. The composition of claim 39, wherein said double stranded break is within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
41. The composition of claim 40, further comprising a template nucleic acid.
42. The composition of claim 40, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
302
43. The composition of claim 42, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of a target position from a sequence of:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ,-2, ΓΧ-3, XIV- 1, or Section VIII.
44. The composition of claim 43, wherein the template nucleic acid is a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ,-2, ΓΧ-3, XIV- 1, or Section VIII.
45. The composition of claim 44, wherein the template nucleic acid is a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ,-2, ΓΧ-3, XIV- 1, or Section VIII.
46. The composition of claim 34, wherein said Cas9 molecule mediates a single stranded break.
47. The composition of claim 34, wherein said gRNA molecule and said second gRNA molecule are configured such that a first and second break are formed in the same strand of the nucleic acid target, e.g., in the case of transcribed sequence, the template strand or the non- template strand.
48. The composition of claim 47, wherein said first and second break flank a target position.
303
49. The composition of claim 48, wherein, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
50. The composition of claim 48, further comprising a template nucleic acid.
51. The composition of claim 49, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
52. The composition of claim 51, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ,-2, ΓΧ-3, XIV- 1, or Section VIII.
53. The composition of claim 51, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ,-2, ΓΧ-3, XIV- 1, or Section VIII.
54. The composition of claim 51, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII- 23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ,-2, ΓΧ-3, XIV- 1, or Section VIII.
304
55. The composition of claim 34, wherein said gRNA molecule and said second gRNA molecule are configured such that a first and second break are formed in different strands of the target.
56. The composition of claim 55, wherein said first and second break flank a target position.
57. The composition of claim 56, wherein, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
58. The composition of claim 55, further comprising a template nucleic acid.
59. The composition of claim 58, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
60. The composition of claim 58, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ,-2, ΓΧ-3, XIV- 1, or Section VIII.
61. The composition of claim 58, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ,-2, ΓΧ-3, XIV- 1, or Section VIII.
62. The composition of claim 58, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at
305 least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ,-2, ΓΧ-3, XIV- 1, or Section VIII.
63. The composition of claim 34, comprising a second Cas9 molecule.
64. The composition of claim 63, wherein one or both of said Cas9 molecule and said second Cas9 molecule are eiCas9 molecules.
65. The composition of claim 64, wherein said eiCas9 molecule is coupled to a payload by a linker and said second eiCas9 molecules is coupled to a second payload by a second linker.
66. The composition of claim 65 wherein said payload and said second payload are the same.
67. The composition of claim 65 wherein said payload and said second payload are different.
68. The composition of claim 65 wherein said linker and said second linker are the same.
69. The composition of claim 65, wherein said linker and said second linker are different, e.g., have different release properties, e.g., different release rates.
70. The composition of claim 65, wherein said payload and said second payload are each described herein, e.g., in Section VI, e.g., in Table VI- 1, VI-2, VI-3, VI-4, VI-5, VI-6, or VI-7.
71. The composition of claim 65, wherein said payload and said second payload can interact, e.g., they are subunits of a protein.
306
72. The composition of claim 63, wherein one of both of said Cas9 molecule and said second Cas9 molecule are eaCas9 molecules.
73. The composition of claim 72, wherein said eACas9 molecule comprises a first cleavage activity and said second eACas9 molecule comprises a second cleavage activity.
74. The composition of claim 73, wherein said cleavage activity and said second cleavage activity are the same, e.g., both are N-terminal RuvC-like domain activity or are both HNH-like domain activity.
75. The composition of claim 73 wherein said cleavage activity and said second cleavage activity are different, e.g., one is N-terminal RuvC-like domain activity and one is HNH-like domain activity.
76. The composition of claim 72, wherein said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for, e.g., NGGNG, NNAGAAW (W = A or T), or NAAR (R = A or G).
77. The composition of claim 63, wherein said Cas9 molecule and said second Cas9 molecule both mediate double stranded breaks.
78. The composition of claim 63, wherein said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein.
79. The composition of claim 77, wherein said gRNA molecule and said second gRNA molecule are configured such that first and second break flank a target position.
80. The composition of claim 79, wherein, one of said first and second double stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
307
81. The composition of claim 77, further comprising a template nucleic acid.
82. The composition of claim 81, wherein the template nucleic acid a nucleotide that corresponds to a nucleotide of the target position.
83. The composition of claim 81, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ,-2, ΓΧ-3, XIV- 1, or Section VIII.
84. The composition of claim 83, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
85. The composition of claim 83, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX-2, IX-3, XIV- 1, or Section VIII.
86. The composition of claim 77, wherein one of said Cas9 molecule and said second Cas9 molecule mediates a double stranded break and the other mediates a single stranded break.
308
87. The composition of claim 77, wherein said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein.
88. The composition of claim 87, wherein said gRNA molecule and said second gRNA molecule are configured such that a first and second break flank a target position.
89. The composition of claim 88, wherein said first and second break flank a target position.
90. The composition of claim 89, wherein, one of said first and second breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
91. The composition of claim 88, further comprising a template nucleic acid.
92. The composition of claim 91, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
93. The composition of claim 91, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
94. The composition of claim 91, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
309
95. The composition of claim 91, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
96. The composition of claim 63, wherein said Cas9 molecule and said second Cas9 molecule both mediate single stranded breaks.
97. The composition of claim 96, wherein said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein.
98. The composition of claim 96, wherein said first and second break flank a target position.
99. The composition of claim 96, wherein, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
100. The composition of claim 96, further comprising a template nucleic acid.
101. The composition of claim 100, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
102. The composition of claim 100, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of:
310 a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
103. The composition of claim 100, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
104. The composition of claim 100, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
105. The composition of claim 96, wherein said gRNA molecule, said second gRNA molecule are configured such that a first and second break are in the same strand.
106. The composition of claim 105, wherein said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another PAM described herein.
107. The composition of claim 105, wherein said gRNA molecule, said second gRNA molecule are configured such that a first and second break flank a target position.
108. The composition of claim 107, wherein, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
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109. The composition of claim 107, further comprising a template nucleic acid.
110. The composition of claim 109, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
111. The composition of claim 109, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
112. The composition of claim 109, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
113. The composition of claim 109, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
114. The composition of claim 96, wherein said first and second break are on the different strands.
312
115. The composition of claim 114, wherein said Cas9 molecule and said second Cas9 molecule are specific for different PAMs, e.g., one is specific for NGG and the other is specific for another PAM, e.g., another Pam described herein.
116. The composition of claim 114, wherein said gRNA molecule, said second gRNA molecule are configured such that a first and second break are on different strands.
117. The composition of claim 114, wherein said gRNA molecule, said second gRNA molecule are configured such that a first and second break flank a target position.
118. The composition of claim 117, wherein said first and second break flank a target position.
119. The composition of claim 117, wherein, one of said first and second single stranded breaks, or both are independently, within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
120. The composition of claim 117, further comprising a template nucleic acid.
121. The composition of claim 117, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
122. The composition of claim 117, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, IX-3, XIV- 1, or Section VIII.
123. The composition of claim 117, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in:
313 a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
124. The composition of claim 117, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
125. A composition, e.g., a pharmaceutical composition, comprising a gRNA molecule and a second gRNA molecule described herein.
126. The composition of claim 125, further comprising a nucleic acid, e.g., a DNA or mRNA, that encodes a Cas9 molecule described herein.
127. The composition of claim 125, further comprising a nucleic acid, e.g., a DNA or RNA, that encodes a second Cas9 molecule described herein.
128. The composition of claim 125, further comprising a template nucleic acid described herein.
129. A composition, e.g., a pharmaceutical composition, comprising, nucleic acid sequence, e.g., a DNA, that encodes one or more gRNA molecules described herein.
130. The composition of claim 129, wherein said nucleic acid comprises a promoter operably linked to the sequence that encodes a gRNA molecule, e.g., a promoter described herein..
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131. The composition of claim 129, wherein said nucleic acid comprises a second promoter operably linked to the sequence that encodes a second gRNA molecule, e.g., a promoter described herein.
132. The composition of claim 131, wherein the promoter and second promoter are different promoters.
133. The composition of claim 131, wherein the promoter and second promoter are the same.
134. The composition of claim 129, wherein the nucleic acid further encodes a Cas9 molecule described herein.
135. The composition of claim 129, wherein the nucleic acid further encodes a second Cas9 molecule described herein.
136. The composition of claim 134, wherein said nucleic acid comprises a promoter operably linked to the sequence that encodes a Cas9 molecule, e.g., a promoter described herein.
137. The composition of claim 135, wherein said nucleic acid comprises a second promoter operably linked to the sequence that encodes a second Cas9 molecule, e.g., a promoter, described herein.
138. The composition of claim 135, wherein the promoter and second promoter are different promoters.
139. The composition of claim 135, wherein the promoter and second promoter are the same.
140. The composition of claim 129, further comprising a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.
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141. A composition, e.g., a pharmaceutical composition, comprising nucleic acid sequence that encodes one or more of
a) a Cas9 molecule,
b) a second Cas9 molecule,
c) a gRNA molecule, and
d) a second gRNA molecule.
142. The composition of claim 141, wherein each of a, b, c and d present are encoded on the same duplex molecule.
143. The composition of claim 141, wherein a first sequence selected from of a, b, c and d is encoded on a first duplex molecule and a second sequence selected from a, b, c, and d is encoded on a second duplex molecule.
144. The composition of claim 141, wherein said nucleic acid encodes:
a and c;
a, c, and d; or
a, b, c, and d.
145. The composition of claim 141, further comprising a Cas9 molecule, e.g., comprising one or more of the Cas9 molecules wherein said nucleic acid does not encode a Cas9 molecule.
146. The composition of claim 141, further comprising an mRNA encoding Cas9 molecule, e.g., comprising one or more mRNAs encoding one or more of the Cas9 molecules wherein said nucleic acid does not encode a Cas9 molecule.
147. The composition of claim 141, further comprising a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.
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148. A nucleic acid of claim 129.
149. A nucleic acid of claim 141.
150. A composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule);and
c) optionally, a template nucleic acid e.g., a template nucleic acid described herein, e.g., in Section IV.
151. A composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.
152. A composition comprising:
a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule) ;
b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.
153. A composition comprising:
317 a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.
154. A method of altering a cell, e.g., altering the structure, e.g., sequence, of a target nucleic acid of a cell, comprising contacting said cell with:
1) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV;
2) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV;
3) a composition comprising:
318 a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; or
4) a composition comprising:
a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.
155. The method of claim 153, wherein a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by, one dosage form, mode of delivery, or formulation.
156. The method of claim 145, wherein:
a) a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by, a first dosage form, a first mode of delivery, or a first formulation; and
b) an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.
157. The method of claim 154, wherein the cell is an animal or plant cell.
158. The method of claim 154, wherein the cell is a mammalian, primate, or human cell.
319
159. The method of claim 154, wherein the cell is a human cell, e.g., a cell from described herein, e.g., in Section VIIA.
160. The method of claim 154, wherein the cell is: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.
161. The method of claim 154, wherein the cell is a human cell, e.g., a cancer cell or other cell characterized by a disease or disorder.
162. The method of claim 155, wherein the target nucleic acid is a chromosomal nucleic acid.
163. The method of claim 155, wherein the target nucleic acid is an organellar nucleic acid.
164. The method of claim 155, wherein the target nucleic acid is a mitochondrial nucleic acid.
165. The method of claim 155, wherein the target nucleic acid is a chloroplast nucleic acid.
166. The method of claim 155, wherein the cell is a cell of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
167. The method of claim 155, wherein the target nucleic acid is the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
168. The method of claim 155, wherein said method comprises: modulating the expression of a gene or inactivating a disease organism.
320
169. The method of claim 155, wherein said cell is a cell characterized by unwanted proliferation, e.g., a cancer cell.
170. The method of claim 155, wherein said cell is a cell characterized by an unwanted genomic component, e.g., a viral genomic component.
171. The method of claim 155, wherein the cell is a cell described herein, e.g., in Section
IIA.
172. The method of claim 155, wherein a control or structural sequence of at least, 2 3, 4, or 5 genes is altered.
173. The method of claim 169, wherein the target nucleic acid is a rearrangement, a kinase, a rearrangement that comprises a kinase, or a tumor suppressor
174. The method of claim 155, comprising cleaving a target nucleic acid within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
175. The method of claim 155, wherein said composition comprises a template nucleic acid.
176. The composition of claim 155, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
177. The composition of claim 155, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
321
178. The composition of claim 155, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
179. The method of claim 155, wherein,
a) a control region, e.g., a cis-acting or tans-acting control region, of a gene is cleaved; b) the sequence of a control region, e.g., a cis-acting or tans-acting control region, of a gene is altered, e.g., by an alteration that modulates, e.g., increases or decreases, expression a gene under control of the control region, e.g., a control sequence is disrupted or a new control sequence is inserted;
c) the coding sequence of a gene is cleaved;
d) the sequence of a transcribed region, e.g., a coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that increases expression of or activity of the gene product is effected, e.g., a mutation is corrected;
e) the sequence of a transcribed region, e.g., the coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that decreases expression of or activity of the gene product is effected, e.g., a mutation is inserted, e.g., the sequence of one or more
nucleotides is altered so as to insert a stop codon;
180. The method of claim 155, wherein a control region or transcribed region, e.g., a coding sequence, of at least 2, 3, 4, 5, or 6 genes are altered.
181. A method of treating a subject, e.g., by altering the structure, e.g., altering the sequence, of a target nucleic acid, comprising administering to the subject, an effective amount of:
1) a composition comprising:
322 a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule) ;
b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule; and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV;
2) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a nucleic acid, e.g. a DNA or mRNA encoding an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV;
3) a composition comprising:
a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) an eaCas9 molecule (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule); and
c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV; or
4) a composition comprising:
a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) nucleic acid, e.g. a DNA or mRNA encoding eaCas9 molecule or (or combination of eaCas9 molecules, e.g., an eaCas9 molecule and a second eaCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
323 c) optionally, a template nucleic acid, e.g., a template nucleic acid described herein, e.g., in Section IV.
182. The method of claim 181, wherein a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation.
183. The method of claim 181, wherein:
a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage form, in a first mode of delivery, or first formulation; and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.
184. The method of claim 181, wherein the subject is an animal or plant.
185. The method of claim 181, wherein the subject is a mammalian, primate, or human.
186. The method of claim 154, wherein the target nucleic acid is the nucleic acid of a human cell, e.g., a cell described herein, e.g., in Section VIIA.
187. The method of claim 181, wherein the target nucleic acid is the nucleic acid of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.
188. The method of claim 181, wherein the target nucleic acid is a chromosomal nucleic acid.
189. The method of claim 181, wherein the target nucleic acid is an organellar nucleic acid.
190. The method of claim 181, wherein the nucleic acid is a mitochondrial nucleic acid.
324
191. The method of claim 181, wherein the nucleic acid is a chloroplast nucleic acid.
192. The method of claim 181, wherein the target nucleic acid is the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
193. The method of claim 181, wherein said method comprises modulating expression of a gene or inactivating a disease organism.
194. The method of claim 181, wherein the target nucleic acid is the nucleic acid of a cell characterized by unwanted proliferation, e.g., a cancer cell.
195. The method of claim 181, wherein said target nucleic acid comprises an unwanted genomic component, e.g., a viral genomic component.
196. The method of claim 181, wherein a control or structural sequence of at least, 2 3, 4, or 5 genes is altered.
197. The method of claim 181, wherein the target nucleic acid is a rearrangement, a kinase, a rearrangement that comprises a kinase, or a tumor suppressor.
198. The method of claim 181, comprising cleaving a target nucleic acid within 10, 20, 30, 40, 50, 100, 150 or 200 nucleotides of a nucleotide of the target position.
199. The method of claim 181, wherein said composition comprises a template nucleic acid.
200. The composition of claim 181, wherein the template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position.
325
201. The composition of claim 181, wherein said template nucleic acid comprises a nucleotide that corresponds to a nucleotide of the target position from a sequence of:
a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
202. The composition of claim 181, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
203. The composition of claim 181, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length, which differs at at least 1 nucleotide, but not more than 5, 10, 20 or 30% of its nucleotides, from a corresponding sequence in:
204. The method of claim 181, wherein,
a) a control region, e.g., a cis-acting or tans-acting control region, of a gene is cleaved; b) the sequence of a control region, e.g., a cis-acting or tans-acting control region, of a gene is altered, e.g., by an alteration that modulates, e.g., increases or decreases, expression a gene under control of the control region, e.g., a control sequence is disrupted or a new control sequence is inserted;
c) the coding sequence of a gene is cleaved;
d) the sequence of a transcribed region, e.g., a coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that increases expression of or activity of the gene product is effected, e.g., a mutation is corrected;
e) the sequence of a transcribed region, e.g., the coding sequence of a gene is altered, e.g., a mutation is corrected or introduced, an alteration that decreases expression of or activity of the gene product is effected, e.g., a mutation is inserted, e.g., the sequence of one or more
nucleotides is altered so as to insert a stop codon;
326
205. The method of claim 181, wherein a control region or transcribed region, e.g., a coding sequence, of at least 2, 3, 4, 5, or 6 genes are altered.
206. A composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule or the gRNA molecule.
207. A composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload which is:
coupled, covalently or non-covalently, the gRNA molecule; or
a fusion partner with the Cas9 molecule.
208. A composition comprising:
a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload which is coupled, covalently or non-covalently, to the Cas9 molecule.
327
209. A composition comprising:
a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA ,encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
c) a payload which is a fusion partner with the Cas9 molecule.
210. A method of delivering a payload to a cell, e.g., by targeting a payload to target nucleic acid, comprising contacting said cell with:
1) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule or the gRNA molecule;
2) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload which is:
coupled, covalently or non-covalently, the gRNA molecule; or
a fusion partner with the Cas9 molecule;
328 3) a composition comprising:
a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload which is coupled, covalently or non-covalently, to the Cas9 molecule; or
4) a composition comprising:
a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA ,encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule) (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
c) a payload which is a fusion partner with the Cas9 molecule.
211. The method of claim 206, wherein a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation.
212. The method of claim 206, wherein:
a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage form, first mode of delivery, or first formulation; and
a Cas9 molecule, or nucleic acid encoding a Cas9 molecule, is delivered in or by a second dosage form, second mode of delivery, or second formulation.
213. The method of claim 206, wherein the cell is an animal or plant cell.
214. The method of claim 206, wherein the cell is a mammalian, primate, or human cell.
329
215. The method of claim 206, wherein the cell is a human cell, e.g., a human cell described herein, e.g., in Section VIIA.
216. The method of claim 206, wherein the cell is: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.
217. The method of claim 206, wherein the cell is a human cell, e.g., a cancer cell, a cell comprising an unwanted genetic element, e.g., all or part of a viral genome.
218. The method of claim 206, wherein the gRNA mediates targeting of a chromosomal nucleic acid.
219. The method of claim 206, wherein the gRNA mediates targeting of a selected genomic signature.
220. The method of claim 206, wherein the gRNA mediates targeting of an organellar nucleic acid.
221. The method of claim 206, wherein gRNA mediates targeting of a mitochondrial nucleic acid.
222. The method of claim 206, wherein the gRNA mediates targeting of a chloroplast nucleic acid.
223. The method of claim 206, wherein the cell is a cell of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
224. The method of claim 206, wherein the gRNA mediates targeting of the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
330
225. The method of claim 206, wherein the payload comprises a payload described herein, e.g., in Section VI.
226. The method of claim 206, wherein said cell is a cell characterized by unwanted proliferation, e.g., a cancer cell.
227. The method of claim 206, wherein said cell is characterized by an unwanted genomic component, e.g., a viral genomic component.
228. The method of claim 206, wherein a control or structural sequence of at least, 2 3, 4, or 5 genes is altered.
229. The method of claim 206, wherein the gRNA targets a selected genomic signature, e.g., a mutation, e.g., a germline or acquired somatic mutation.
230. The method of claim 206, wherein the gRNA targets a rearrangement, a kinase, a rearrangement that comprises a kinase, or tumor suppressor.
231. The method of claim 206, wherein the gRNA targets a cancer cell, e.g., a cancer cell disclosed herein, e.g., in Section VIIA.
232. The method of claim 206, wherein the gRNA targets a cell which has been infected with a virus.
233. A method of treating a subject, e.g., by targeting a payload to target nucleic acid, comprising administering to the subject, an effective amount of:
1) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
331 b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload coupled, covalently or non-covalently, to a complex of the gRNA molecule and the Cas9 molecule, e.g., coupled to the Cas9 molecule;
2) a composition comprising:
a) a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a nucleic acid, e.g. a DNA or mRNA encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload which is:
coupled, covalently or non-covalently, the gRNA molecule; or
is a fusion partner with the Cas9 molecule;
3) a composition comprising:
a) a nucleic acid, e.g., a DNA, which encodes a gRNA molecule (or combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule);
b) a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule); and
c) a payload which is coupled, covalently or non-covalently, to the Cas9 molecule; or
4) a composition comprising:
a) nucleic acid, e.g., a DNA, which encodes a gRNA molecule or ( or
combination of gRNA molecules, e.g., a gRNA molecule and a second gRNA molecule); b) nucleic acid, e.g. a DNA or mRNA ,encoding a Cas9 molecule, e.g., an eiCas9 molecule (or combination of Cas9 molecules, e.g., an eiCas9 molecule and a second eiCas9 molecule), (wherein the gRNA molecule encoding nucleic acid and the eaCas9 molecule encoding nucleic acid can be on the same or different molecules); and
c) a payload which is a fusion partner with the Cas9 molecule.
332
234. The method of claim 233, wherein a gRNA molecule or nucleic acid encoding a gRNA molecule, and an eaCas9 molecule, or nucleic acid encoding an eaCas9 molecule, are delivered in or by one dosage form, mode of delivery, or formulation.
235. The method of claim 233, wherein:
a gRNA molecule or nucleic acid encoding a gRNA molecule is delivered in or by a first dosage, mode of delivery form or formulation; and
a Cas9 molecule, or nucleic acid encoding a Cas9 molecule, is delivered in or by a second dosage form, mode of delivery, or formulation.
236. The method of claim 233, wherein the subject is an animal or plant cell.
237. The method of claim 233, wherein the subject is a mammalian, primate, or human cell.
238. The method of claim 233, wherein the gRNA mediates targeting of a human cell, e.g., a human cell described herein, e.g., in Section VIIA.
239. The method of claim 233, wherein the gRNA mediates targeting of: a somatic cell, germ cell, prenatal cell, e.g., zygotic, blastocyst or embryonic, blasotcyst cell, a stem cell, a mitotically competent cell, a meiotically competent cell.
240. The method of claim 233, wherein the gRNA mediates targeting of a cancer cell or a cell comprising an unwanted genomic element, e.g., all or part of a viral genome.
241. The method of claim 233, wherein the gRNA mediates targeting of a chromosomal nucleic acid.
242. The method of claim 233, wherein the gRNA mediates targeting of a selected genomic signature.
333
243. The method of claim 233, wherein the gRNA mediates targeting of an organellar nucleic acid.
244. The method of claim 233, wherein gRNA mediates targeting of a mitochondrial nucleic acid.
245. The method of claim 233, wherein the gRNA mediates targeting of a chloroplast nucleic acid.
246. The method of claim 233, wherein the gRNA mediates targeting of the nucleic acid of a disease causing organism, e.g., of a disease causing organism, e.g., a virus, bacterium, fungus, protozoan, or parasite.
247. The method of claim 233, wherein the gRNA targets a cell characterized by unwanted proliferation, e.g., a cancer cell, e.g., a cancer cell from Section VIIA, e.g., from Table VII-11.
248. The method of claim 233, wherein the gRNA targets a cell characterized by an unwanted genomic component, e.g., a viral genomic component.
249. The method of claim 233, wherein a control element, e.g., a promoter or enhancer, is targeted.
250. The method of claim 233, wherein the gRNA targets a rearrangement, a kinase, a rearrangement that comprises a kinase, or a tumor suppressor.
251. The method of claim 233, wherein the gRNA targets a selected genomic signature, e.g., a mutation, e.g., a germline or acquired somatic mutation.
334
252. The method of claim 233, wherein the gRNA targets a rearrangement, a kinase, a rearrangement that comprises a kinase, or a tumor suppressor.
253. The method of claim 233, wherein the gRNA targets a cancer cell.
254. The method of claim 233, wherein the gRNA targets a cell which has been infected with a virus.
255. The method of claim 233, wherein at least one eaCas9 molecule and a payload are administered.
256. The method of claim 233, wherein the payload comprises a payload described herein, e.g., in Section VI.
257. A reaction mixture comprising a composition described herein and a cell.
258. The composition of claim 155, wherein the template nucleic acid is or comprises a fragment of 10 to 500, 10 to 400, 10 to 300, 10 to 200 nucleotides in length from a sequence in: a gene, or a gene from a pathway, described herein, e.g., in Section VIIB, e.g., in Table VII-13, VII-14, VII-15, VII-16, VII-17, VII-18, VII-19, VII-20, VII, 21, VII-22, VII-23, VII-24, VII-25, IX- 1, IX- 1 A, ΓΧ-2, ΓΧ-3, XIV- 1, or Section VIII.
335
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