WO2024168253A1 - Distribution d'un système de recombinaison guidé par arn - Google Patents

Distribution d'un système de recombinaison guidé par arn Download PDF

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WO2024168253A1
WO2024168253A1 PCT/US2024/015176 US2024015176W WO2024168253A1 WO 2024168253 A1 WO2024168253 A1 WO 2024168253A1 US 2024015176 W US2024015176 W US 2024015176W WO 2024168253 A1 WO2024168253 A1 WO 2024168253A1
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sequence
protein
rna
nucleic acid
target
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Christian HINDERER
Zhenhua HE
Alex KARNAL
Le Cong
Chengkun WANG
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Possible Medicines Llc
The Board Of Trustees Of The Leland Stanford Junior University
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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
    • A—HUMAN NECESSITIES
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to genetic modification systems utilizing RNA-guided recombination-editing systems using phage recombination enzymes as well as methods, vectors, nucleic acid compositions, and kids thereof.
  • BACKGROUND OF THE INVENTION [0004]
  • the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) system originally found in bacteria and archaea as part of the immune system to defend against invading viruses, forms the basis for genome editing technologies that can be programmed to target specific stretches of a genome or other DNA for editing precise locations. While various CRISPR-based tools are available, the majority are geared towards editing short sequences.
  • the invention relates to methods and compositions for modifying an organism or a non-human organism by manipulation of a target sequence, which comprises delivering in a cell, an RNA guided-recombination system or composition comprising: (i) a nucleic acid molecule comprising a guide RNA sequence that is complementary to a target DNA sequence; wherein the target DNA sequence comprises a genomic DNA sequence in the cell, and (ii) a recombination protein, wherein the recombination protein comprises a nuclease, a single stranded DNA annealing protein (SSAP) or a combination thereof; or, (iii) nucleic acid molecule(s) encoding or delivering the nucleic acid molecule, and/or the recombination protein for expression in vivo in a cell; or
  • the method also includes a Cas protein.
  • the method includes a donor nucleic acid.
  • the target DNA sequence comprises a genomic sequence of albumin (ALB), AAVS1, HSP90AA1, DYNLT1, ACTB, BCAP31, HIST1H2BK, CLTA, ROSA26, PCSK, or RAB11A.
  • the method also includes a recruitment system comprising at least one aptamer sequence; and an aptamer binding protein functionally linked to the recombination protein as part of a fusion protein.
  • the at least one aptamer sequence is an RNA aptamer sequence or a peptide aptamer sequence.
  • the nucleic acid molecule or nucleic acid molecules additionally comprise the at least one RNA aptamer sequence.
  • the at least one RNA aptamer sequences comprise the same sequence.
  • the aptamer binding protein comprises an MS2 coat protein, or a functional derivative or variant thereof.
  • the at least one peptide aptamer sequence is conjugated to the Cas protein.
  • the at least one peptide aptamer sequence comprises between 1 and 24 peptide aptamer sequences.
  • the aptamer sequences comprise the same sequence.
  • the recombination protein N-terminus is linked to the aptamer binding protein C-terminus.
  • the recombination protein comprises a microbial recombination protein or active portion thereof.
  • the Cas protein is catalytically inactive (less than 5% nuclease activity as compared with a wild type or non-mutated Cas protein) or catalytically dead.
  • the Cas protein comprises a Cas9, Cas12a, or Cas12f.
  • the single stranded DNA annealing protein and the Cas protein are functionally linked to each other and comprise a fusion protein.
  • the RNA guided recombination system is delivered via a viral vector system and a lipid-based system.
  • the viral vector system is an AAV viral vector system.
  • the viral vector system is an AAV8 viral vector system.
  • the lipid- based system is a lipid nanoparticle (LNP).
  • the invention provides a system or composition for delivering into an organism or a non-human organism: (i) a nucleic acid molecule comprising a guide RNA sequence that is complementary to a target DNA sequence; wherein the target DNA sequence comprises a genomic DNA sequence in the cell, and (ii) a recombination protein, wherein the recombination protein comprises a nuclease, a single stranded DNA annealing protein (SSAP) or a combination thereof; or, (iii) nucleic acid molecule(s) encoding or delivering (i), and/or (ii) for expression in vivo in a cell; or, (iv) vector(s) containing the nucleic acid molecule(s) of (iii) for expression in vivo in a cell; wherein the system or composition comprises a delivery system comprising a viral vector system and/or a lipid-based delivery system.
  • SSAP single stranded DNA annealing protein
  • the system or composition comprises a donor nucleic acid.
  • the system or composition comprises a donor nucleic acid, wherein the donor nucleic acid comprises a sequence that encodes GLP-1 or a GLP-1 analog.
  • the system or composition targets a DNA sequence which comprises a genomic sequence of albumin (ALB), AAVS1, HSP90AA1, DYNLT1, ACTB, BCAP31, HIST1H2BK, CLTA, ROSA26, PCSK, or RAB11A.
  • the system or composition comprises a recruitment system comprising at least one aptamer sequence; and an aptamer binding protein functionally linked to the recombination protein as part of a fusion protein.
  • the at least one D M2 ⁇ 19143850.1 3 PATENT Docket No. J0217-99008 aptamer sequence is an RNA aptamer sequence or a peptide aptamer sequence.
  • the nucleic acid molecule or nucleic acid molecules comprise the at least one RNA aptamer sequence.
  • the at least one RNA aptamer sequences comprise the same sequence.
  • the aptamer binding protein comprises an MS2 coat protein, or a functional derivative or variant thereof.
  • the at least one peptide aptamer sequence is conjugated to the Cas protein.
  • the at least one peptide aptamer sequence comprises between 1 and 24 peptide aptamer sequences.
  • the aptamer sequences comprise the same sequence.
  • the recombination protein N-terminus is linked to the aptamer binding protein C-terminus.
  • recombination protein comprises a microbial recombination protein or active portion thereof.
  • the Cas protein comprises a Cas9, Cas12a, or Cas12f.
  • the Cas protein is catalytically inactive (less than 5% nuclease activity as compared with a wild type or non-mutated Cas protein) or catalytically dead.
  • the single stranded DNA annealing protein and the Cas protein are functionally linked to each other and comprise a fusion protein.
  • the RNA guided recombination system is delivered via a viral vector system and a lipid-based system.
  • the viral vector system comprises an AAV viral vector system.
  • viral vector system comprises an AAV8 viral vector system.
  • the invention provides a kit comprising components of the delivery system or composition. [0024] Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C.
  • FIG. 1 Experimental design for Milestone 1. Milestone 1 compared the blood serum levels of untreated mice and mice treated with AAV delivery alone, LNP delivery alone, and AAV + LNP delivery. [0030] FIG.2. Experimental design for Milestone 2. Milestone 2 compared the blood serum levels of mice treated with LNP delivery alone and AAV + LNP delivery.
  • FIG.3. Test articles for Milestone 1.
  • FIG.4. Test articles for Milestone 2.
  • FIG.5. Reagents used for Milestones 1 and 2.
  • FIG.6 Instruments used for Milestones 1 and 2.
  • FIG.7. Drug administration information for Milestone 1.
  • FIG.8. Drug administration information for Milestone 2.
  • FIG.9. Blood GLP-1 measurement dilution factors.
  • FIG.12. Final state of mice in Milestone 1.
  • FIG.14 Serum concentrations of GLP-1 over time.
  • FIG.15 Detailed description of serum concentrations of GLP-1.
  • FIG.16 Schematic of serum concentrations of GLP-1 over time.
  • FIG. 17. Robust in vivo dulaglutide expression from the albumin locus using SAFE editing. Safe editing, which comprises AAV and LNP delivered dCas9-SSAP + sgRNA + hGLP1 donor template, achieved over 500nM serum dulaglutide 14 days after treatment. Expression exceeded AAV gene transfer with TBG promoter and resulted in a 650-fold therapeutic serum concentration of dulaglutide.
  • AAV viral delivery systems carrying GLP-1 donor template is injected into mice with LNP delivery systems carrying dCas9 +SSAP mRNA and Alb MS2 gRNA and injected into mice, which were studied over a 14 day period (see FIG.17).
  • DETAILED DESCRIPTION OF THE INVENTION [0047]
  • the present invention is directed to a system and the components for DNA editing.
  • the disclosed system is based on CRISPR targeting and homology directed repair by phage recombination enzymes.
  • the disclosed system is an RNA guided recombination system. The system results in superior recombination efficiency and accuracy at a kilobase scale.
  • the present invention is also directed to nucleic acids encoding an RNA guided recombination system and vectors encoding an RNA guided recombination system.
  • nucleic acids encoding an RNA guided recombination system and vectors encoding an RNA guided recombination system.
  • components thereof, and delivery of such components including methods, materials, delivery vehicles, vectors, particles, AAV, and making and using thereof, including as to amounts and formulations, all useful in the practice of the instant invention, reference is made to: U.S. Pat. Nos. 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,889,418 and 8,895,308; US Patent Publications US 2014-0310830 (U.S. application Ser. No.
  • 61/915,150, 61/915,301, 61/915,267 and 61/915,260 each filed Dec. 12, 2013; 61/757,972 and 61/768,959, filed on Jan. 29, 2013 and Feb. 25, 2013; 61/835,936, 61/836,127, 61/836,101, D M2 ⁇ 19143850.1 7 PATENT Docket No. J0217-99008 61/836,080, 61/835,973, and 61/835,931, filed Jun. 17, 2013; 62/010,888 and 62/010,879, both filed Jun. 11, 2014; 62/010,329 and 62/010,441, each filed Jun.
  • provisional patent application 61/939,242 filed Feb.12, 2014.
  • CRISPR clustered, regularly interspaced, short palindromic repeats
  • Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis.
  • Ran et al. described an approach that combined a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks.
  • the authors reported providing a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
  • the structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface.
  • the recognition lobe is essential for binding sgRNA and DNA
  • the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and non- complementary strands of the target DNA, respectively.
  • the nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes.
  • the authors showed that targeted sequencing of 295 dCas9 binding sites in D M2 ⁇ 19143850.1 12 PATENT Docket No. J0217-99008 mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels.
  • the authors proposed a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.
  • Hsu 2014 is a review article that discusses generally CRISPR-Cas9 history from yogurt to genome editing, including genetic screening of cells, that is in the information, data and findings of the applications in the lineage of this specification filed prior to Jun. 5, 2014.
  • the general teachings of Hsu 2014 do not involve the specific models, animals of the instant specification.
  • the terms "comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures.
  • the singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise.
  • the present invention also contemplates other embodiments "comprising,” “consisting of' and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
  • each intervening number there between with the same degree of precision is explicitly contemplated.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • a cell has been "genetically modified,” “transformed,” or “transfected” by exogenous DNA, e.g., a recombinant expression vector, when such DNA has been introduced inside the cell.
  • exogenous DNA e.g., a recombinant expression vector
  • the presence of the exogenous DNA results in permanent or transient genetic change.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations. D M2 ⁇ 19143850.1 13 PATENT Docket No.
  • a "vector” or "expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, e.g., an "insert,” may be attached or incorporated so as to bring about the replication of the attached segment in a cell.
  • the CRISPR-Cas or CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g.
  • RNA(s) as that term is herein used (e.g., RNA(s) to guide Cas9, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus.
  • RNA(s) to guide Cas9, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).
  • target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence is located in the nucleus or cytoplasm of a cell, and may include nucleic acids in or from mitochondrial, organelles, vesicles, liposomes or particles present within the cell.
  • NLSs are not preferred.
  • direct repeats may be identified in silico by searching for repetitive motifs that fulfill any or all of the following criteria: 1. found in a 2 Kb window of genomic sequence flanking the type II CRISPR locus; 2. span from 20 to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3.
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence.
  • the degree of complementarity between a guide sequence and its corresponding D M2 ⁇ 19143850.1 14 PATENT Docket No. J0217-99008 target sequence, when optimally aligned using a suitable alignment algorithm is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • any suitable algorithm for aligning sequences include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego
  • a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. In some embodiments the guide sequence is 10-30 nucleotides long. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to form a CRISPR complex may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein.
  • cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • the degree of complementarity between a guide sequence and its corresponding target sequence can be about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%;
  • a guide or RNA or sgRNA can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length; or guide or RNA or sgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length; and advantageously tracr RNA is 30 or 50 nucleotides in length.
  • an aspect of the invention is to reduce off-target interactions, e.g., reduce the guide interacting with a target sequence having low complementarity.
  • the invention involves mutations that result in the D M2 ⁇ 19143850.1 15 PATENT Docket No. J0217-99008 CRISPR-Cas system being able to distinguish between target and off-target sequences that have greater than 80% to about 95% complementarity, e.g., 83%-84% or 88-89% or 94-95% complementarity (for instance, distinguishing between a target having 18 nucleotides from an off- target of 18 nucleotides having 1, 2 or 3 mismatches).
  • the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or 98% or 98.5% or 99% or 99.5% or 99.9%, or 100%.
  • Off target is less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80% complementarity between the sequence and the guide, with it advantageous that off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity between the sequence and the guide.
  • the escorted guide RNA (capable of guiding Cas to a target locus) may comprise (1) a guide sequence capable of hybridizing to a genomic target locus in the eukaryotic cell; (2) a tracr sequence; and (3) a tracr mate sequence. All (1) to (3) may reside in a single RNA, i.e. an sgRNA (arranged in a 5′ to 3′ orientation), or the tracr RNA may be a different RNA than the RNA containing the guide and tracr sequence. The tracr hybridizes to the tracr mate sequence and directs the CRISPR/Cas complex to the target sequence.
  • the methods according to the invention as described herein comprehend inducing one or more mutations in a eukaryotic cell (in vitro, i.e. in an isolated eukaryotic cell) as herein discussed comprising delivering to cell a vector as herein discussed.
  • the mutation(s) can include the introduction, deletion, or substitution of one or more nucleotides at each target sequence of cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • the mutations can include the introduction, deletion, or substitution of 1-75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • the mutations can include the introduction, deletion, or substitution of 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • the mutations can include the introduction, deletion, or substitution of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • the mutations include the introduction, deletion, or substitution of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, D M2 ⁇ 19143850.1 16 PATENT Docket No. J0217-99008 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • the mutations can include the introduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • the mutations can include the introduction, deletion, or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides at each target sequence of said cell(s) via the guide(s) RNA(s) or sgRNA(s).
  • concentration of Cas mRNA and guide RNA delivered it will be important to control the concentration of Cas mRNA and guide RNA delivered.
  • Optimal concentrations of Cas mRNA and guide RNA can be determined by testing different concentrations in a cellular or non-human eukaryote animal model and using deep sequencing the analyze the extent of modification at potential off-target genomic loci.
  • Cas nickase mRNA for example S.
  • a Cas protein can comprise a modified form of a wild type Cas protein.
  • the modified form of the wild type Cas protein can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the Cas protein.
  • the modified form of the Cas protein can have less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity of the wild-type Cas protein (e.g., Cas9 from S. pyogenes).
  • the modified form of Cas protein can have no substantial nucleic acid-cleaving activity.
  • a Cas protein is a modified form that has no substantial nucleic acid-cleaving activity, it can be referred to as enzymatically inactive and/or “dead” (abbreviated by “d”).
  • a dead Cas protein (e.g., dCas12f, dCas9) can bind to a target polynucleotide but may not cleave the target polynucleotide.
  • a dead Cas protein is a dead Cas9 protein.
  • a dCas9 polypeptide can associate with a single guide RNA (sgRNA) to activate or repress transcription of target DNA.
  • sgRNAs can be introduced into cells expressing the engineered chimeric receptor polypeptide. In some cases, such cells contain one or more different sgRNAs that target the same nucleic acid. In other cases, the sgRNAs target different nucleic acids in the cell.
  • the nucleic acids targeted by the guide RNA can be any that are expressed in a cell D M2 ⁇ 19143850.1 17 PATENT Docket No. J0217-99008 such as an immune cell.
  • the nucleic acids targeted may be a gene involved in immune cell regulation.
  • the nucleic acid is associated with cancer.
  • the nucleic acid associated with cancer can be a cell cycle gene, cell response gene, apoptosis gene, or phagocytosis gene.
  • the recombinant guide RNA can be recognized by a CRISPR protein, a nuclease-null CRISPR protein, variants thereof, or derivatives thereof.
  • Enzymatically inactive can refer to a polypeptide that can bind to a nucleic acid sequence in a polynucleotide in a sequence-specific manner, but may not cleave a target polynucleotide.
  • An enzymatically inactive site-directed polypeptide can comprise an enzymatically inactive domain (e.g. nuclease domain).
  • Enzymatically inactive can refer to no activity.
  • Enzymatically inactive can refer to substantially no activity.
  • Enzymatically inactive can refer to essentially no activity.
  • Enzymatically inactive can refer to an activity less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, less than 9%, or less than 10% activity compared to a wild-type exemplary activity (e.g., nucleic acid cleaving activity, wild-type Cas9 activity).
  • a wild-type exemplary activity e.g., nucleic acid cleaving activity, wild-type Cas9 activity.
  • One or a plurality of the nuclease domains (e.g., RuvC, HNH) of a Cas protein can be deleted or mutated so that they are no longer functional or comprise reduced nuclease activity.
  • a Cas protein comprising at least two nuclease domains (e.g., Cas9)
  • the resulting Cas protein can generate a single-strand break at a CRISPR RNA (crRNA) recognition sequence within a double-stranded DNA but not a double-strand break.
  • crRNA CRISPR RNA
  • Such a nickase can cleave the complementary strand or the non-complementary strand, but may not cleave both.
  • the resulting Cas protein can have a reduced or no ability to cleave both strands of a double-stranded DNA.
  • An example of a mutation that can convert a Cas9 protein into a nickase is a D10A (aspartate to alanine at position 10 of Cas9) mutation in the RuvC domain of Cas9 from S. pyogenes.
  • H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) in the HNH domain of Cas9 from S. pyogenes can convert the Cas9 into a nickase.
  • An example of a mutation that can convert a Cas9 protein into a dead Cas9 is a D10A (aspartate to alanine at position 10 of Cas9) mutation in the RuvC domain and H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) in the HNH domain of Cas9 from S. pyogenes.
  • a dead Cas protein can comprise one or more mutations relative to a wild-type version of the protein.
  • the mutation can result in less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nucleic acid-cleaving activity in one or more of the plurality of nucleic acid- cleaving domains of the wild-type Cas protein.
  • the mutation can result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the complementary strand of the target nucleic acid but reducing its ability to cleave the non-complementary strand of the target nucleic acid.
  • the mutation can result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the non-complementary strand of the target nucleic acid but reducing its ability to cleave the complementary strand of the target nucleic acid.
  • the mutation can result in one or more of the plurality of nucleic acid-cleaving domains lacking the ability to cleave the complementary strand and the non-complementary strand of the target nucleic acid.
  • the residues to be mutated in a nuclease domain can correspond to one or more catalytic residues of the nuclease.
  • residues in the wild type exemplary S. pyogenes Cas9 polypeptide such as Asp10, His840, Asn854 and Asn856 can be mutated to inactivate one or more of the plurality of nucleic acid-cleaving domains (e.g., nuclease domains).
  • the residues to be mutated in a nuclease domain of a Cas protein can correspond to residues Asp10, His840, Asn854 and Asn856 in the wild type S.
  • residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987 can be mutated.
  • D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and/or D986A can be suitable.
  • a D10A mutation can be combined with one or more of H840A, N854A, or N856A mutations to produce a Cas9 protein substantially lacking DNA cleavage activity (e.g., a dead Cas9 protein).
  • a H840A mutation can be combined with one or more of D10A, N854A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity.
  • a N854A mutation can be combined with one or more of H840A, D10A, or N856A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity.
  • a N856A mutation can be combined with one or more of H840A, N854A, or D10A mutations to produce a site-directed polypeptide substantially lacking DNA cleavage activity.
  • two nucleic acid molecules comprising a guide RNA sequence may be utilized.
  • the two nucleic acid molecules may have the same or different guide RNA sequences, thus complementary to the same or different target DNA sequence.
  • the guide RNA sequences of the two nucleic acid molecules are complementary to a target DNA sequences at opposite ends (e.g., 3' or 5') and/or on opposite strands of the insert location.
  • the Cas protein are Cas12f, which can also be referred to as Cms1
  • CRISPR nucleases are a class of CRISPR nucleases that have certain desirable properties relative to other CRISPR nucleases such as Cas9 nucleases.
  • the Cas12f polypeptides interact with specific guide RNAs (gRNAs), which direct the Cas12f endonuclease to a specific target site, at which site the Cas12f endonuclease introduces a double-stranded break that can be repaired by a DNA repair process such that the DNA sequence is modified. Since the specificity is provided by the guide RNA, the Cas12f polypeptide is universal and can be used with different guide RNAs to target different genomic sequences. Cas12f endonucleases have certain advantages over the Cas nucleases (e.g., Cas9) traditionally used with CRISPR arrays.
  • Cas12f-associated CRISPR arrays are processed into mature crRNAs without the requirement of an additional trans-activating crRNA (tracrRNA).
  • Cas12f-crRNA complexes can cleave target DNA preceded by a short protospacer-adjacent motif (PAM) that is often T-rich, in contrast to the G-rich PAM following the target DNA for many Cas9 systems.
  • PAM protospacer-adjacent motif
  • Cas12f nucleases can introduce a staggered DNA double-stranded break.
  • the methods disclosed herein can be used to target and modify specific chromosomal sequences and/or introduce exogenous sequences at targeted locations in the genome of eukaryotic and prokaryotic cells.
  • Cas12f polypeptides comprise at least one RNA recognition and/or RNA binding domain. RNA recognition and/or RNA binding domains interact with guide RNAs. Typically the guide RNA comprises a region with a stem-loop structure that interacts with the Cas12f polypeptide. Cas12f polypeptides can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains.
  • nuclease domains i.e., DNase or RNase domains
  • a D M2 ⁇ 19143850.1 20 PATENT Docket No. J0217-99008 Cas12f polypeptide, or a polynucleotide encoding a Cas12f polypeptide comprises: an RNA- binding portion that interacts with the DNA-targeting RNA, and an activity portion that exhibits site-directed enzymatic activity, such as a RuvC endonuclease domain.
  • Cas12f polypeptides can be wild type Cas12f polypeptides, modified Cas12f polypeptides, or a fragment of a wild type or modified Cas12f polypeptide.
  • the Cas12f polypeptide can be modified to increase nucleic acid binding affinity and/or specificity, alter an enzymatic activity, and/or change another property of the protein.
  • nuclease i.e., DNase, RNase
  • the Cas12f polypeptide can be truncated to remove domains that are not essential for the function of the protein.
  • the Cas12f polypeptide can be derived from a wild type Cas12f polypeptide or fragment thereof.
  • the Cas12f polypeptide can be derived from a modified Cas12f polypeptide.
  • the amino acid sequence of the Cas12f polypeptide can be modified to alter one or more properties (e.g., nuclease activity, affinity, stability, etc.) of the protein.
  • domains of the Cas12f polypeptide not involved in RNA-guided cleavage can be eliminated from the protein such that the modified Cas12f polypeptide is smaller than the wild type Cas12f polypeptide.
  • the nuclease domain can be modified using well-known methods, such as site-directed mutagenesis, PCR-mediated mutagenesis, and total gene synthesis, as well as other methods known in the art.
  • Cas12f proteins with inactivated nuclease domains can be used to modulate gene expression without modifying DNA sequences.
  • Non-limiting examples of genetic modifications to Cas12f that result in inactivated nuclease domains include D225A, D401A, and/or E324A.
  • a dCas12f protein may be targeted to particular regions of a genome such as promoters for a gene or genes of interest through the use of appropriate gRNAs.
  • the dCas12f protein can bind to the desired region of DNA and may interfere with RNA polymerase binding to this region of DNA and/or with the binding of transcription factors to this region of DNA.
  • the dCas12f protein may be fused to a repressor domain to further downregulate the expression of a gene or genes whose expression is regulated by interactions of RNA polymerase, transcription factors, or other transcriptional regulators with the region of chromosomal DNA targeted by the gRNA.
  • the dCas12f D M2 ⁇ 19143850.1 21 PATENT Docket No.
  • J0217-99008 protein may be fused to an activation domain to effect an upregulation of a gene or genes whose expression is regulated by interactions of RNA polymerase, transcription factors, or other transcriptional regulators with the region of chromosomal DNA targeted by the gRNA.
  • the dCas12f polypeptides disclosed herein can further comprise at least one nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • an NLS comprises a stretch of basic amino acids. Nuclear localization signals are known in the art (see, e.g., Lange et al., J. Biol. Chem. (2007) 282:5101- 5105).
  • the NLS can be located at the N-terminus, the C-terminus, or in an internal location of the dCas12f polypeptide.
  • the dCas12f polypeptide can further comprise at least one cell-penetrating domain.
  • the cell-penetrating domain can be located at the N-terminus, the C- terminus, or in an internal location of the protein.
  • the dCas12f polypeptide disclosed herein can further comprise at least one plastid targeting signal peptide, at least one mitochondrial targeting signal peptide, or a signal peptide targeting the dCas12f polypeptide to both plastids and mitochondria.
  • Plastid, mitochondrial, and dual-targeting signal peptide localization signals are known in the art (see, e.g., Nassoury and Morse (2005) Biochim Biophys Acta 1743:5-19; Kunze and Berger (2015) Front Physiol 6:259; Herrmann and Neupert (2003) IUBMB Life 55:219-225; Soll (2002) Curr Opin Plant Biol 5:529- 535; Carrie and Small (2013) Biochim Biophys Acta 1833:253-259; Carrie et al.
  • the plastid, mitochondrial, or dual-targeting signal peptide can be located at the N-terminus, the C-terminus, or in an internal location of the Cas12f polypeptide.
  • the Cas12f polypeptide can also comprise at least one marker domain.
  • Non-limiting examples of marker domains include fluorescent proteins, purification tags, and epitope tags.
  • the marker domain can be a fluorescent protein.
  • suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g. YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g. EBFP, EBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g.
  • the marker domain can be a purification tag and/or an epitope tag.
  • Exemplary tags include, but are not limited to, glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6 ⁇ His, biotin carboxyl carrier protein (BCCP), and calmodulin.
  • GST glutathione-S-transferase
  • CBP chitin binding protein
  • TRX thioredoxin
  • poly(NANP) poly(NANP)
  • TAP tandem affinity purification
  • the Cas12f polypeptide may be part of a protein-RNA complex comprising a guide RNA.
  • the guide RNA interacts with the Cas12f polypeptide to direct the Cas12f polypeptide to a specific target site, wherein the 5′ end of the guide RNA can base pair with a specific protospacer sequence of the nucleotide sequence of interest in the plant genome, whether part of the nuclear, plastid, and/or mitochondrial genome.
  • the term “DNA- targeting RNA” refers to a guide RNA that interacts with the Cas12f polypeptide and the target site of the nucleotide sequence of interest in the genome of a plant cell.
  • a DNA-targeting RNA, or a DNA polynucleotide encoding a DNA-targeting RNA can comprise: a first segment comprising a nucleotide sequence that is complementary to a sequence in the target DNA, and a second segment that interacts with a Cas12f polypeptide.
  • the polynucleotides encoding Cas12f polypeptides disclosed herein can be used to isolate corresponding sequences from other prokaryotic or eukaryotic organisms, or from metagenomically-derived sequences whose native host organism is unclear or unknown. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology or identity to the sequences set forth herein.
  • Sequences isolated based on their sequence identity to the entire Cas12f sequences set forth herein or to variants and fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed Cas12f sequences. “Orthologs” is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, D M2 ⁇ 19143850.1 23 PATENT Docket No.
  • Cas12f endonuclease activity refers to CRISPR endonuclease activity wherein, a guide RNA (gRNA) associated with a Cas12f polypeptide causes the Cas12f -gRNA complex to bind to a pre- determined nucleotide sequence that is complementary to the gRNA; and wherein Cas12f activity can introduce a double-stranded break at or near the site targeted by the gRNA. In certain embodiments, this double-stranded break may be a staggered DNA double-stranded break.
  • gRNA guide RNA
  • a “staggered DNA double-stranded break” can result in a double strand break with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 nucleotides of overhang on either the 3′ or 5′ ends following cleavage.
  • the Cas12f polypeptide introduces a staggered DNA double-stranded break with a 5′ overhang.
  • the double strand break can occur at or near the sequence to which the DNA-targeting RNA (e.g., guide RNA) sequence is targeted.
  • Cas12f nuclease activity is intended the binding of a pre-determined DNA sequence as mediated by a guide RNA.
  • Cas12f nuclease activity can further comprise double-strand break induction.
  • fragment is intended a portion of the polynucleotide or a portion of the amino acid sequence.
  • Variants is intended to mean substantially similar sequences.
  • a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5′ and/or 3′ end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide.
  • a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively.
  • variants of a particular polynucleotide of the invention will have at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.
  • D M2 ⁇ 19143850.1 24 PATENT Docket No. J0217-99008 [00106] Fusion proteins are provided herein comprising a Fusion proteins are provided herein comprising a Cas12f polypeptide, or a fragment or variant thereof, and an effector domain.
  • the Cas12f polypeptide can be directed to a target site by a guide RNA, at which site the effector domain can modify or effect the targeted nucleic acid sequence.
  • the effector domain can be a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.
  • the fusion protein can further comprise at least one additional domain chosen from a nuclear localization signal, plastid signal peptide, mitochondrial signal peptide, signal peptide capable of protein trafficking to multiple subcellular locations, a cell- penetrating domain, or a marker domain, any of which can be located at the N-terminus, C- terminus, or an internal location of the fusion protein.
  • the Cas12f polypeptide can be located at the N-terminus, the C-terminus, or in an internal location of the fusion protein.
  • the Cas12f polypeptide can be directly fused to the effector domain, or can be fused with a linker.
  • the linker sequence fusing the Cas12f polypeptide with the effector domain can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 amino acids in length.
  • the linker can range from 1-5, 1-10, 1-20, 1-50, 2-3, 3-10, 3-20, 5-20, or 10-50 amino acids in length.
  • the Cas12f polypeptide of the fusion protein can be derived from a wild type Cas12f protein.
  • the Cas12f -derived protein can be a modified variant or a fragment.
  • the Cas12f polypeptide can be modified to contain a nuclease domain (e.g. a RuvC or RuvC-like domain) with reduced or eliminated nuclease activity.
  • the Cas12f -derived polypeptide can be modified such that the nuclease domain is deleted or mutated such that it is no longer functional (i.e., the nuclease activity is absent).
  • fusion can refer to a protein and/or nucleic acid comprising one or more non-native sequences (e.g., moieties).
  • a fusion can comprise one or more of the same non- native sequences.
  • a fusion can comprise one or more of different non-native sequences.
  • a fusion can be a chimera.
  • a fusion can comprise a nucleic acid affinity tag.
  • a fusion can comprise a barcode.
  • a fusion can comprise a peptide affinity tag.
  • a fusion can provide for subcellular localization of the site-directed polypeptide (e.g., a nuclear localization signal (NLS) for targeting to the nucleus, a mitochondrial localization signal for targeting to the mitochondria, a chloroplast localization signal for targeting to a chloroplast, an endoplasmic reticulum (ER) retention signal, and the like).
  • a fusion can provide a non-native sequence (e.g., affinity tag) that can be used to D M2 ⁇ 19143850.1 25 PATENT Docket No. J0217-99008 track or purify.
  • a fusion can be a small molecule such as biotin or a dye such as alexa fluor dyes, Cyanine3 dye, Cyanine5 dye.
  • a fusion can refer to any protein with a functional effect.
  • a fusion protein can comprise methyltransferase activity, demethylase activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity or glycosylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, remodelling activity, protease activity, oxidoreductase activity, transferase activity
  • An effector protein can modify a genomic locus.
  • a fusion protein can be a fusion in a Cas protein.
  • An fusion protein can be a non-native sequence in a Cas protein.
  • the system further comprises a recruitment system comprising at least one aptamer sequence and an aptamer binding protein functionally linked to the recombination protein as part of a fusion protein.
  • the aptamer sequence is an RNA aptamer sequence.
  • the nucleic acid molecule comprising the guide RNA also comprises one or more RNA aptamers, or distinct RNA secondary structures or sequences that can recruit and bind another molecular species, an adaptor molecule, such as a nucleic acid or protein.
  • an adaptor molecule such as a nucleic acid or protein.
  • CRISPR systems are compatible with guide RNA insertions and extensions, including but not limited to SpCas9, SaCas9, and LbCasl2a (aka Cpfl).
  • the RNA aptamers can be naturally occurring or synthetic oligonucleotides that have been engineered through repeated rounds of in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) to bind to a specific target molecular species.
  • the nucleic acid comprises two or more aptamer sequences.
  • the aptamer sequences may be the same or different and may target the same or different adaptor proteins.
  • the nucleic acid comprises two aptamer sequences.
  • Any RNA aptamer/ aptamer binding protein pair known may be selected and used in connection with the present invention (see, e.g., Jayasena, S.D., Clinical Chemistry, 1999.45(9): D M2 ⁇ 19143850.1 26 PATENT Docket No. J0217-99008 p. 1628-1650; Gelinas, et al., Current Opinion in Structural Biology, 2016. 36: p.
  • RNA aptamer binding, or adaptor, proteins exist, including a diverse array of bacteriophage coat proteins.
  • coat proteins include but are not limited to: MS2, Q ⁇ , F2, GA, fr, JP501, Ml2, RI 7, BZ13, JP34, JP500, KUI, Ml 1, MXI, TW18, VK, SP, Fl, ID2, NL95, TW19, AP205, ⁇ Cb5, ⁇ Cb8r, ⁇ Cbl2r, ⁇ Cb23r, 7s and PRRI.
  • the RNA aptamer binds MS2 bacteriophage coat protein or a functional derivative, fragment or variant thereof.
  • MS2 binding RNA aptamers commonly have a simple stem-loop structure, classically defined by a 19 nucleotide RNA molecule with a single bulged adenine on the 5' leg of the stem (Witherall G.W., et al., (1991) Prog. Nucleic Acid Res. Mol. Biol., 40, 185-220, incorporated herein by reference).
  • MCP MS2 coat protein
  • RNA aptamer sequence known to bind the MS2 bacteriophage coat protein (MCP) may be utilized in connection with the present invention to bind to fusion proteins comprising MS2.
  • the MS2 RNA aptamer sequence comprises: AACAUGAGGAUCACCCAUGUCUGCAG, AGCAUGAGGAUCACCCAUGUCUGCAG, or AGCGUGAGGAUCACCCAUGCCUGCAG.
  • N-proteins (Nut-utilization site proteins) of bacteriophages contain arginine-rich conserved RNA recognition motifs of ⁇ 20 amino acids, referred to as N peptides.
  • the RNA aptamer may bind a phage N peptide or a functional derivative, fragment or variant thereof.
  • the phage N peptide is the lambda or P22 phage N peptide or a functional derivative, fragment or variant thereof.
  • the N peptide is lambda phage N22 peptide, or a functional derivative, fragment or variant thereof.
  • the N22 peptide comprises an amino acid sequence with at least 70% similarity to the ammo acid sequence GNARTRRRERRAEKQAQWKAAN.
  • N22 peptide, the 22 amino acid RNA binding domain of the 11. bacteriophage antiterminator protein N (11.N-(1-22) or 11.N peptide) is capable of specifically binding to specific stem-loop structures, including but not limited to the BoxB stem- loop. See, for example Cilley and Williamson, RNA 1997; 3(1):57-67, incorporated herein by reference.
  • the N22 peptide RNA aptamer sequence comprises a nucleotide sequence with at least 70% similarity to an RNA sequence selected from the group consisting of GCCCUGAAAAAGGGC, GCCCUGAAGAAGGGC, GCGCUGAAAAAGCGC, GCCCUGACAAAGGGC, and GCGCUGACAAAGCGC.
  • the N peptide is the P22 phage N peptide, or a functional derivative, fragment or variant thereof.
  • the P22 phage N peptide comprises an amino acid sequence with at least 70% similarity to the amino acid sequence GNAKTRRHERRRKLAIERDTI.
  • the P22 phage N peptide RNA aptamer sequence comprises a sequence with at least 70% similarity to an RNA sequence selected from the group consisting of GCGCUGACAAAGCG and CCGCCGACAACGCGG.
  • different aptamer/aptamer binding protein pairs can be selected to bring together a combination of recombination proteins and functions.
  • the aptamer sequence is a peptide aptamer sequence.
  • the peptide aptamers can be naturally occurring or synthetic peptides that are specifically recognized by an affinity agent.
  • Such aptamers include, but are not limited to, a c-Myc affinity tag, an HA affinity tag, a His affinity tag, an S affinity tag, a methionine-His affinity tag, an RGD-His affinity tag, a 7x His tag, a FLAG octapeptide, a strep tag or strep tag II, a VS tag, or a VSV-G epitope.
  • Corresponding aptamer binding proteins are well-known in the art and include, for example, primary antibodies, biotin, affimers, single domain antibodies, and antibody mimetics.
  • An exemplary peptide aptamer includes a GCN4 peptide (Tanenbaum et al., Cell 2014; 159(3):635-646, incorporated herein by reference). Antibodies, or GCN4 binding protein can be used as the aptamer binding proteins.
  • the peptide aptamer sequence is conjugated to the Cas protein.
  • the peptide aptamer sequence may be fused to the Cas in any orientation (e.g., N-terminus to Cterminus, C-terminus to N-terminus, N-terminus to N-terminus).
  • the peptide aptamer is fused to the C-terminus of the Cas protein.
  • peptide aptamer sequences may be conjugated to the Cas protein.
  • the aptamer sequences may be the same or different and may target the same or different aptamer binding proteins.
  • 1 to 24 tandem repeats of the same peptide aptamer sequence are conjugated to the Cas protein.
  • between 4 and 18 tandem repeats are conjugated to the Cas protein.
  • the individual aptamers may be separated by a linker region. Suitable linker regions are known in the art. The linker may be flexible or configured to allow the binding of affinity agents to adjacent aptamers without or with decreased steric hindrance.
  • the linker sequences may provide an unstructured or linear region of the polypeptide, for example, with the inclusion of one or more glycine and/or serine residues.
  • the linker sequences can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in length.
  • the fusion protein comprises a recombination protein functionally linked to an aptamer binding protein.
  • the recombination protein comprises a microbial recombination protein.
  • the recombination protein comprises a recombinase.
  • the recombination protein comprises a nuclease.
  • the recombination protein comprises an endonuclease. In certain embodiments, the recombination protein comprises 5' -3' exonuclease activity . In certain embodiments, the recombination protein comprises 3 '-5' exonuclease activity . In certain embodiments, the recombination protein comprises ssDNA binding activity . In certain embodiments, the recombination protein comprises ssDNA annealing activity . [00122]
  • the bacteriophage A-encoded genetic recombination machinery named the 11, red system, comprises the exo and bet genes, assisted by the gam gene, together designated 11, red genes.
  • Exo is a 5'-3' exonuclease which targets dsDNA and Bet is a ssDNA-binding protein.
  • Bet functions include protecting ssDNA from degradation and promoting annealing of complementary ssDNA strands.
  • Another bacteriophage system found in E. coli is the Rae prophage system, comprising recE and recT genes which are functionally similar to exo and bet .
  • the microbial recombination protein may be RecE, RecT, lambda exonuclease (Exo), Bet protein (betA, redB), exonuclease gp6, single-stranded DNA-binding protein gp2.5, or a derivative or variant thereof.
  • Recombination proteins and functional fragments thereof useful in the invention include nucleases, ssDNA-binding proteins (SSBs), and ssDNA annealing proteins (SSAPs) .
  • SSBs ssDNA-binding proteins
  • SSAPs ssDNA annealing proteins
  • E. coli proteins such as ExoI (xonA; D M2 ⁇ 19143850.1 29 PATENT Docket No.
  • SSBs ssDNA binding proteins
  • Useful SSBs include, without limitation, SSBs of prokaryotes, bacteriophage, eukaryotes, mammals, mitochondria, and viruses . While SSBs are found in every organism, the proteins themselves share surprisingly little sequence similarity, and may differ in subunit composition and oligomerization states. SSB proteins may comprise certain structural features .
  • oligonucleotide/oligosaccharide-binding (OB) domains to bind ssDNA through a combination of electrostatic and base-stacking interactions with the phosphodiester backbone and nucleotide bases .
  • Another feature is oligomerization that brings together DNA-binding OB folds .
  • Eukaryotic SSBs are regulated by phosphorylation on serine and threonine residues . Tyrosine phosphorylation of microbial SSBs is observed in taxonomically distant bacteria and substantially increases affinity for ssDNA.
  • the human mitochondrial ssDNAbinding protein is structurally similar to SSB from Escherichia coli (EcoSSB), but lacks the Cterminal disordered domain.
  • Eukaryotic replication protein A (RP A) shares function, but not sequence homology with bacterial SSB.
  • the herpes simplex virus (HSV-1) SSB, ICP8, is a nuclear protein that, along other replication proteins is required for viral DNA replication. [00125] Without being bound by theory, it is thought that exonuclease activities and ssDNA binding activities of the recombination proteins of the invention uncover and protect single stranded regions of template and target DNAs, thereby facilitating recombination.
  • targeting can be cooperative, involving target directed CRISPR-mediated nicking, cleaving, or binding of chromosomal DNA coordinated with recombination directed by homology arms designed into donor DNAs .
  • off-target effects are minimized .
  • homology with the HR donor DNA is absent and nick repair may be favored.
  • SSAPs Single stranded DNA annealing proteins
  • phage encoded SSAPs are recognized to encode their own SSAP recombinases which substitute for classic RecA proteins while functioning with host proteins to control DNA metabolism .
  • Steczkiewiz classified SSAPs into seven families (RecA, Gp2.5, RecT/Red ⁇ , Erf, Rad52/22, Sak3, and Sak4) organized into three superfamilies including prokaryotes, eukaryotes, and phage (Steczkiewicz et al., 2021, Front. Microbial 12:644622).
  • a microbial recombination protein is RecE or RecT, or a derivative or variant thereof.
  • RecE and RecT are functionally equivalent proteins or polypeptides which possess substantially similar function to wild type RecE and RecT.
  • RecE and RecT derivatives or variants include biologically active amino acid sequences similar to the wild-type sequences but differing due to amino acid substitutions, additions, deletions, truncations, post-translational modifications, or other modifications.
  • the derivatives may improve translation, purification, biological half-life, activity, or eliminate or lessen any undesirable side effects or reactions.
  • the derivatives or variants may be naturally occurring polypeptides, synthetic or chemically synthesized polypeptides or genetically engineered peptide polypeptides.
  • a Cas protein as used herein can be a wildtype or a modified form of a Cas protein.
  • a Cas protein can be an active variant, inactive variant, or fragment of a wild type or modified Cas protein.
  • a Cas protein can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof relative to a wild-type version of the Cas protein.
  • a Cas protein can be a polypeptide with at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a wild type exemplary Cas protein.
  • a Cas protein can be a polypeptide with at most about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% sequence identity and/or sequence similarity to a wild type exemplary Cas protein.
  • Variants or fragments can comprise at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity or sequence similarity to a wild type or modified Cas protein or a portion thereof. Variants or fragments can be targeted to a nucleic acid locus in complex with a guide nucleic acid while lacking nucleic acid cleavage activity.
  • a Cas protein can be a fusion protein.
  • a Cas protein can be fused to a cleavage domain, an epigenetic modification domain, a transcriptional activation domain, or a transcriptional repressor domain.
  • a Cas protein can also be fused to a heterologous polypeptide providing increased or decreased stability.
  • the fused domain or heterologous polypeptide can be located at the N-terminus, the C-terminus, or internally within the Cas protein.
  • a microbial recombination protein may be linked to either terminus of an aptamer binding protein in any orientation (e.g., N-terminus to C-terminus, C-terminus to N- terminus, N-terminus to Nterminus).
  • a microbial recombination protein N- terminus is linked to the aptamer binding protein C-terminus.
  • the overall fusion protein from N- to C-terminus comprises the aptamer binding protein (N- to C-terminus) linked to the microbial recombination protein (N- to C-terminus).
  • formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.
  • the tracr sequence which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence.
  • the nucleic acid molecule encoding a Cas is advantageously codon optimized Cas.
  • a codon optimized sequence is in this instance a sequence optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in humans), or for another eukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 human codon optimized sequence in WO 2014/093622 (PCT/US2013/074667). Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species other than human, or for codon optimization for specific organs is known. [00133] In some embodiments, an enzyme coding sequence encoding a Cas is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, or non- human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, D M2 ⁇ 19143850.1 32 PATENT Docket No. J0217-99008 or non-human mammal or primate.
  • processes for modifying the germ line genetic identity of human beings and/or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes may be excluded.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000).
  • codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available.
  • one or more codons e.g.1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • the Cas sequence is fused to one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs.
  • the Cas comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. zero or at least one or more NLS at the amino-terminus and zero or at one or more NLS at the carboxy terminus).
  • NLS NLS at the amino-terminus and the carboxy terminus.
  • each may be selected independently of the others, such that a single NLS D M2 ⁇ 19143850.1 33 PATENT Docket No. J0217-99008 may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies.
  • the Cas comprises at most 6 NLSs.
  • an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.
  • NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV; the NLS from nucleoplasmin (e.g.
  • the one or more NLSs are of sufficient strength to drive accumulation of the Cas in a detectable amount in the nucleus of a eukaryotic cell.
  • strength of nuclear localization activity may derive from the number of NLSs in the Cas, the particular NLS(s) used, or a combination of these factors.
  • Detection of accumulation in the nucleus may be performed by any suitable technique.
  • a detectable marker may be fused to the Cas, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g. a stain specific for the nucleus such as DAPI).
  • Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of CRISPR complex formation (e.g. assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by CRISPR complex formation and/or Cas D M2 ⁇ 19143850.1 34 PATENT Docket No. J0217-99008 enzyme activity), as compared to a control no exposed to the Cas or complex, or exposed to a Cas lacking the one or more NLSs. In other embodiments, no NLS is required.
  • an assay for the effect of CRISPR complex formation e.g. assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by CRISPR complex formation and/or Cas D M2 ⁇ 19143850.1 34 PATENT Docket
  • the term “crRNA” or “guide RNA” or “single guide RNA” or “sgRNA” or “one or more nucleic acid components” of a Type II CRISPR-Cas locus effector protein comprises any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence.
  • the degree of complementarity when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • any suitable algorithm for aligning sequences include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina,
  • a guide sequence within a nucleic acid-targeting guide RNA
  • a guide sequence may direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence
  • the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid-targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by Surveyor assay as described herein.
  • preferential targeting e.g., cleavage
  • cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • Other assays are possible, and will occur to those skilled in the art.
  • a guide sequence, and hence a nucleic acid-targeting guide RNA may be selected to target any target nucleic acid sequence.
  • the target sequence may be DNA.
  • the target sequence D M2 ⁇ 19143850.1 35 PATENT Docket No. J0217-99008 may be any RNA sequence.
  • the target sequence may be a sequence within a RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double stranded RNA (dsRNA), non coding RNA (ncRNA), long non-coding RNA (lncRNA), and small cytoplasmatic RNA (scRNA).
  • the target sequence may be a sequence within a RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA.
  • the target sequence may be a sequence within a RNA molecule selected from the group consisting of ncRNA, and lncRNA. In some more embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule.
  • a nucleic acid-targeting guide RNA is selected to reduce the degree secondary structure within the RNA-targeting guide RNA. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleic acid-targeting guide RNA participate in self-complementary base pairing when optimally folded.
  • Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res.9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
  • a guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat (DR) sequence and a guide sequence or spacer sequence.
  • the guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence.
  • the direct repeat sequence may be located upstream (i.e., 5′) from the guide sequence or spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (i.e., 3′) from the guide sequence or spacer sequence.
  • the crRNA comprises a stem loop. In certain embodiments, the direct repeat sequence forms a stem loop.
  • tracrRNA sequence or analogous terms includes any polynucleotide sequence that has sufficient complementarity with a crRNA sequence to hybridize.
  • degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences.
  • Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence.
  • the degree of complementarity between the tracr sequence and the tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • a guide sequence may be selected to target any target sequence.
  • the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, for the S.
  • a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG where NNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome.
  • a unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMNNNNNNNNNNNXGG where NNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome.
  • S pyogenes Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNNXGG where NNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome.
  • thermophiles CRISPR1 Cas9 a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW where NNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome.
  • a unique target sequence in a genome may include an S. thermophiles CRISPR1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW where NNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome.
  • a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG where NNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome.
  • a unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMNNNNNNNNNNNXGGXG where NNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome.
  • a guide sequence is selected to reduce the degree secondary structure within the guide sequence. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the guide sequence participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy.
  • mFold as described by Zuker and Stiegler (Nucleic Acids Res.9 (1981), 133-148).
  • Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
  • a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence.
  • degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences.
  • Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self- complementarity within either the tracr sequence or tracr mate sequence.
  • the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.
  • the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin.
  • the transcript or transcribed polynucleotide sequence has at least two or more hairpins.
  • the transcript has two, three, four or five hairpins.
  • the transcript has at most five hairpins. D M2 ⁇ 19143850.1 38 PATENT Docket No.
  • sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1.
  • sequences (4) to (6) are used in combination with Cas9 from S. pyogenes.
  • the tracr sequence is a separate transcript from a transcript comprising the tracr mate sequence.
  • it may be preferred in a CRISPR complex that the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme.
  • candidate tracrRNA may be subsequently predicted by sequences that fulfill any or all of the following criteria: 1. sequence homology to direct repeats (motif search in Geneious with up to 18-bp mismatches); 2. presence of a predicted Rho- independent transcriptional terminator in direction of transcription; and 3. stable hairpin secondary D M2 ⁇ 19143850.1 39 PATENT Docket No. J0217-99008 structure between tracrRNA and direct repeat. In some embodiments, 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.
  • chimeric synthetic guide RNAs designs may incorporate at least 12 bp of duplex structure between the direct repeat and tracrRNA.
  • Donor nucleic acids can be single-strand or double-stranded DNA and comprise (1) various lengths of homology arms (HA) to match a genomic target region, and (2) a transgene, i.e. knock-in sequence or replacement sequence etc. There is no limit to the sized of the transgene .
  • the target genomic DNA sequence can comprise a gene, the mutation of which contributes to a particular disease in combination with mutations in other genes.
  • multifactorial or polygenic diseases include, but are not limited to, asthma, diabetes, epilepsy, hypertension, bipolar disorder, and schizophrenia.
  • Certain developmental abnormalities also can be inherited in a multifactorial or polygenic pattern and include, for example, cleft lip/palate, congenital heart defects, and neural tube defects.
  • the method of altering a target genomic DNA sequence can be used to delete nucleic acids from a target sequence in a cell by cleaving the target sequence and allowing the cell to repair the cleaved sequence in the absence of an exogenously provided donor nucleic acid molecule.
  • Deletion of a nucleic acid sequence in this manner can be used in a variety of applications, such as, for example, to remove disease-causing trinucleotide repeat sequences in neurons, to create gene knock-outs or knock-downs, and to generate mutations for disease models in research.
  • the term "donor nucleic acid molecule" refers to a nucleotide sequence that is inserted into the target DNA (e.g., genomic DNA).
  • the donor DNA may include, for example, a gene or part of a gene, a sequence encoding a tag or localization sequence, or a regulating element.
  • the donor nucleic acid molecule may be of any length. In some embodiments, the donor nucleic acid molecule is between 10 and 10,000 nucleotides in length. For example, between about 100 and 5,000 nucleotides in length, between about 200 and 2,000 nucleotides in length, between about 500 and 1,000 nucleotides in length, between about 500 and 5,000 nucleotides in length, between about 1,000 and 5,000 nucleotides in length, or between about 1,000 and 10,000 nucleotides in length. D M2 ⁇ 19143850.1 40 PATENT Docket No.
  • the CRISPR system is derived advantageously from a type II CRISPR system.
  • one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • the CRISPR system is a type II CRISPR system and the Cas enzyme is Cas9, which catalyzes DNA cleavage.
  • Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12, Cas12a, Cas12f (also known as Csm1), Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof.
  • the Cas9 protein may be an ortholog of an organism of a genus which includes but is not limited to Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter. Species of an organism of such a genus can be as otherwise herein discussed.
  • chimeric enzymes may comprise fragments of CRISPR enzyme orthologs of an organism which includes but is not limited to Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter.
  • a chimeric enzyme can comprise a first fragment and a second fragment, and the fragments can be of CRISPR enzyme orthologs of organisms of genuses herein mentioned or of species herein mentioned; advantageously the fragments are from CRISPR enzyme orthologs of different species [00158]
  • the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9.
  • the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence.
  • the CRISPR enzyme directs cleavage D M2 ⁇ 19143850.1 41 PATENT Docket No.
  • a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • D10A aspartate-to-alanine substitution
  • pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
  • Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A.
  • two or more catalytic domains of Cas9 may be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity.
  • a D10A mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity.
  • a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the DNA cleavage activity of the non-mutated form of the enzyme; an example can be when the DNA cleavage activity of the mutated form is nil or negligible as compared with the non-mutated form.
  • mutations may be made at any or all residues corresponding to positions 10, 762, 840, 854, 863 and/or 986 of SpCas9 (which may be ascertained for instance by standard sequence comparison tools).
  • any or all of the following mutations are envisioned in SpCas9: D10A, E762A, H840A, N854A, N863A and/or D986A; as well as conservative substitution for any of the replacement amino acids is also envisaged.
  • the same (or conservative substitutions of these mutations) at corresponding positions in other Cas9s are also envisioned.
  • residues corresponding to SpCas9 D10 and H840 are also envisioned.
  • a Cas enzyme may be identified Cas9 as this can refer to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the type II CRISPR system.
  • the Cas9 enzyme is from, or is derived from, spCas9 (S. pyogenes Cas9) or saCas9 (S. aureus Cas9).
  • StCas9 refers to wild type Cas9 from S. thermophilus, the protein sequence of which is given in the SwissProt database under accession number G3ECR1. Similarly, D M2 ⁇ 19143850.1 42 PATENT Docket No.
  • J0217-99008 S pyogenes Cas9 or spCas9 is included in SwissProt under accession number Q99ZW2.
  • Applicants mean that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as described herein.
  • the terms Cas and CRISPR enzyme are generally used herein interchangeably, unless otherwise apparent.
  • residue numberings used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes.
  • this invention includes many more Cas9s from other species of microbes, such as SpCas9, SaCa9, St1Cas9 and so forth.
  • Enzymatic action by Cas9 derived from Streptococcus pyogenes or any closely related Cas9 generates double stranded breaks at target site sequences which hybridize to 20 nucleotides of the guide sequence and that have a protospacer-adjacent motif (PAM) sequence (examples include NGG/NRG or a PAM that can be determined as described herein) following the 20 nucleotides of the target sequence.
  • PAM protospacer-adjacent motif
  • CRISPR activity through Cas9 for site-specific DNA recognition and cleavage is defined by the guide sequence, the tracr sequence that hybridizes in part to the guide sequence and the PAM sequence.
  • More aspects of the CRISPR system are described in Karginov and Hannon, The CRISPR system: small RNA-guided defence in bacteria and archaea, Mol Cell 2010, Jan. 15; 37(1): 7.
  • the type II CRISPR locus from Streptococcus pyogenes SF370 which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers, about 30 bp each).
  • DSB targeted DNA double-strand break
  • tracrRNA hybridizes to the direct repeats of pre-crRNA, which is then processed into mature crRNAs containing individual spacer sequences.
  • the mature crRNA:tracrRNA complex directs Cas9 to the DNA target consisting of the protospacer and the corresponding PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA.
  • Cas9 mediates cleavage of target DNA upstream of PAM to create a DSB within the protospacer.
  • a pre-crRNA array consisting of a single spacer flanked by two direct repeats (DRs) is also encompassed by the term “tracr-mate sequences”).
  • Cas9 may be constitutively present or inducibly present or conditionally present or administered or delivered. Cas9 optimization may be used to enhance D M2 ⁇ 19143850.1 43 PATENT Docket No. J0217-99008 function or to develop new functions, one can generate chimeric Cas9 proteins. And Cas9 may be used as a generic DNA binding protein. [00162] Aspects of the invention relate to the expression of the gene product being decreased or a donor polynucleotide being further introduced into the DNA molecule encoding the gene product or an intervening sequence being excised precisely by allowing the two 5′ overhangs to reanneal and ligate or the activity or function of the gene product being altered or the expression of the gene product being increased.
  • the gene product is a protein. Only sgRNA pairs creating 5′ overhangs with less than 8 bp overlap between the guide sequences (offset greater than ⁇ 8 bp) were able to mediate detectable indel formation. Importantly, each guide used in these assays is able to efficiently induce indels when paired with wildtype Cas9, indicating that the relative positions of the guide pairs are the most important parameters in predicting double nicking activity.
  • Cas9H840A Since Cas9n and Cas9H840A nick opposite strands of DNA, substitution of Cas9n with Cas9H840A with a given sgRNA pair should have resulted in the inversion of the overhang type; but no indel formation is observed as with Cas9H840A indicating that Cas9H840A is a CRISPR enzyme substantially lacking all DNA cleavage activity (which is when the DNA cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the DNA cleavage activity of the non-mutated form of the enzyme; whereby an example can be when the DNA cleavage activity of the mutated form is nil or negligible as compared with the non-mutated form, e.g., when no indel formation is observed as with Cas9H840A in the eukaryotic system in contrast to the biochemical or prokaryotic systems).
  • a recombination donor, or template is also provided.
  • a recombination donor may be a component of another vector as described herein, contained in a separate vector, or provided as a separate polynucleotide.
  • a recombination donor is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a CRISPR enzyme as a part of a CRISPR complex.
  • a donor polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In some D M2 ⁇ 19143850.1 44 PATENT Docket No. J0217-99008 embodiments, the donor polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence.
  • a donor polynucleotide When optimally aligned, a donor polynucleotide might overlap with one or more nucleotides of a target sequences (e.g. about or more than about 1, 5, 10, 15, 20, or more nucleotides). In some embodiments, when a donor sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the donor polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence. [00164] In prokaryotic hosts such as E.
  • homologous gene replacements can be effected with bacteriophage l Red homologous recombination systems which comprise a bacteriophage l exonuclease, a bacteriophage l Beta protein, a single-stranded DNA annealing protein (SSAP) which facilitates annealing of complementary DNA strands, and a DNA donor (Murphy, 2016).
  • Bacteriophage l Red homologous recombination systems have been combined with CRISPR-Cas9 systems in prokaryotes to effect recombination at target sequences in bacterial genomes (Jiang et al., 2013; Wang et al., 2016).
  • these aspects employ a single stranded DNA annealing protein (SSAP) system.
  • SSAPs include RecT, Red ⁇ , ERF and Rad52.
  • the system or composition comprises: a nucleic acid molecule comprising a guide RNA sequence that is complementary to a target DNA sequence and a recombination protein .
  • the system or composition is capable of promoting R-loop formation .
  • the system or composition is capable of recombination.
  • the system or composition is free of CRISPR proteins.
  • the recombination protein comprises a microbial recombination protein.
  • the recombination protein comprises a viral recombination protein.
  • the recombination protein comprises a eukaryotic recombination protein.
  • the recombination protein comprises a mitochondrial recombination protein .
  • the recombination protein comprises a single stranded DNA annealing protein (SSAP), a nuclease, or a combination of two or more thereof.
  • the system or composition does not comprise a Cas9.
  • the system or composition does not comprise a Cas. In certain embodiments, the system or composition does not comprise a CRISPR. D M2 ⁇ 19143850.1 45 PATENT Docket No. J0217-99008 [00167]
  • the recombination protein may be functionally linked as a fusion protein or chimera or chimeric molecule to a nuclease. In some embodiments, the recombination protein may be functionally linked as a fusion protein or chimera or chimeric molecule to an endonuclease. In some embodiments, the recombination protein may be functionally linked as a fusion protein or chimera or chimeric molecule to an exonuclease.
  • the recombination protein may be functionally linked as a fusion protein or chimera or chimeric molecule to a nuclease and/or a Cas or dCas. In some embodiments, the recombination protein may be functionally linked as a fusion protein or chimera or chimeric molecule to a endonuclease and/or a Cas or dCas. In some embodiments, the recombination protein may be functionally linked as a fusion protein or chimera or chimeric molecule to a exonuclease and/or a Cas or dCas.
  • the recombination protein may be expressed independently from, not a fusion protein with a nuclease. In some embodiments, the recombination protein may be expressed independently from, not a fusion protein with an endonuclease. In some embodiments, the recombination protein may be expressed independently from, not a fusion protein with an exonuclease. In some embodiments, the recombination protein may be expressed independently from, not a fusion protein with a nuclease and/or a Cas or dCas.
  • the recombination protein may be functionally linked as a fusion protein or chimera or chimeric molecule to an aptamer and/or aptamer binding protein. In some embodiments, the recombination protein may be expressed independently, not as a fusion protein, with an aptamer and/or aptamer binding protein. In some embodiments, the recombination protein may be functionally linked as a fusion protein or chimera or chimeric molecule to a nuclease and/or Cas or dCas and/or to an aptamer and/or aptamer binding protein.
  • the recombination protein may be expressed independently from, not a fusion protein with a nuclease and/or a Cas or dCas and/or an aptamer and/or aptamer binding protein.
  • the aptamer and/or aptamer binding protein is an MCP protein.
  • the recombination protein may be an SSAP.
  • nuclease refers to an agent, such as a protein or small molecule, that is capable of cleaving phosphodiester bonds that join nucleotide residues in a nucleic acid molecule.
  • the nuclease is but woven, e.g., an enzyme that is capable of binding to a nucleic acid molecule and cleaving phosphodiester bonds linking nucleotide residues in the nucleic acid molecule.
  • the nuclease may be an endonuclease, which D M2 ⁇ 19143850.1 46 PATENT Docket No. J0217-99008 cleaves a phosphodiester bond in a polynucleotide strand, or an exonuclease, which cleaves a phosphodiester bond at the end of a polynucleotide strand.
  • the nuclease is a site-specific nuclease that binds to and/or cleaves a particular phosphodiester bond within a particular nucleotide sequence, which is also referred to herein as a "recognition sequence", "nuclease target site", or "target site”.
  • the nuclease is an RNA-guided (i.e., RNA-programmable) nuclease that complexes (e.g., binds) to RNA having a sequence complementary to the target site, thereby providing sequence specificity of the nuclease.
  • the nuclease recognizes a single-stranded target site, while in other embodiments, the nuclease recognizes a double-stranded target site, e.g., a double-stranded DNA target site.
  • Target sites for many naturally occurring nucleases for example many naturally occurring DNA restriction nucleases, are well known to those skilled in the art.
  • DNA nucleases such as EcoRI, HindIII or BamHI recognize palindromic double-stranded DNA target sites that are 4 to 10 base pairs in length and cut each of the two DNA strands at specific positions within the target site.
  • Some endonucleases symmetrically cleave a double-stranded nucleic acid target site, i.e., cleave both strands at the same position, such that the ends comprise base-paired nucleotides, also referred to herein as blunt ends.
  • Other endonucleases cleave double-stranded nucleic acid target sites asymmetrically, i.e., each strand is cleaved at a different position such that the ends contain unpaired nucleotides.
  • Unpaired nucleotides at the ends of a double-stranded DNA molecule are also referred to as "overhangs", e.g., “5 '-overhangs” or “3' -overhangs”, depending on whether the unpaired nucleotide forms the 5 'or 3' end of the corresponding DNA strand.
  • the ends of a double-stranded DNA molecule that terminate in unpaired nucleotides are also referred to as sticky ends, so they can "stick" to the ends of other double-stranded DNA molecules that contain complementary unpaired nucleotides.
  • Nuclease proteins typically comprise a "binding domain” that mediates interaction of the protein with a nucleic acid substrate (in some cases also specifically binding to a target site) and a "cleavage domain” that catalyzes the cleavage of phosphodiester bonds within the nucleic acid backbone.
  • the nuclease protein is capable of binding and cleaving a nucleic acid molecule in a monomeric form, while in other embodiments, the nuclease protein must dimerize or otherwise cleave a target nucleic acid molecule.
  • Binding and cleavage domains of naturally occurring nucleases, as well as mode binding and cleavage domains that can be fused to create nucleases, are well known to those of skill in the art.
  • a zinc finger or transcriptional activator-like element can be used as a D M2 ⁇ 19143850.1 47 PATENT Docket No. J0217-99008 binding domain to specifically bind a desired target site and fused or conjugated to a cleavage domain, such as the cleavage domain of fokl, to create an engineered nuclease that cleaves the target site.
  • exonuclease examples include exonuclease I, exonuclease II, exonnuclease III, exonuclease IV, exonuclease V, exonuclease VII, exonuclease VIII, lambda exonuclease, Xrn1, mung bean nuclease, TREX2, exonuclease T, T7 exonuclease, strandase exonuclease, 3’-5’ exophosphodiesterase, and Bal31 nuclease.
  • the system can be thought of as comprising a guide nucleic acid that promotes R-loop formation by binding to target DNA and a recombination protein that promotes recombination between the target nucleic acids and donor nucleic acids.
  • the guide RNA and the recombination protein are effectively linked .
  • the linkage is covalent.
  • the linkage is noncovalent.
  • the guide nucleic acid comprises and aptamer sequence and the recombination protein comprises or is joined to an aptamer binding domain.
  • the invention involves vectors, e.g.
  • a “vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • a vector is capable of replication when associated with the proper control elements.
  • the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector is another type of vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g.
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • operably linked is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • the vector(s) can include the regulatory element(s), e.g., promoter(s).
  • the vector(s) can comprise Cas encoding sequences, and/or a single, but possibly also can comprise at least 3 or 8 or 16 or 32 or 48 or 50 guide RNA(s) (e.g., sgRNAs) encoding sequences, such as 1-2, 1-3, 1-4 1-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s) (e.g., sgRNAs).
  • guide RNA(s) e.g., sgRNAs
  • a promoter for each RNA there can be a promoter for each RNA (e.g., sgRNA), advantageously when there are up to about 16 RNA(s) (e.g., sgRNAs); and, when a single vector provides for more than 16 RNA(s) (e.g., sgRNAs), one or more promoter(s) can drive expression of more than one of the RNA(s) (e.g., sgRNAs), e.g., when there are 32 RNA(s) (e.g., sgRNAs), each promoter can drive expression of two RNA(s) (e.g., sgRNAs), and when there are 48 RNA(s) (e.g., sgRNAs), each promoter can drive expression of three RNA(s) (e.g., sgRNAs).
  • RNA(s) e.g., sgRNA(s) for a suitable exemplary vector such as D M2 ⁇ 19143850.1 49 PATENT Docket No. J0217-99008 AAV
  • a suitable promoter such as the U6 promoter, e.g., U6-sgRNAs.
  • the packaging limit of AAV is ⁇ 4.7 kb.
  • the length of a single U6-sgRNA (plus restriction sites for cloning) is 361 bp.
  • the skilled person can readily fit about 12-16, e.g., 13 U6-sgRNA cassettes in a single vector.
  • This can be assembled by any suitable means, such as a golden gate strategy used for TALE assembly (www.genome-engineering.org/taleffectors).
  • the skilled person can also use a tandem guide strategy to increase the number of U6- sgRNAs by approximately 1.5 times, e.g., to increase from 12-16, e.g., 13 to approximately 18- 24, e.g., about 19 U6-sgRNAs.
  • promoter-RNAs e.g., U6-sgRNAs
  • a further means for increasing the number of promoters and RNAs, e.g., sgRNA(s) in a vector is to use a single promoter (e.g., U6) to express an array of RNAs, e.g., sgRNAs separated by cleavable sequences.
  • a further means for increasing the number of promoter-RNAs, e.g., sgRNAs in a vector is to express an array of promoter-RNAs, e.g., sgRNAs separated by cleavable sequences in the intron of a coding sequence or gene; and, in this instance it is advantageous to use a polymerase II promoter, which can have increased expression and enable the transcription of long RNA in a tissue specific manner.
  • a polymerase II promoter which can have increased expression and enable the transcription of long RNA in a tissue specific manner.
  • AAV may package U6 tandem sgRNA targeting up to about 50 genes.
  • vector(s) e.g., a single vector, expressing multiple RNAs or guides or sgRNAs under the control or operatively or functionally linked to one or more promoters- especially as to the numbers of RNAs or guides or sgRNAs discussed herein, without any undue experimentation.
  • the guide RNA(s), e.g., sgRNA(s) encoding sequences and/or Cas encoding sequences can be functionally or operatively linked to regulatory element(s) and hence the regulatory element(s) drive expression.
  • the promoter(s) can be constitutive promoter(s) and/or conditional promoter(s) and/or inducible promoter(s) and/or tissue specific promoter(s).
  • the promoter can be selected from the group consisting of RNA polymerases, pol I, pol II, pol III, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) D M2 ⁇ 19143850.1 50 PATENT Docket No. J0217-99008 promoter, the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 ⁇ promoter.
  • An advantageous promoter is the promoter is U6.
  • one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites.
  • a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors.
  • RNA(s) of the CRISPR System can be delivered to a transgenic Cas9 animal or mammal, e.g., an animal or mammal that constitutively or inducibly or conditionally expresses Cas9; or an animal or mammal that is otherwise expressing Cas9 or has cells containing Cas9, such as by way of prior administration thereto of a vector or vectors that code for and express in vivo Cas9.
  • a transgenic Cas9 animal or mammal e.g., an animal or mammal that constitutively or inducibly or conditionally expresses Cas9; or an animal or mammal that is otherwise expressing Cas9 or has cells containing Cas9, such as by way of prior administration thereto of a vector or vectors that code for and express in vivo Cas9.
  • two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector.
  • CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5′ with respect to (“upstream” of) or 3′ with respect to (“downstream” of) a second element.
  • the coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction.
  • a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g.
  • a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In some embodiments, one or more insertion sites (e.g.
  • a vector comprises an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell.
  • a vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site.
  • the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these.
  • a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell.
  • a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences.
  • a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein.
  • CRISPR enzyme or CRISPR enzyme mRNA or CRISPR guide RNA or RNA(s) can be delivered separately; and advantageously at least one of these is delivered via a nanoparticle complex.
  • CRISPR enzyme mRNA can be delivered prior to the guide RNA to give time for CRISPR enzyme to be expressed.
  • CRISPR enzyme mRNA might be administered 1-12 hours (preferably around 2-6 hours) prior to the administration of guide RNA.
  • CRISPR enzyme mRNA and guide RNA can be administered together.
  • a second booster dose of guide RNA can be administered 1-12 hours (preferably around 2-6 hours) after the initial administration of CRISPR enzyme mRNA+guide RNA. Additional administrations of CRISPR enzyme mRNA and/or guide RNA might be useful to achieve the most efficient levels of genome modification.
  • the invention provides methods for using one or more elements of a CRISPR system.
  • the CRISPR complex of the invention provides an effective means for modifying a target polynucleotide.
  • the CRISPR complex of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in a multiplicity of cell types.
  • the CRISPR complex of the invention has a D M2 ⁇ 19143850.1 52 PATENT Docket No. J0217-99008 broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis.
  • An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide.
  • the guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.
  • this invention provides a method of cleaving a target polynucleotide.
  • the method comprises modifying a target polynucleotide using a CRISPR complex that binds to the target polynucleotide and effect cleavage of said target polynucleotide.
  • the CRISPR complex of the invention when introduced into a cell, creates a break (e.g., a single or a double strand break) in the genome sequence.
  • the method can be used to cleave a disease gene in a cell.
  • the break created by the CRISPR complex can be repaired by a repair processes such as the error prone non- homologous end joining (NHEJ) pathway or the high fidelity homology-directed repair (HDR).
  • NHEJ error prone non- homologous end joining
  • HDR high fidelity homology-directed repair
  • an exogenous polynucleotide donor can be introduced into the genome sequence.
  • the HDR process is used modify genome sequence.
  • an exogenous polynucleotide donor comprising a sequence to be integrated flanked by an upstream sequence and a downstream sequence is introduced into a cell.
  • the upstream and downstream sequences share sequence similarity with either side of the site of integration in the chromosome.
  • a donor polynucleotide can be DNA, e.g., a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • the exogenous polynucleotide donor comprises a sequence to be integrated (e.g., a mutated gene).
  • the sequence for integration may be a sequence endogenous or exogenous to the cell.
  • sequences to be integrated examples include polynucleotides encoding a protein or a non- coding RNA (e.g., a microRNA).
  • the sequence for integration may be operably linked to an appropriate control sequence or sequences.
  • the sequence to be integrated may provide a regulatory function.
  • the upstream and downstream sequences in the exogenous polynucleotide donor are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide.
  • the upstream sequence is a nucleic acid sequence that shares sequence similarity with the genome sequence upstream of the targeted site for integration.
  • the downstream sequence is a nucleic acid sequence that shares sequence similarity with D M2 ⁇ 19143850.1 53 PATENT Docket No. J0217-99008 the chromosomal sequence downstream of the targeted site of integration.
  • the upstream and downstream sequences in the exogenous polynucleotide donor can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the targeted genome sequence.
  • the upstream and downstream sequences in the exogenous polynucleotide donor have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the targeted genome sequence.
  • the upstream and downstream sequences in the exogenous polynucleotide donor have about 99% or 100% sequence identity with the targeted genome sequence.
  • An upstream or downstream sequence may comprise from about 20 bp to about 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp.
  • the exemplary upstream or downstream sequence have about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000 bp.
  • the exogenous polynucleotide donor may further comprise a marker.
  • a marker may make it easy to screen for targeted integrations. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers.
  • the exogenous polynucleotide donor of the invention can be constructed using recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996).
  • a double stranded break is introduced into the genome sequence by the CRISPR complex, the break is repaired via homologous recombination an exogenous polynucleotide donor such that the donor is integrated into the genome.
  • the presence of a double- stranded break facilitates integration of the donor.
  • this invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell. The method comprises increasing or decreasing expression of a target polynucleotide by using a CRISPR complex that binds to the polynucleotide.
  • a target polynucleotide can be inactivated to effect the modification of the expression in a cell. For example, upon the binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the coded protein is not produced, or the sequence does not function as the wild-type sequence does.
  • a protein or microRNA coding sequence may be inactivated such that the protein or microRNA or pre-microRNA transcript is not produced.
  • a control sequence can be inactivated such that it no longer functions as a control sequence. As used herein, D M2 ⁇ 19143850.1 54 PATENT Docket No.
  • control sequence refers to any nucleic acid sequence that effects the transcription, translation, or accessibility of a nucleic acid sequence.
  • Examples of a control sequence include, a promoter, a transcription terminator, and an enhancer are control sequences.
  • the target polynucleotide of a CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell.
  • the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell.
  • the target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA).
  • a gene product e.g., a protein
  • a non-coding sequence e.g., a regulatory polynucleotide or a junk DNA.
  • target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway-associated gene or polynucleotide.
  • target polynucleotides include a disease associated gene or polynucleotide.
  • a “disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non-disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease.
  • a disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease.
  • the transcribed or translated products may be known or unknown, and may be at a normal or abnormal level.
  • the target polynucleotide of a CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell.
  • the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell.
  • the target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA).
  • the target polynucleotide of a CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell.
  • the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell.
  • the target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA).
  • the target can be a control element or a regulatory element or a promoter or an enhancer or a silencer.
  • the promoter may, in some embodiments, be in the region of +200 bp or even +1000 bp from the TTS.
  • the regulatory region may be an enhancer. D M2 ⁇ 19143850.1 55 PATENT Docket No. J0217-99008
  • the enhancer is typically more than +1000 bp from the TTS. More in particular, expression of eukaryotic protein-coding genes generally is regulated through multiple cis-acting transcription- control regions. Some control elements are located close to the start site (promoter-proximal elements), whereas others lie more distant (enhancers and silencers) Promoters determine the site of transcription initiation and direct binding of RNA polymerase II.
  • promoter- proximal elements occur within ⁇ 200 base pairs of the start site. Several such elements, containing up to ⁇ 20 base pairs, may help regulate a particular gene. Enhancers, which are usually ⁇ 100-200 base pairs in length, contain multiple 8- to 20-bp control elements. They may be located from 200 base pairs to tens of kilobases upstream or downstream from a promoter, within an intron, or downstream from the final exon of a gene.
  • Promoter-proximal elements and enhancers may be cell-type specific, functioning only in specific differentiated cell types. However, any of these regions can be the target sequence and are encompassed by the concept that the target can be a control element or a regulatory element or a promoter or an enhancer or a silencer.
  • the target sequence should be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognized by the CRISPR complex.
  • PAM protospacer adjacent motif
  • the precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence).
  • the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell.
  • the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or D M2 ⁇ 19143850.1 56 PATENT Docket No. J0217-99008 decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro.
  • the method comprises sampling a cell or population of cells from a human or non-human animal, and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant. For re-introduced cells it is particularly preferred that the cells are stem cells.
  • the CRISPR complex may comprise a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence, wherein said guide sequence may be linked to a tracr mate sequence which in turn may hybridize to a tracr sequence.
  • the invention relates to the engineering and optimization of systems, methods and compositions used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that relate to the CRISPR-Cas system and components thereof.
  • the Cas enzyme is Cas9.
  • nucleic acid molecule(s) encoding CRISPR-Cas9 or an ortholog or homolog thereof may be codon-optimized for expression in a eukaryotic cell.
  • a eukaryote can be as herein discussed.
  • Nucleic acid molecule(s) can be engineered or non-naturally occurring.
  • the CRISPR-Cas9 effector protein may comprise one or more mutations. The mutations may be artificially introduced mutations and may include but are not limited to one or more mutations in a catalytic domain, to provide a nickase, for example.
  • catalytic domains with reference to a Cas enzyme may include but are not limited to RuvC I, RuvC II, RuvC III, and HNH domains.
  • the CRISPR-Cas9 effector protein may be used as a generic nucleic acid binding protein with fusion to or being operably linked to a functional domain.
  • Exemplary functional domains may include but are not limited to translational initiator, translational activator, D M2 ⁇ 19143850.1 57 PATENT Docket No.
  • the CRISPR-Cas9 effector protein may have cleavage activity.
  • the Cas9 effector protein may direct cleavage of one or both nucleic acid strands at the location of or near a target sequence, such as within the target sequence and/or within the complement of the target sequence or at sequences associated with the target sequence.
  • the Cas9 effector protein may direct cleavage of one or both DNA or RNA strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • the cleavage may be blunt, i.e., generating blunt ends.
  • the cleavage may be staggered, i.e., generating sticky ends.
  • the cleavage may be a staggered cut with a 5′ overhang, e.g., a 5′ overhang of 1 to 5 nucleotides.
  • the cleavage may be a staggered cut with a 3′ overhang, e.g., a 3′ overhang of 1 to 5 nucleotides.
  • a vector encodes a nucleic acid-targeting Cas protein that may be mutated with respect to a corresponding wild-type enzyme such that the mutated nucleic acid-targeting Cas protein lacks the ability to cleave one or both DNA or RNA strands of a target polynucleotide containing a target sequence.
  • two or more catalytic domains of Cas may be mutated to produce a mutated Cas substantially lacking all RNA cleavage activity.
  • corresponding catalytic domains of a Cas9 effector protein may also be mutated to produce a mutated Cas9 lacking all DNA cleavage activity or having substantially reduced DNA cleavage activity.
  • a nucleic acid-targeting effector protein may be considered to substantially lack all RNA cleavage activity when the RNA cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the nucleic acid cleavage activity of the non-mutated form of the enzyme; an example can be when the nucleic acid cleavage activity of the mutated form is nil or negligible as compared with the non-mutated form.
  • An effector protein may be identified with reference to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the Type II CRISPR system.
  • the effector protein is a Type II protein such as Cas9.
  • a Type II protein such as Cas9.
  • Applicants mean that the derived enzyme is largely based, in the sense of having a high degree of D M2 ⁇ 19143850.1 58 PATENT Docket No. J0217-99008 sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as known in the art or as described herein.
  • Cas9 may be constitutively present or inducibly present or conditionally present or administered or delivered. Cas9 optimization may be used to enhance function or to develop new functions, one can generate chimeric Cas proteins. And Cas may be used as a generic nucleic acid binding protein.
  • nucleic acid-targeting complex comprising a guide RNA hybridized to a target sequence and complexed with one or more nucleic acid-targeting effector proteins
  • cleavage of one or both DNA or RNA strands in or near e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from
  • sequence(s) associated with a target locus of interest refers to sequences near the vicinity of the target sequence (e.g.
  • a codon optimized sequence is in this instance a sequence optimized for expression in a eukaryote, e.g., humans (i.e.
  • codon optimizing coding nucleic acid molecule(s), especially as to effector protein e.g., Cas9 is within the ambit of the skilled artisan). Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species other than human, or for codon optimization for specific organs is known.
  • an enzyme coding sequence encoding a DNA-targeting Cas protein is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate.
  • processes for modifying the germ line genetic identity of human beings and/or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes may be excluded.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • codons e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons
  • Various species exhibit particular bias for certain codons of a particular amino acid.
  • Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules.
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis.
  • genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways.
  • codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.), are also available.
  • one or more codons e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons in a sequence encoding a DNA/RNA-targeting Cas protein corresponds to the most frequently used codon for a particular amino acid.
  • the invention provides methods for using one or more elements of a nucleic acid-targeting system.
  • the nucleic acid-targeting complex of the invention provides an effective means for modifying a target DNA (double stranded, linear or super-coiled).
  • the nucleic acid-targeting complex of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target DNA in a multiplicity of cell types.
  • modifying e.g., deleting, inserting, translocating, inactivating, activating
  • a target DNA in a multiplicity of cell types.
  • the nucleic acid-targeting complex of the invention has a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis.
  • An exemplary nucleic acid-targeting complex comprises a DNA targeting effector protein complexed with a guide RNA hybridized to a target sequence within the target locus of interest.
  • the method may comprise allowing a nucleic acid-targeting complex to bind to the target DNA and thereby modifying the target DNA, wherein the nucleic D M2 ⁇ 19143850.1 60 PATENT Docket No. J0217-99008 acid-targeting complex comprises a nucleic acid-targeting effector protein complexed with a guide RNA hybridized to a target sequence within said target DNA or RNA.
  • the invention provides a method of modifying expression of DNA in a eukaryotic cell.
  • the method comprises allowing a nucleic acid-targeting complex to bind to the DNA such that said binding results in increased or decreased expression of said DNA; wherein the nucleic acid-targeting complex comprises a nucleic acid-targeting effector protein complexed with a guide RNA.
  • the invention provides for methods of modifying a target DNA in a eukaryotic cell, which may be in vivo, ex vivo or in vitro.
  • the method comprises sampling a cell or population of cells from a human or non- human animal, and modifying the cell or cells. Culturing may occur at any stage ex vivo.
  • the cell or cells may even be re-introduced into the non-human animal or plant.
  • the cells are stem cells.
  • the invention relates to the engineering and optimization of systems, methods and compositions used for the control of gene expression involving DNA or RNA sequence targeting, that relate to the nucleic acid-targeting system and components thereof.
  • An advantage of the present methods is that the CRISPR system minimizes or avoids off-target binding and its resulting side effects.
  • TALEs CRISPR-Cas system
  • novel CRISPR systems described herein or components thereof or nucleic acid molecules thereof (including, for instance HDR donor) or nucleic acid molecules encoding or providing components thereof may be delivered by a delivery system herein described both generally and in detail.
  • Vector delivery e.g., plasmid, viral delivery:
  • the CRISPR enzyme for instance a Cas9, and/or any of the present RNAs, for instance a guide RNA, can be delivered using any suitable vector, e.g., plasmid or viral vectors, such as adeno associated virus (AAV), lentivirus, adenovirus or other viral vector types, or combinations thereof.
  • Cas9 and one or more guide RNAs can be packaged into one or more vectors, e.g., plasmid or viral vectors.
  • the vector e.g., plasmid or viral vector is delivered to the tissue of interest by, for example, an intramuscular D M2 ⁇ 19143850.1 61 PATENT Docket No. J0217-99008 injection, while other times the delivery is via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery may be either via a single dose, or multiple doses.
  • the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choice, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc.
  • Such a dosage may further contain, for example, a carrier (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, a pharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), a pharmaceutically-acceptable excipient, and/or other compounds known in the art.
  • a carrier water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.
  • a pharmaceutically-acceptable carrier e.g., phosphate-buffered saline
  • a pharmaceutically-acceptable excipient e.g., phosphate-buffered saline
  • the dosage may further contain one or more pharmaceutically acceptable salts such as, for example, a mineral acid salt such as a hydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and the salts of organic acids such as acetates, propionates, malonates, benzoates, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, gels or gelling materials, flavorings, colorants, microspheres, polymers, suspension agents, etc. may also be present herein.
  • one or more other conventional pharmaceutical ingredients such as preservatives, humectants, suspending agents, surfactants, antioxidants, anticaking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc. may also be present, especially if the dosage form is a reconstitutable form.
  • suitable exemplary ingredients include microcrystalline cellulose, carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin and a combination thereof.
  • the delivery is via an adenovirus, which may be at a single booster dose containing at least 1 ⁇ 10 5 particles (also referred to as particle units, pu) of adenoviral vector.
  • the dose is at least about 1 ⁇ 10 6 particles (for example, about 1 ⁇ 10 6 -1 ⁇ 10 12 particles), at least about 1 ⁇ 10 7 particles, at least about 1 ⁇ 10 8 particles (e.g., about 1 ⁇ 10 8 -1 ⁇ 10 11 particles or about 1 ⁇ 10 8 -1 ⁇ 10 12 particles), and at least about 1 ⁇ 10 0 particles (e.g., about 1 ⁇ 10 9 -1 ⁇ 10 10 particles or about 1 ⁇ 10 9 -1 ⁇ 10 12 particles), or even at least about D M2 ⁇ 19143850.1 62 PATENT Docket No. J0217-99008 1 ⁇ 10 10 particles (e.g., about 1 ⁇ 10 10 -1 ⁇ 10 12 particles) of the adenoviral vector.
  • 1 ⁇ 10 6 particles for example, about 1 ⁇ 10 6 -1 ⁇ 10 12 particles
  • at least about 1 ⁇ 10 7 particles at least about 1 ⁇ 10 8 particles (e.g., about 1 ⁇ 10 8 -1 ⁇ 10 11 particles or about 1 ⁇ 10 8 -1 ⁇ 10 12 particles)
  • the dose comprises no more than about 1 ⁇ 10 14 particles, no more than about 1 ⁇ 10 13 particles, no more than about 1 ⁇ 10 12 particles, no more than about 1 ⁇ 10 11 particles, no more than about 1 ⁇ 10 10 particles (e.g., no more than about 1 ⁇ 10 9 articles).
  • the dose may contain a single dose of adenoviral vector with, for example, about 1 ⁇ 10 6 particle units (pu), about 2 ⁇ 10 6 pu, about 4 ⁇ 10 6 pu, about 1 ⁇ 10 7 pu, about 2 ⁇ 10 7 pu, about 4 ⁇ 10 7 pu, about 1 ⁇ 10 8 pu, about 2 ⁇ 10 8 pu, about 4 ⁇ 10 8 pu, about 1 ⁇ 10 9 pu, about 2 ⁇ 10 9 pu, about 4 ⁇ 10 9 pu, about 1 ⁇ 10 10 pu, about 2 ⁇ 10 10 pu, about 4 ⁇ 10 10 pu, about 1 ⁇ 10 11 pu, about 2 ⁇ 10 11 pu, about 4 ⁇ 10 11 pu, about 1 ⁇ 10 12 pu, about 2 ⁇ 10 12 pu, or about 4 ⁇ 10 12 pu of adenoviral vector.
  • adenoviral vector with, for example, about 1 ⁇ 10 6 particle units (pu), about 2 ⁇ 10 6 pu, about 4 ⁇ 10 6 pu, about 1 ⁇ 10 7 pu, about 2 ⁇ 10 7 pu, about 4 ⁇ 10 7 pu, about 1 ⁇ 10 8 pu, about 2 ⁇ 10 8 pu, about 4 ⁇ 10
  • the adenovirus is delivered via multiple doses.
  • the delivery is via an AAV.
  • a therapeutically effective dosage for in vivo delivery of the AAV to a human is believed to be in the range of from about 20 to about 50 ml of saline solution containing from about 1 ⁇ 10 10 to about 1 ⁇ 10 10 functional AAV/ml solution. The dosage may be adjusted to balance the therapeutic benefit against any side effects.
  • the AAV dose is generally in the range of concentrations of from about 1 ⁇ 10 5 to 1 ⁇ 10 50 genomes AAV, from about 1 ⁇ 10 8 to 1 ⁇ 10 20 genomes AAV, from about 1 ⁇ 10 10 to about 1 ⁇ 10 16 genomes, or about 1 ⁇ 10 11 to about 1 ⁇ 10 16 genomes AAV.
  • a human dosage may be about 1 ⁇ 10 13 genomes AAV.
  • concentrations may be delivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of a carrier solution.
  • Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. See, for example, U.S. Pat.
  • the delivery is via a plasmid.
  • the dosage should be a sufficient amount of plasmid to elicit a response.
  • suitable quantities of plasmid DNA in plasmid compositions can be from about 0.1 to about 2 mg, or from about 1 ⁇ g to about 10 ⁇ g per 70 kg individual.
  • Plasmids of the invention will generally comprise (i) a promoter; (ii) a sequence encoding a CRISPR enzyme, operably linked to said promoter; (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator downstream of D M2 ⁇ 19143850.1 63 PATENT Docket No. J0217-99008 and operably linked to (ii).
  • the plasmid can also encode the RNA components of a CRISPR complex, but one or more of these may instead be encoded on a different vector.
  • the doses herein are based on an average 70 kg individual.
  • RNA molecules of the invention are delivered in liposome or lipofectin formulations and the like and can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos.5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference.
  • RNA delivery is a useful method of in vivo delivery. It is possible to deliver Cas9 and gRNA (and, for instance, HR repair donor) into cells using liposomes or nanoparticles.
  • delivery of the CRISPR enzyme, such as a Cas9 and/or delivery of the RNAs of the invention may be in RNA form and via microvesicles, liposomes or nanoparticles.
  • Cas9 mRNA and gRNA can be packaged into liposomal particles for delivery in vivo.
  • RNA molecules into the liver.
  • Means of delivery of RNA also envisioned include delivery of RNA via nanoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y., Bogatyrev, S., Langer, R.
  • exosome-mediated delivery of siRNA in vitro and in vivo (“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc. 2012 December; 7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012 Nov.15.) describe how exosomes are promising tools for drug delivery across different biological barriers and can be harnessed for delivery of siRNA in vitro and in vivo. Their approach is to generate targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand. The exosomes are then purify and characterized from transfected cell supernatant, then RNA is loaded into the exosomes. Delivery or administration according to the invention can be performed with exosomes, in particular but not limited to the brain.
  • Vitamin E may be conjugated with CRISPR Cas and delivered to the brain along with high density lipoprotein (HDL), for example in a similar manner as was done by Uno et al. (HUMAN GENE THERAPY 22:711-719 (June 2011)) for delivering short-interfering RNA (siRNA) to the brain.
  • Mice were infused via Osmotic minipumps (model 1007D; Alzet, Cupertino, Calif.) filled with phosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE/HDL and connected with Brain Infusion Kit 3 (Alzet).
  • a brain-infusion cannula was placed about 0.5 mm posterior to the bregma at midline for infusion into the dorsal third ventricle.
  • Uno et al. found that as little as 3 nmol of Toc-siRNA with HDL could induce a target reduction in comparable degree by the same ICV infusion method.
  • a similar dosage of CRISPR Cas conjugated to ⁇ -tocopherol and co-administered with HDL targeted to the brain may be contemplated for humans in the present invention, for example, about 3 nmol to about 3 ⁇ mol of CRISPR Cas targeted to the brain may be contemplated. Zou et al.
  • a similar dosage of CRISPR Cas expressed in a lentiviral vector targeted to the brain may be contemplated for humans in the present invention, for example, about 10-50 ml of CRISPR Cas targeted to the brain in a lentivirus having a titer of 1 ⁇ 10 9 transducing units (TU)/ml may be contemplated.
  • Cas9 and one or more guide RNA can be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, U.S. Pat. No. 8,454,972 (formulations, doses for adenovirus), U.S. Pat.
  • the route of administration, formulation and dose can be as in U.S. Pat. No.5,846,946 and as in clinical studies involving plasmids.
  • Doses may be based on or extrapolated to an average 70 kg individual (e.g. a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed.
  • the viral vectors can be injected into the tissue of interest.
  • the expression of Cas9 can be driven by a cell-type specific promoter.
  • liver-specific expression might use the Albumin promoter and neuron-specific expression (e.g. for targeting CNS disorders) might use the Synapsin I promoter.
  • AAV is advantageous over other viral vectors for a couple of reasons: Low toxicity (this may be due to the purification method not requiring ultra- centrifugation of cell particles that can activate the immune response). Low probability of causing insertional mutagenesis because it doesn't integrate into the host genome.
  • the AAV can be AAV1, AAV2, AAV5 or any combination thereof.
  • AAV8 is useful for delivery to the liver.
  • Non-limiting examples of the AAV types that can be used in combination or alone include: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-rh10, AAV-DJ, AAV-DJ/8, AAV-PHP.eB, AAV2-retro, AAV2- QuadYF, AAV2.7m8, AAV2/1, AAV2/5, AAV2/6, AAV2/7, AAV2/8, AAV2/9, AAV PHP.S, AAV2/6.2, AAV2/Rh8.
  • RNAs may be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other viral vector types, or combinations thereof.
  • AAV adeno associated virus
  • lentivirus lentivirus
  • adenovirus lentivirus
  • adenovirus lentivirus
  • the viral vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the viral delivery is via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery may be either via a single dose, or multiple doses.
  • the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector chose, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc. D M2 ⁇ 19143850.1 67 PATENT Docket No.
  • Such a dosage may further contain, for example, a carrier (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, a pharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), a pharmaceutically-acceptable excipient, and/or other compounds known in the art.
  • a carrier water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.
  • a pharmaceutically-acceptable carrier e.g., phosphate-buffered saline
  • a pharmaceutically-acceptable excipient e.g., phosphate-buffered saline
  • the dosage may further contain one or more pharmaceutically acceptable salts such as, for example, a mineral acid salt such as a hydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and the salts of organic acids such as acetates, propionates, malonates, benzoates, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, gels or gelling materials, flavorings, colorants, microspheres, polymers, suspension agents, etc. may also be present herein.
  • one or more other conventional pharmaceutical ingredients such as preservatives, humectants, suspending agents, surfactants, antioxidants, anticaking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc. may also be present, especially if the dosage form is a reconstitutable form.
  • suitable exemplary ingredients include microcrystalline cellulose, carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin and a combination thereof.
  • the delivery is via an adenovirus, which may be at a single booster dose containing at least 1 ⁇ 10 5 particles (also referred to as particle units, pu) of adenoviral vector.
  • the dose is at least about 1 ⁇ 10 6 particles (for example, about 1 ⁇ 10 6 -1 ⁇ 10 12 particles), at least about 1 ⁇ 10 10 particles, at least about 1 ⁇ 10 8 particles (e.g., about 1 ⁇ 10 8 -1 ⁇ 10 11 particles or about 1 ⁇ 10 8 -1 ⁇ 10 12 particles), and at least about 1 ⁇ 10 0 particles (e.g., about 1 ⁇ 10 9 -1 ⁇ 10 10 particles or about 1 ⁇ 10 9 -1 ⁇ 10 12 particles), or even at least about 1 ⁇ 10 10 particles (e.g., about 1 ⁇ 10 10 -1 ⁇ 10 12 particles) of the adenoviral vector.
  • 1 ⁇ 10 6 particles for example, about 1 ⁇ 10 6 -1 ⁇ 10 12 particles
  • at least about 1 ⁇ 10 10 particles at least about 1 ⁇ 10 8 particles (e.g., about 1 ⁇ 10 8 -1 ⁇ 10 11 particles or about 1 ⁇ 10 8 -1 ⁇ 10 12 particles)
  • at least about 1 ⁇ 10 0 particles e.g., about 1 ⁇ 10 9 -1 ⁇ 10 10 particles or about 1 ⁇ 10 9
  • the dose comprises no more than about 1 ⁇ 10 14 particles, no more than about 1 ⁇ 10 13 particles, no more than about 1 ⁇ 10 12 particles, no more than about 1 ⁇ 10 11 particles, and no more than about 1 ⁇ 10 10 particles (e.g., no more than about 1 ⁇ 10 9 articles).
  • the dose may contain a single dose of adenoviral vector with, for example, about 1 ⁇ 10 6 particle units (pu), about 2 ⁇ 10 6 pu, about 4 ⁇ 10 6 pu, about 1 ⁇ 10 7 pu, about 2 ⁇ 10 7 pu, about 4 ⁇ 10 7 pu, about 1 ⁇ 10 8 pu, about 2 ⁇ 10 8 pu, about D M2 ⁇ 19143850.1 68 PATENT Docket No.
  • adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel, et. al., granted on Jun.4, 2013; incorporated by reference herein, and the dosages at col 29, lines 36-58 thereof.
  • the adenovirus is delivered via multiple doses.
  • the delivery is via an AAV.
  • a therapeutically effective dosage for in vivo delivery of the AAV to a human is believed to be in the range of from about 20 to about 50 ml of saline solution containing from about 1 ⁇ 10 10 to about 1 ⁇ 10 10 functional AAV/ml solution. The dosage may be adjusted to balance the therapeutic benefit against any side effects.
  • the AAV dose is generally in the range of concentrations of from about 1 ⁇ 10 5 to 1 ⁇ 10 50 genomes AAV, from about 1 ⁇ 10 8 to 1 ⁇ 10 20 genomes AAV, from about 1 ⁇ 10 10 to about 1 ⁇ 10 16 genomes, or about 1 ⁇ 10 11 to about 1 ⁇ 10 16 genomes AAV.
  • a human dosage may be about 1 ⁇ 10 13 genomes AAV. Such concentrations may be delivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of a carrier solution. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. See, for example, U.S. Pat. No.8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col.27, lines 45-60. [00231] The doses herein are based on an average 70 kg individual. The frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), or scientist skilled in the art.
  • mice used in experiments are about 20 g. From that which is administered to a 20 g mouse, one can extrapolate to a 70 kg individual.
  • the delivery is via a plasmid.
  • the dosage should be a sufficient amount of plasmid to elicit a response.
  • suitable quantities of plasmid DNA in plasmid compositions can be from about 0.1 to about 2 mg, or from about 1 ⁇ g to about 10 ⁇ g.
  • Several types of particle delivery systems and/or formulations are known to be useful in a diverse spectrum of biomedical applications. In general, a particle is defined as a small object that behaves as a whole unit with respect to its transport and properties.
  • Particles are further classified according to diameter Coarse particles cover a range between 2,500 and 10,000 nanometers. Fine particles are sized between 100 and 2,500 nanometers. Ultrafine particles, or D M2 ⁇ 19143850.1 69 PATENT Docket No. J0217-99008 nanoparticles, are generally between 1 and 100 nanometers in size. The basis of the 100-nm limit is the fact that novel properties that differentiate particles from the bulk material typically develop at a critical length scale of under 100 nm. [00234] As used herein, a particle delivery system/formulation is defined as any biological delivery system/formulation which includes a particle in accordance with the present invention. A particle in accordance with the present invention is any entity having a greatest dimension (e.g.
  • inventive particles have a greatest dimension of less than 100 microns ( ⁇ m). In some embodiments, inventive particles have a greatest dimension of less than 10 In some embodiments, inventive particles have a greatest dimension of less than 2000 nanometers (nm). In some embodiments, inventive particles have a greatest dimension of less than 1000 nanometers (nm). In some embodiments, inventive particles have a greatest dimension of less than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. Typically, inventive particles have a greatest dimension (e.g., diameter) of 500 nm or less. In some embodiments, inventive particles have a greatest dimension (e.g., diameter) of 250 nm or less.
  • inventive particles have a greatest dimension (e.g., diameter) of 200 nm or less. In some embodiments, inventive particles have a greatest dimension (e.g., diameter) of 150 nm or less. In some embodiments, inventive particles have a greatest dimension (e.g., diameter) of 100 nm or less. Smaller particles, e.g., having a greatest dimension of 50 nm or less are used in some embodiments of the invention. In some embodiments, inventive particles have a greatest dimension ranging between 25 nm and 200 nm. [00235] Particle characterization (including e.g., characterizing morphology, dimension, etc.) is done using a variety of different techniques.
  • TEM electron microscopy
  • SEM atomic force microscopy
  • AFM dynamic light scattering
  • XPS X-ray photoelectron spectroscopy
  • XRD powder X-ray diffraction
  • FTIR Fourier transform infrared spectroscopy
  • MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • NMR nuclear magnetic resonance
  • Characterization may be made as to native particles (i.e., preloading) or after loading of the cargo (herein cargo refers to one or more RNAs and/or vectors encoding the same, and may include additional components, carriers and/or excipients) to provide particles of an optimal size for delivery for any in vitro, ex vivo and/or in vivo application of the present invention.
  • particle dimension (e.g., diameter) characterization is based on measurements using dynamic laser scattering (DLS). D M2 ⁇ 19143850.1 70 PATENT Docket No.
  • Particles delivery systems within the scope of the present invention may be provided in any form, including but not limited to solid, semi-solid, emulsion, or colloidal particles.
  • any of the delivery systems described herein including but not limited to, e.g., lipid-based systems, liposomes, micelles, microvesicles, exosomes, or gene gun may be provided as particle delivery systems within the scope of the present invention.
  • CRISPR enzyme mRNA and guide RNA may be delivered simultaneously using nanoparticles or lipid envelopes.
  • nanoparticle refers to any particle having a diameter of less than 1000 nm.
  • nanoparticles of the invention have a greatest dimension (e.g., diameter) of 500 nm or less.
  • nanoparticles of the invention have a greatest dimension ranging between 25 nm and 200 nm.
  • nanoparticles of the invention have a greatest dimension of 100 nm or less.
  • nanoparticles of the invention have a greatest dimension ranging between 35 nm and 60 nm.
  • Nanoparticles encompassed in the present invention may be provided in different forms, e.g., as solid nanoparticles (e.g., metal such as silver, gold, iron, titanium), non-metal, lipid- based solids, polymers), suspensions of nanoparticles, or combinations thereof.
  • Metal, dielectric, and semiconductor nanoparticles may be prepared, as well as hybrid structures (e.g., core-shell nanoparticles).
  • Nanoparticles made of semiconducting material may also be labeled quantum dots if they are small enough (typically sub 10 nm) that quantization of electronic energy levels occurs. Such nanoscale particles are used in biomedical applications as drug carriers or imaging agents and may be adapted for similar purposes in the present invention.
  • Nanoparticles with one half hydrophilic and the other half hydrophobic are termed Janus particles and are particularly effective for stabilizing emulsions. They can self-assemble at water/oil interfaces and act as solid surfactants.
  • Su X, Fricke J, Kavanagh D G, Irvine D J In vitro and in vivo mRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles” Mol Pharm. 2011 Jun.
  • nanoparticles based on self assembling bioadhesive polymers are contemplated, which may be applied to oral delivery of peptides, intravenous delivery of peptides and nasal delivery of peptides, all to the brain.
  • Other embodiments, such as oral absorption and ocular deliver of hydrophobic drugs are also contemplated.
  • the molecular envelope technology involves an engineered polymer envelope which is protected and delivered to the site of the disease (see, e.g., Mazza, M. et al. ACSNano, 2013.7(2): 1016-1026; Siew, A., et al. Mol Pharm, 2012. 9(1):14-28; Lalatsa, A., et al.
  • nanoparticles that can deliver RNA to a cancer cell to stop tumor growth developed by Dan Anderson's lab at MIT may be used/and or adapted to the CRISPR Cas system of the present invention.
  • the Anderson lab developed fully automated, combinatorial systems for the synthesis, purification, characterization, and formulation of new biomaterials and nanoformulations.
  • US patent application 20110293703 relates to lipidoid compounds are also particularly useful in the administration of polynucleotides, which may be applied to deliver the CRISPR Cas system of the present invention.
  • the aminoalcohol lipidoid compounds are combined D M2 ⁇ 19143850.1 72 PATENT Docket No. J0217-99008 with an agent to be delivered to a cell or a subject to form microparticles, nanoparticles, liposomes, or micelles.
  • the agent to be delivered by the particles, liposomes, or micelles may be in the form of a gas, liquid, or solid, and the agent may be a polynucleotide, protein, peptide, or small molecule.
  • the minoalcohol lipidoid compounds may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form the particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.
  • US Patent Publication No. 0110293703 also provides methods of preparing the aminoalcohol lipidoid compounds. One or more equivalents of an amine are allowed to react with one or more equivalents of an epoxide-terminated compound under suitable conditions to form an aminoalcohol lipidoid compound of the present invention.
  • all the amino groups of the amine are fully reacted with the epoxide-terminated compound to form tertiary amines. In other embodiments, all the amino groups of the amine are not fully reacted with the epoxide-terminated compound to form tertiary amines thereby resulting in primary or secondary amines in the aminoalcohol lipidoid compound. These primary or secondary amines are left as is or may be reacted with another electrophile such as a different epoxide-terminated compound.
  • amines may be fully functionalized with two epoxide-derived compound tails while other molecules will not be completely functionalized with epoxide-derived compound tails.
  • a diamine or polyamine may include one, two, three, or four epoxide-derived compound tails off the various amino moieties of the molecule resulting in primary, secondary, and tertiary amines. In certain embodiments, all the amino groups are not fully functionalized.
  • two of the same types of epoxide-terminated compounds are used.
  • two or more different epoxide-terminated compounds are used.
  • the synthesis of the aminoalcohol lipidoid compounds is performed with or without solvent, and the synthesis may be performed at higher temperatures ranging from 30.-100 C., preferably at approximately 50.-90 C.
  • the prepared aminoalcohol lipidoid compounds may be optionally purified.
  • the mixture of aminoalcohol lipidoid compounds may be purified to yield an aminoalcohol lipidoid compound with a particular number of epoxide-derived compound tails.
  • the mixture may be purified to yield a particular stereo- or regioisomer.
  • the aminoalcohol lipidoid compounds may also be alkylated using an alkyl halide (e.g., methyl iodide) or other alkylating agent, and/or they may be acylated.
  • US Patent Publication No.0110293703 also provides libraries of aminoalcohol lipidoid compounds prepared by the inventive methods. These aminoalcohol lipidoid compounds may be prepared and/or screened using high-throughput techniques involving liquid handlers, robots, microtiter plates, computers, etc.
  • the aminoalcohol lipidoid compounds are screened for their ability to transfect polynucleotides or other agents (e.g., proteins, peptides, small molecules) into the cell.
  • US Patent Publication No.20130302401 relates to a class of poly(beta-amino alcohols) (PBAAs) has been prepared using combinatorial polymerization.
  • PBAAs poly(beta-amino alcohols)
  • the inventive PBAAs may be used in biotechnology and biomedical applications as coatings (such as coatings of films or multilayer films for medical devices or implants), additives, materials, excipients, non-biofouling agents, micropatterning agents, and cellular encapsulation agents.
  • these PBAAs When used as surface coatings, these PBAAs elicited different levels of inflammation, both in vitro and in vivo, depending on their chemical structures.
  • the large chemical diversity of this class of materials allowed us to identify polymer coatings that inhibit macrophage activation in vitro. Furthermore, these coatings reduce the recruitment of inflammatory cells, and reduce fibrosis, following the subcutaneous implantation of carboxylated polystyrene microparticles. These polymers may be used to form polyelectrolyte complex capsules for cell encapsulation.
  • the invention may also have many other biological applications such as antimicrobial coatings, DNA or siRNA delivery, and stem cell tissue engineering.
  • US Patent Publication No.20130302401 may be applied to the system of the present invention.
  • lipid nanoparticles are contemplated.
  • an antitransthyretin small interfering RNA encapsulated in lipid nanoparticles may be applied to the system of the present invention.
  • Doses of about 0.01 to about 1 mg per kg of body weight administered intravenously are contemplated.
  • Medications to reduce the risk of infusion-related reactions are contemplated, such as dexamethasone, acetampinophen, diphenhydramine or cetirizine, and ranitidine are contemplated.
  • Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200 and colipids D M2 ⁇ 19143850.1 74 PATENT Docket No. J0217-99008 disteroylphosphatidyl choline, cholesterol, and PEG-DMG may be formulated RNA instead of siRNA (see, e.g., Novobrantseva, Molecular Therapy—Nucleic Acids (2012) 1, e4; doi:10.1038/mtna.2011.3) using a spontaneous vesicle formation procedure.
  • the component molar ratio may be about 50/10/38.5/1.5 (DLin-KC2-DMA or C12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG).
  • the final lipid:siRNA weight ratio may be ⁇ 12:1 and 9:1 in the case of DLin-KC2-DMA and C12-200 lipid nanoparticles (LNPs), respectively.
  • the formulations may have mean particle diameters of ⁇ 80 nm with >90% entrapment efficiency. A 3 mg/kg dose may be contemplated.
  • LNPs have been shown to be highly effective in delivering siRNAs to the liver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol.
  • Negatively charged polymers such as siRNA oligonucleotides may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge. However, at physiological pH values, the LNPs exhibit a low surface charge compatible with longer circulation times.
  • ionizable cationic lipids have been focused upon, namely 1,2-dilineoyl-3-dimethylammonium- propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2- D M2 ⁇ 19143850.1 75 PATENT Docket No.
  • DLinKDMA dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane
  • DLinKC2-DMA 1,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane
  • LNP siRNA systems containing these lipids exhibit remarkably different gene silencing properties in hepatocytes in vivo, with potencies varying according to the series DLinKC2- DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII gene silencing model (see, e.g., Rosin et al, Molecular Therapy, vol.19, no.12, pages 1286-2200, December 2011).
  • a dosage of 1 ⁇ g/ml levels may be contemplated, especially for a formulation containing DLinKC2-DMA.
  • Preparation of LNPs and CRISPR Cas encapsulation may be used/and or adapted from Rosin et al, Molecular Therapy, vol.19, no.12, pages 1286-2200, December 2011).
  • the cationic lipids 1,2- dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N- dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3- o-[2′′-(methoxypolyethyleneglycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), and R-3-[(w-
  • Cholesterol may be purchased from Sigma (St Louis, Mo.).
  • the specific CRISPR Cas RNA may be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios).
  • 0.2% SP-DiOC18 Invitrogen, Burlington, Canada
  • Encapsulation may be performed by dissolving lipid mixtures comprised of cationic lipid:DSPC:cholesterol:PEG-c-DOMG (40:10:40:10 molar ratio) in ethanol to a final lipid concentration of 10 mmol/1.
  • This ethanol solution of lipid may be added drop-wise to 50 mmol/1 citrate, pH 4.0 to form multilamellar vesicles to produce a final concentration of 30% ethanol vol/vol.
  • Large unilamellar vesicles may be formed following extrusion of multilamellar vesicles through two stacked 80 nm Nuclepore polycarbonate filters using the Extruder (Northern Lipids, Vancouver, Canada).
  • Encapsulation may be achieved by adding RNA dissolved at 2 mg/ml in 50 mmol/1 citrate, pH 4.0 containing 30% ethanol vol/vol drop-wise to extruded preformed large unilamellar vesicles and incubation at 31° C. for 30 minutes with constant mixing to a final RNA/lipid weight ratio of 0.06/1 wt/wt. Removal of ethanol and neutralization of formulation buffer were performed D M2 ⁇ 19143850.1 76 PATENT Docket No. J0217-99008 by dialysis against phosphate-buffered saline (PBS), pH 7.4 for 16 hours using Spectra/Por 2 regenerated cellulose dialysis membranes.
  • PBS phosphate-buffered saline
  • Nanoparticle size distribution may be determined by dynamic light scattering using a NICOMP 370 particle sizer, the vesicle/intensity modes, and Gaussian fitting (Nicomp Particle Sizing, Santa Barbara, Calif.). The particle size for all three LNP systems may be ⁇ 70 nm in diameter.
  • siRNA encapsulation efficiency may be determined by removal of free siRNA using VivaPureD MiniH columns (Sartorius Stedim Biotech) from samples collected before and after dialysis. The encapsulated RNA may be extracted from the eluted nanoparticles and quantified at 260 nm.
  • siRNA to lipid ratio was determined by measurement of cholesterol content in vesicles using the Cholesterol E enzymatic assay from Wako Chemicals USA (Richmond, Va.).
  • PEGylated liposomes (or LNPs) can also be used for delivery.
  • Preparation of large LNPs may be used/and or adapted from Rosin et al, Molecular Therapy, vol.19, no.12, pages 1286-2200, December 2011.
  • a lipid premix solution (20.4 mg/ml total lipid concentration) may be prepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at 50:10:38.5 molar ratios.
  • Sodium acetate may be added to the lipid premix at a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA).
  • the lipids may be subsequently hydrated by combining the mixture with 1.85 volumes of citrate buffer (10 mmol/1, pH 3.0) with vigorous stirring, resulting in spontaneous liposome formation in aqueous buffer containing 35% ethanol.
  • the liposome solution may be incubated at 37° C. to allow for time-dependent increase in particle size. Aliquots may be removed at various times during incubation to investigate changes in liposome size by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK).
  • the liposomes should their size, effectively quenching further growth.
  • RNA may then be added to the empty liposomes at an siRNA to total lipid ratio of approximately 1:10 (wt:wt), followed by incubation for 30 minutes at 37° C. to form loaded LNPs. The mixture may be subsequently dialyzed overnight in PBS and filtered with a 0.45- ⁇ m syringe filter.
  • Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic D M2 ⁇ 19143850.1 77 PATENT Docket No. J0217-99008 phospholipid bilayer. Liposomes have gained considerable attention as drug delivery carriers because they are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol.
  • BBB blood brain barrier
  • Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Although liposome formation is spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol.2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).
  • liposomes may be added to liposomes in order to modify their structure and properties.
  • either cholesterol or sphingomyelin may be added to the liposomal mixture in order to help stabilize the liposomal structure and to prevent the leakage of the liposomal inner cargo.
  • liposomes are prepared from hydrogenated egg phosphatidylcholine or egg phosphatidylcholine, cholesterol, and dicetyl phosphate, and their mean vesicle sizes were adjusted to about 50 and 100 nm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol.2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).
  • Conventional liposome formulation is mainly comprised of natural phospholipids and lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside. Since this formulation is made up of phospholipids only, liposomal formulations have encountered many challenges, one of the ones being the instability in plasma. Several attempts to overcome these challenges have been made, specifically in the manipulation of the lipid membrane. One of these attempts focused on the manipulation of cholesterol.
  • DSPC 1,2-distearoryl-sn-glycero-3-phosphatidyl choline
  • nucleic acid molecule e.g., DNA, RNA
  • liposomes may be contemplated for in vivo administration in liposomes.
  • the system may be administered in liposomes, such as a stable nucleic-acid-lipid particle (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005).
  • SNALP stable nucleic-acid-lipid particle
  • Daily intravenous injections of about 1, 3 or 5 mg/kg/day of a specific CRISPR Cas targeted in a SNALP are contemplated.
  • the daily treatment may be over about three days and then weekly for about five weeks.
  • a specific CRISPR Cas encapsulated SNALP administered by intravenous injection to at doses of abpit 1 or 2.5 mg/kg are also contemplated (see, e.g., Zimmerman et al., Nature Letters, Vol.441, 4 May 2006).
  • the SNALP formulation may contain the lipids 3-N-[(wmethoxypoly(ethylene glycol) 2000) carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3- aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a 2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al., Nature Letters, Vol.441, 4 May 2006).
  • PEG-C-DMA 1,2-dilinoleyloxy-N,N-dimethyl-3- aminopropane
  • DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine
  • cholesterol in a 2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al.,
  • SNALPs stable nucleic-acid-lipid particles
  • the SNALP liposomes may be prepared by formulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio of Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA.
  • a SNALP may comprise synthetic cholesterol (Sigma- Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, D M2 ⁇ 19143850.1 79 PATENT Docket No.
  • a SNALP may comprise synthetic cholesterol (Sigma- Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG- cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g., Judge, J. Clin. Invest. 119:661-673 (2009)).
  • Formulations used for in vivo studies may comprise a final lipid/RNA mass ratio of about 9:1.
  • DLin-KC2-DMA amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]- dioxolane
  • DLin-KC2-DMA amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]- dioxolane
  • a preformed vesicle with the following lipid composition may be contemplated: amino lipid, di stearoylphosphatidylcholine (DSPC), cholesterol and (R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethylene glycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10, respectively, and a FVII siRNA/total lipid ratio of approximately 0.05 (w/w).
  • the particles may be extruded up to three times through 80 nm membranes prior to adding the CRISPR Cas RNA.
  • Particles containing the highly potent amino lipid 16 may be used, in which the molar ratio of the four lipid components 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) which may be further optimized to enhance in vivo activity.
  • delivery of editing systems or components comprises delivery of a ribonucleoprotein (RNP) complex.
  • RNP ribonucleoprotein
  • targeting nucleic acids including but not limited to gRNAs, dgRNAs can be provided in complexes, such as without limitation, complexes comprising Cas9 or dCas9.
  • an RNP complex comprises a guide nucleic acid and a Cas9 fusion protein, such as without limitation a complex comprising dCas9-SSAP .
  • an RNP complex comprises a guide nucleic acid and a recombination protein, e.g., an SSAP or SSB, which may be adapted or modified to bind to the guide nucleic acid.
  • the guide nucleic acid and the recombination protein or Cas9 fusion protein comprise binding elements that promote complex formation .
  • a recombination protein comprises an MCP domain and a D M2 ⁇ 19143850.1 80 PATENT Docket No. J0217-99008 guide RNA comprises an MS2 aptamer, whereby binding of the MS2 aptamer to the MCP domain produces an RNP.
  • the guide RNA sequence binds to the target genomic DNA sequence in the cell genome
  • the Cas protein associates with the guide RNA and may induce a double strand break or single strand nick in the target genomic DNA sequence and the aptamer recruits the microbial recombination proteins to the target genomic DNA sequence through the aptamer binding protein of the fusion protein, thereby altering the target genomic DNA sequence in the cell.
  • the nucleic acid molecule comprising a guide RNA sequence, the Cas9 protein, and the fusion protein are first expressed in the cell.
  • the cell is in an organism or host, such that introducing the disclosed systems, compositions, vectors into the cell comprises administration to a subject.
  • the method may comprise providing or administering to the subject, in vivo, or by transplantation of ex vivo treated cells, systems, compositions, vectors of the present system.
  • a "subject" may be human or non-human and may include, for example, animal strains or species used as "model systems" for research purposes, such a mouse model as described herein. Likewise, subject may include either adults or juveniles (e.g. , children). Moreover, subject may mean any living organism, preferably a mammal (e.g.
  • human or non-human that may benefit from the administration of compositions contemplated herein.
  • mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
  • non-mammals include, but are not limited to, birds, fish and the like.
  • the mammal is a human.
  • the terms “providing”, “administering,” “introducing,” are used interchangeably herein and refer to the placement of the systems of the invention into a subject by a method or route which results in at least partial localization of the system to a desired site.
  • the systems can be administered by any appropriate route which results in delivery to a desired location in the subject.
  • D M2 ⁇ 19143850.1 81 PATENT Docket No. J0217-99008 [00270]
  • the systems and methods described herein may be used to correct one or more defects or mutations in a gene (referred to as "gene correction").
  • the target genomic DNA sequence encodes a defective version of a gene
  • the system further comprises a donor nucleic acid molecule which encodes a wild-type or corrected version of the gene.
  • the target genomic DNA sequence is a "disease-associated" gene.
  • disease-associated gene refers to any gene or polynucleotide whose gene products are expressed at an abnormal level or in an abnormal form in cells obtained from a disease-affected individual as compared with tissues or cells obtained from an individual not affected by the disease.
  • a disease-associated gene may be expressed at an abnormally high level or at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease.
  • a disease-associated gene also refers to a gene, the mutation or genetic variation of which is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease.
  • genes responsible for such "single gene” or “monogenic” diseases include, but are not limited to, adenosine deaminase, a-1 antitrypsin, cystic fibrosis transmembrane conductance regulator (CFTR), ⁇ -hemoglobin (HBB), oculocutaneous albinism II (OCA2), Huntingtin (HTT), dystrophia myotonica-protein kinase (DMPK), low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), neurofibromin I (NFI), polycystic kidney disease I (PKD I), polycystic kidney disease 2 (PKD2), coagulation factor VIII (F8), dystrophin (DMD), phosphate
  • the invention provides knock-ins of large transgenes at therapeutically relevant loci in the human genome .
  • the locus provides cell or tissue-specific expression.
  • the invention comprises insertion of nucleic acids into the albumin (ALB) locus.
  • ALB locus provides for liver targeting in human hepatocytes, is highly expressed and in a liver-specific manner.
  • the invention comprises insertion of nucleic acids into the AAVSI locus.
  • the AAVSI locus is a safe-harbor locus for gene therapy that is well D M2 ⁇ 19143850.1 82 PATENT Docket No. J0217-99008 expressed in certain tissue types and can be used in a wide variety of treatments, with low expression in liver.
  • US Patent Publication 2018/0214490 Al describes gene therapy for lysosomal storage diseases, including targeting transgenes to safe harbo" loci such as the AAVSI, HPRT and CCR5 genes in human cells, and Rosa26 in murine cells.
  • US Patent 9267154 describes integration of exogenous nucleic acid sequences into the PPPIR12C locus, which is widely expressed in most tissues . describes cell-specific expression by targeting transgenes (e.g., encoding chimeric antigen receptors (CARs)) to the T-cell receptor a constant (TRAC) locus.
  • transgenes e.g., encoding chimeric antigen receptors (CARs)
  • CARs chimeric antigen receptors
  • exemplary and non-limiting loci that can be targeted according to the invention include ALB, HSP90AA1, DYN2TI, ACRB, BCAP31, HIST1H2BK, CLTA, and RABIIA.
  • Exemplary, non-limiting guides for targeting ALB include the following: sgRNA sg3 GTAAATATCTACTAAGACAA id G C G C [00274]
  • An exemplary, non-limiting fusion protein comprising an MCP aptamer binding sequence, linker, and SSAP protein and encoding nucleotide sequence (RNA) is shown in the following table.
  • the SSAP comprises RecT. D M2 ⁇ 19143850.1 83 PATENT Docket No.
  • validation of the genome edit is performed using the screening method Tracking of Indels by Decomposition (TIDE), which uses the trace file generated in Sanger sequencing and a simple algorithm to quantify editing efficiency by insertion and deletion frequency.
  • TIDE Tracking of Indels by Decomposition
  • the disclosed invention is utilized to deliver GLP-1 or a GLP-1 analog to provide therapy for type II diabetes, among others.
  • a GLP-1 analog is defined as a molecule having a modification including one or more amino acid substitutions, deletions, D M2 ⁇ 19143850.1 84 PATENT Docket No. J0217-99008 inversions, or additions when compared with GLP-1.
  • GLP-1 analogs known in the art include, e.g., GLP-1(7-34) and GLP-1(7-35), GLP-1(7-36), Val 8 -GLP-1(7-37), Gln 9 -GLP-1(7-37), D-Gln 9 - GLP-1(7-37), Thr 16 -Lys 18 -GLP-1(7-37), and Lys 18 -GLP-1(7-37), disclosed in U.S. Pat.
  • GLP-1 Glucagon-like peptide-1
  • the invention is utilized to deliver a long-acting GLP-1 analog, such as, but not limited to dulaglutide (also referred to herein as GLP1-IgG4Fc or hGLP1-Protracted) or liraglutide.
  • dulaglutide also referred to herein as GLP1-IgG4Fc or hGLP1-Protracted
  • liraglutide Glucagon-like peptide-1 (GLP-1), which is involved in insulin resistance, regulates insulin secretion in the pancreas.
  • DPP4 dipeptidyl peptidase-4
  • GLP-1 decompose glycoprotein-1
  • sitagliptin, a known DPP4 inhibitor, and the like are used as medicines for treating type 2 diabetes.
  • these medicines must be taken every day, and the use thereof with respect to type 2 diabetes accompanying kidney disorders is limited because of side effects.
  • various other side effects including allergic reactions caused by the medicine have been reported (Lenski M et al., “Effects of DPP-4 inhibition on cardiac metabolism and function in mice.” J. Mol.
  • the disclosed systems and methods overcome challenges encountered during conventional gene editing, including low efficiency and off-target events, particularly with kilobase-scale nucleic acids.
  • the disclosed systems and methods improve the efficiency of gene editing.
  • the disclosed systems and methods can have an increase in efficiency over conventional CRISPR-Cas9 systems and methods, as shown in Example 1 and Figure 17.
  • the improvement in efficiency is accompanied by a reduction in off-target events.
  • Another aspect of increasing the overall accuracy of a gene editing system is reducing the on-target insertion-deletions (indels), a byproduct of HDR editing.
  • kits containing one or more reagents or other components useful, necessary, or sufficient for practicing any of the methods described herein.
  • kits may include CRISPRreagents (Cas protein, guide RNA, vectors, compositions, etc.), recombination reagents (recombination protein-aptamer binding protein fusion protein, the aptamer sequence, vectors, compositions, etc.) transfection or administration reagents, negative and positive control samples (e.g., cells, donor DNA), cells, containers housing one or more D M2 ⁇ 19143850.1 85 PATENT Docket No.
  • Milestone 1 consisted of 6 groups of mice, consisting of 3 mice per group.
  • the experimental design for Milestone 1 is outlined in Figure 1.
  • the test article information for Milestone 1 is outlined in Figure 3.
  • Milestone 2 consisted of 3 groups of mice, consisting of 3 mice per group.
  • the experimental design for Milestone 2 is outlined in Figure 2.
  • the test article information for Milestone 2 is outlined in Figure 4.
  • Reagents used during experimentation are outlined in Figure 5 and instruments used during experimentation are outlined in Figure 6.
  • the AAV was stored at -80°C and thawed on ice.
  • the LNP was stored at 4°C.
  • the AAV and LNP were diluted according to the calculated dosage based on information provided in Figure 8.
  • the diluted AAV and LNP were transferred on ice to the animal facilities. D M2 ⁇ 19143850.1 87 PATENT Docket No. J0217-99008 [00317]
  • the diluted AAV was injected intravenously into mice grouped according to Figure 8.
  • the diluted AAV was injected first, followed by the diluted LNP, 4 hours later.
  • Prior to injection, the diluted LNP was placed at room temperature for 5 minutes and mixed.
  • Animal blood collection [00321] 50 ⁇ L of whole blood was collected from the retro-orbital venous plexus of mice through a 0.3 mm capillary. After standing at 4°C for 30 minutes, the blood was centrifuges at 7500 rpm for 10 min at 4°C. The upper layer of serum was carefully drawn and stored at -80°C. [00322] Milestone 1: The mouse serum was collected in the first and second groups at 2 days, 7 days, and 14 days after administration. [00323] All mice from group 6 of milestone 1 died after administration. [00324] Milestone 2: The mouse serum was collected in group 3 at 2 days, 7 days, and 14 days after administration.
  • mice [00325] Animal liver collection: [00326] On day 14 after administration, the mice were euthanized and livers were harvested. The livers of each mouse was divided into 3 EP tubes for quick freezing with liquid nitrogen and stored at -80°C. [00327] Milestone 1: Livers were collected from the mice in groups 1, 3, 4, and 5. [00328] Milestone 2: Livers were collected from the mice in groups 1, 2, and 3. [00329] Blood GLP-1 measurement (ELISA, EXGLPHS-35K, Millipore): [00330] The dilution factor of the test was carried out according to Figure 9 to ensure all samples were within detection range.

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Abstract

La présente invention concerne des systèmes de modification génétique utilisant des systèmes d'édition de recombinaison guidés par ARN utilisant des enzymes de recombinaison de phage. La manipulation d'une séquence cible implique l'administration d'un système d'édition de recombinaison guidée par ARN avec un système de vecteur viral et un système d'administration à base de lipide possédant une molécule d'acide nucléique avec une séquence d'ARN guide qui est complémentaire d'une séquence d'ADN cible et d'une protéine de recombinaison avec une nucléase, une protéine de recuit d'ADN monocaténaire (SSAP) ou une combinaison de celles-ci pour l'expression dans une cellule.
PCT/US2024/015176 2023-02-10 2024-02-09 Distribution d'un système de recombinaison guidé par arn WO2024168253A1 (fr)

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