WO2016164356A1 - Chemically modified guide rnas for crispr/cas-mediated gene regulation - Google Patents

Chemically modified guide rnas for crispr/cas-mediated gene regulation Download PDF

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Publication number
WO2016164356A1
WO2016164356A1 PCT/US2016/026028 US2016026028W WO2016164356A1 WO 2016164356 A1 WO2016164356 A1 WO 2016164356A1 US 2016026028 W US2016026028 W US 2016026028W WO 2016164356 A1 WO2016164356 A1 WO 2016164356A1
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WIPO (PCT)
Prior art keywords
modified
sgrna
cell
target
nucleotide sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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PCT/US2016/026028
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English (en)
French (fr)
Inventor
Matthew H. PORTEUS
Ayal HENDEL
Joe Clark
Rasmus O. BAK
Daniel E. Ryan
Douglas J. Dellinger
Robert Kaiser
Joel Myerson
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Agilent Technologies Inc
Leland Stanford Junior University
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Agilent Technologies Inc
Leland Stanford Junior University
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First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=55910340&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2016164356(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Priority to EP16720220.9A priority Critical patent/EP3280803B1/en
Priority to CN201680032647.XA priority patent/CN107787367B/zh
Priority to AU2016246450A priority patent/AU2016246450B2/en
Priority to JP2017552447A priority patent/JP6873911B2/ja
Priority to ES16720220T priority patent/ES2884838T3/es
Priority to CA2981715A priority patent/CA2981715A1/en
Priority to CN202111196588.9A priority patent/CN114231527A/zh
Priority to KR1020247008357A priority patent/KR102888521B1/ko
Priority to KR1020177031965A priority patent/KR102648489B1/ko
Application filed by Agilent Technologies Inc, Leland Stanford Junior University filed Critical Agilent Technologies Inc
Publication of WO2016164356A1 publication Critical patent/WO2016164356A1/en
Priority to US15/724,073 priority patent/US11306309B2/en
Anticipated expiration legal-status Critical
Priority to US17/694,336 priority patent/US20220195426A1/en
Priority to US17/694,361 priority patent/US11851652B2/en
Priority to US17/694,310 priority patent/US11535846B2/en
Priority to AU2022204254A priority patent/AU2022204254B2/en
Priority to US18/408,504 priority patent/US20240401034A1/en
Ceased legal-status Critical Current

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Definitions

  • Genome editing with engineered nucleases is a breakthrough technology for modifying essentially any genomic sequence of interest (Porteus, M.H. & Carroll, D., Nature Biotechnology 23, 967-973 (2005)). This technology exploits engineered nucleases to generate site-specific double-strand breaks (DSBs) followed by resolution of DSBs by endogenous cellular repair mechanisms.
  • DSBs site-specific double-strand breaks
  • the outcome can be either mutation of a specific site through mutagenic nonhomologous end-joining (NHEJ), creating insertions or deletions (in/dels) at the site of the break, or precise change of a genomic sequence through homologous recombination (HR) using an exogenously introduced donor template (Hendel et al., Trends in Biotechnology 33, 132-140 (2015).
  • NHEJ nonhomologous end-joining
  • HR homologous recombination
  • CRISPR clustered regularly interspaced palindromic repeat
  • Cas RNA-guided nuclease
  • sgRNA short guide RNA
  • sgRNAs for genome editing can consist of 100 nucleotides (nt) of which 20 nt at the 5' end hybridize to a target DNA sequence by means of Watson-Crick base pairing and guide the Cas endonuclease to cleave the target genomic DNA.
  • the CRISPR/Cas system has also been adapted for sequence-specific control of gene expression, e.g., inhibition or activation of gene expression.
  • target genes can be repressed or activated (Qi et al, Cell, 2013, 152(5): 1173-7783, Perez-Pinera et al, Nat Methods, 2013, 10(10):973-976, Maeder et al, Nat Methods, 2013, 10(10):977-979, Gilbert et al, Cell, 2014, 159:647-661, O'Connell et al., Nature, 2014, 516:263-266).
  • the present invention provides methods for inducing (e.g., initiating, modulating, enhancing, etc.) gene regulation of a target nucleic acid in a cell.
  • the invention includes using modified single guide RNAs (sgRNAs) that enhance genome editing and/or inhibition or activation of gene expression of a target nucleic acid in a primary cell (e.g., cultured in vitro for use in ex vivo therapy) or in a cell in a subject such as a human (e.g., for use in in vivo therapy).
  • the present invention also provides methods for preventing or treating a disease in a subject by enhancing precise genome editing to correct a mutation in a target gene associated with the disease.
  • the present invention can be used with any cell type and at any gene locus that is amenable to nuclease-mediated genome editing technology.
  • the present invention provides a method for inducing gene regulation of a target nucleic acid in a primary cell, the method comprising:
  • a modified single guide RNA comprising a first nucleotide sequence that is complementary to the target nucleic acid and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, wherein one or more of the nucleotides in the first nucleotide sequence and/or the second nucleotide sequence are modified nucleotides; and
  • modified sgRNA guides the Cas polypeptide to the target nucleic acid
  • the present invention provides a method for enhancing genome editing of a target DNA in a primary cell, the method comprising:
  • a modified single guide RNA comprising a first nucleotide sequence that is complementary to the target DNA and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, wherein one or more of the nucleotides in the first nucleotide sequence and/or the second nucleotide sequence are modified nucleotides; and
  • modified sgRNA guides the Cas polypeptide to the target DNA
  • modified sgRNA enhances genome editing of the target DNA (e.g., via increased stability of the modified sgRNA and/or increased specificity of the modified sgRNA for the target DNA) relative to a corresponding unmodified sgRNA.
  • the present invention provides a method for preventing or treating a genetic disease in a subject, the method comprising:
  • sgRNA single guide RNA
  • the modified sgRNA comprises a first nucleotide sequence that is complementary to the target gene and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, and wherein one or more of the nucleotides in the first nucleotide sequence and/or the second nucleotide sequence are modified nucleotides.
  • Cas CRISPR-associated protein
  • FIGS. 1 A-G show that synthesized and chemically modified sgRNAs facilitate high levels of DNA cleavage in vitro and high frequencies of in/dels in a human cell line (K562).
  • FIG. 1 A shows the sequence and schematic of the secondary structure of the IL2RG sgRNA loaded into Cas9 and bound to its genomic target site. Nucleotides with chemical modifications are marked with white flags.
  • FIG. IB depicts structures of chemical modifications incorporated during chemical synthesis of sgRNAs (Table 1 for sequences).
  • FIG. 1C shows gene disruption by mutagenic NUEJ as measured by deep sequencing of PCR amplicons.
  • FIG. ID shows gene addition by HR at the three loci IL2RG, HBB, and CCR5 in K562 cells induced by Cas9 in combination with synthetic sgRNAs. The synthetic sgRNAs were delivered at 1 ⁇ g (light shade) or 20 ⁇ g (dark shade) per 1 million cells.
  • Cas9 was expressed from a plasmid (2 ⁇ g) and for HR experiments 5 ⁇ g of GFP-encoding donor plasmid was included.
  • FIG. IE shows specificity of targeted cleavage mediated by synthetic sgRNAs as performed in FIG. 1C for 20 ⁇ g of sgRNA. In/del frequencies were measured by deep sequencing of PCR amplicons of the targeted genomic loci and three bio
  • Cas9 protein was pre-complexed with a 2.5 molar excess of the indicated synthetic IL2RG sgRNAs and nucleofected into 1 million K562 cells at the indicated amounts.
  • FIGS. 2A-2C show that chemically modified sgRNAs facilitate high rates of gene disruption in primary human T cells and CD34 + hematopoietic stem and progenitor cells (HSPCs).
  • HSPCs hematopoietic stem and progenitor cells
  • 1 million primary human T cells were nucleofected with 10 ⁇ g of the indicated synthetic sgRNAs and either 15 ⁇ g Cas9 mRNA or 1 ⁇ g Cas9-encoding plasmid (FIG. 2A). 1 ⁇ g plasmid encoding both the sgRNA and Cas9 protein was included for comparison.
  • stimulated T cells were nucleofected as above, but with 15 ⁇ g Cas9 protein pre-complexed with a 2.5 molar excess of the indicated synthetic CCR5 sgRNAs.
  • FIG. 2B stimulated T cells were nucleofected as above, but with 15 ⁇ g Cas9 protein pre-complexed with a 2.5 molar excess of the indicated synthetic CCR5 sgRNAs.
  • In/del frequencies were measure by TIDE analysis as above.
  • FIG. 1 Bars represent average in/del
  • FIG. 3 shows Cas9 cleavage of dsDNA targets directed by chemically modified sgRNAs in vitro. Bars indicate percent yield of cleavage products of target DNA fragments (see, FIG. 4) treated with Cas9 protein and sgRNA. Average values +SEM for three independent syntheses of each sgRNA are shown.
  • FIG. 4 shows Cas9 cleavage of dsDNA targets directed by chemically modified sgRNAs in vitro. Cleavage products from biochemical cleavage of dsDNA targets were assayed on DNA 7500 LabChips on a Bioanalyzer 2200. Representative gels are shown for each target, and additional replicates are included in the results plotted in FIG. 3.
  • FIG. 5 illustrates the specificity of targeted cleavage mediated by synthetic sgRNAs. Target specificity was assessed as in FIG. IE using Illumina deep sequencing, but with the samples from FIG. 1C nucleofected with 1 ⁇ g sgRNA.
  • FIG. 7 provides a schematic experimental outline of staggered delivery of sgRNA and Cas9 mRNA. Schematic overview of the experiment yielding data for FIG. 1G. K562 cells were nucleofected at the indicated time points with Cas9 mRNA and/or sgRNAs targeting IL2RG. Genomic DNA was extracted 72 hrs after nucleofection of the last component and in/del frequencies were measured by TIDE using a mock-treated sample as a reference control.
  • FIG. 10 shows high RNA nucleofection efficiencies in primary human T cells. Stimulated T cells from three different donors were nucleofected with GFP mRNA three days after stimulation. Expression of GFP was measured three days after nucleofection by flow cytometry. GFP expression in nucleofected cells (grey) is shown relative to mock-transfected cells (black).
  • FIG. 11 shows that increasing CCR5 sgRNA and Cas9 mRNA amounts in T cell nucleofection yielded similar in/del frequencies.
  • Stimulated T cells were nucleofected with the indicated amounts of the MSP CCR5 sgRNA and Cas9 mRNA.
  • FIG. 12 shows similar in/del frequencies in CD4+, CD8+, and total T cell populations.
  • Stimulated T cells were nucleofected with CCR5 MSP sgRNA and Cas9 mRNA and subsequently sorted into CD4+ and CD8+ subpopulations.
  • FIG. 14 shows lower frequencies of cell death in T cells nucleofected with synthetic sgRNAs and Cas9 mRNA compared to Cas9 plasmid.
  • 1 million stimulated T cells were nucleofected 10 ⁇ g of of the indicated synthetic sgRNAs and either 15 ⁇ g Cas9 mRNA or 1 ⁇ g Cas9-encoding plasmid.
  • 1 ⁇ g of plasmid encoding both the CCR5 sgRNA and Cas9 protein was included for comparison (sgRNA plasmid).
  • Three days after nucleofection, cells were stained with the LIVE/DEAD cell staining. Bars represent average percentages of dead cells for three different T cell donors +SEM, n 6.
  • FIG. 16 shows CCR5 disruption in unstimulated T cells.
  • Unstimulated human T cells from three different donors were nucleofected on the day of isolation with the MS sgRNA and Cas9 mRNA.
  • FIG. 17 illustrates in/del frequencies in mobilized PB CD34+ HSPCs for IL2RG and HBB.
  • genomic DNA was extracted and in/del frequencies were measured by the T7 assay.
  • FIGS. 18A-B show high CCR5 gene modification frequencies in primary human T cells and CD34+ HSPCs using two sgRNAs.
  • Stimulated T cells from three different donors and PB mobilized CD34+ HSPCs were nucleofected in triplicate with both the 'D' and 'Q' sgRNA together with Cas9 mRNA.
  • gDNA was extracted three days after nucleofection and the modified region of CCR5 was PCR-amplified using a pair of primers generating a 2.1kb amplicon for non-modified alleles (FIG. 18 A).
  • PCR amplicons were subcloned into a plasmid for transformation, and individual colonies representing individual alleles were sequenced, referenced against the expected genomic sequence, and categorized according to the allelic genotype (FIG. 18B).
  • FIG. 19 shows that MS-modified sgRNAs perform better than unmodified sgRNAs in CD34+ HSPCs.
  • CD34+ HSPCs were nucleofected with 30 ⁇ g Cas9 protein complexed with a 2.5 molar excess of the indicated synthetic HBB sgRNAs.
  • FIG. 20 shows that modified sgRNAs can be used for efficient multiplexed genome editing.
  • 1 million K562 cells were nucleofected with 15 ⁇ g Cas9 mRNA and either 5 ⁇ g CCR5, HBB, or IL2RG MS-modified sgRNAs or all three sgRNAs (multiplexed) (3x5 ⁇ g).
  • FIG. 21 shows PCR products spanning the CCR5 target site after homologous recombination using chemically modified CCR5 sgRNAs, Cas9 mRNA and a CCR5 ssODN.
  • sgRNAs chemically modified single guide RNAs having enhanced activity during gene regulation ⁇ e.g., genome editing, inhibition of gene expression, and activation of gene expression) compared to corresponding unmodified sgRNAs.
  • the present invention provides methods for gene regulation of a target nucleic acid in a cell by introducing a chemically modified sgRNA that hybridizes to the target nucleic acid together with either a Cas nuclease ⁇ e.g., Cas9 polypeptide) or a variant or fragment thereof, an mRNA encoding a Cas nuclease ⁇ e.g., Cas9 polypeptide) or a variant or fragment thereof, or a recombinant expression vector comprising a nucleotide sequence encoding a Cas nuclease ⁇ e.g., Cas9 polypeptide) or a variant or fragment thereof.
  • the present invention provides methods for preventing or treating a genetic disease in a subject by administering a sufficient amount of the chemically modified sgRNA to correct a genetic mutation associated with the disease.
  • Oligonucleotides that are not commercially available can be chemically synthesized, e.g., according to the solid phase phosphoramidite triester method first described by Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), using an automated synthesizer, as described in Van Devanter et. al, Nucleic Acids Res. 12:6159-6168 (1984). Purification of oligonucleotides is performed using any art-recognized strategy, e.g., native acrylamide gel electrophoresis or anion-exchange high performance liquid chromatography (HPLC) as described in Pearson and Reanier, J. Chrom. 255: 137-149 (1983).
  • HPLC high performance liquid chromatography
  • primary cell refers to a cell isolated directly from a multicellular organism. Primary cells typically have undergone very few population doublings and are therefore more representative of the main functional component of the tissue from which they are derived in comparison to continuous (tumor or artificially immortalized) cell lines. In some cases, primary cells are cells that have been isolated and then used immediately. In other cases, primary cells cannot divide indefinitely and thus cannot be cultured for long periods of time in vitro.
  • the term "genome editing” refers to a type of genetic engineering in which DNA is inserted, replaced, or removed from a target DNA, e.g., the genome of a cell, using one or more nucleases and/or nickases.
  • the nucleases create specific double-strand breaks (DSBs) at desired locations in the genome, and harness the cell's endogenous mechanisms to repair the induced break by homology-directed repair (HDR) (e.g., homologous recombination) or by nonhomologous end joining (NHEJ).
  • HDR homology-directed repair
  • NHEJ nonhomologous end joining
  • two nickases can be used to create two single-strand breaks on opposite strands of a target DNA, thereby generating a blunt or a sticky end.
  • Any suitable nuclease can be introduced into a cell to induce genome editing of a target DNA sequence including, but not limited to, CRISPR-associated protein (Cas) nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, other endo- or exo-nucleases, variants thereof, fragments thereof, and combinations thereof.
  • Cas CRISPR-associated protein
  • ZFNs zinc finger nucleases
  • TALENs transcription activator-like effector nucleases
  • meganucleases other endo- or exo-nucleases, variants thereof, fragments thereof, and combinations thereof.
  • nuclease-mediated genome editing of a target DNA sequence can be "induced” or “modulated” (e.g., enhanced) using the modified single guide RNAs (sgRNAs) described herein in combination with Cas nucleases (e.g., Cas9 polypeptides or Cas9 mRNA), e.g., to improve the efficiency of precise genome editing via homology-directed repair (HDR).
  • sgRNAs modified single guide RNAs
  • Cas nucleases e.g., Cas9 polypeptides or Cas9 mRNA
  • HDR homologous recombination
  • nonhomologous end joining refers to a pathway that repairs double-strand DNA breaks in which the break ends are directly ligated without the need for a homologous template.
  • nucleic acid refers to deoxyribonucleic acids (DNA), ribonucleic acids (RNA) and polymers thereof in either single-, double- or multi-stranded form.
  • the term includes, but is not limited to, single-, double- or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and/or pyrimidine bases or other natural, chemically modified, biochemically modified, non-natural, synthetic or derivatized nucleotide bases.
  • a nucleic acid can comprise a mixture of DNA, RNA and analogs thereof.
  • nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms (SNPs), and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al, J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8:91-98 (1994)).
  • the term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
  • nucleotide analog or “modified nucleotide” refers to a nucleotide that contains one or more chemical modifications (e.g., substitutions), in or on the nitrogenous base of the nucleoside (e.g., cytosine (C), thymine (T) or uracil (U), adenine (A) or guanine (G)), in or on the sugar moiety of the nucleoside (e.g., ribose, deoxyribose, modified ribose, modified deoxyribose, six-membered sugar analog, or open-chain sugar analog), or the phosphate.
  • substitutions e.g., substitutions
  • polypeptide or "nucleotide sequence encoding a polypeptide” means the segment of DNA involved in producing a polypeptide chain.
  • the DNA segment may include regions preceding and following the coding region (leader and trailer) involved in the transcription/translation of the gene product and the regulation of the transcription/translation, as well as intervening sequences (introns) between individual coding segments (exons).
  • polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
  • amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.
  • variable refers to a form of an organism, strain, gene, polynucleotide, polypeptide, or characteristic that deviates from what occurs in nature.
  • complementarity refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or other non- traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • Substantially complementary refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%. 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • stringent conditions refers to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences.
  • Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.
  • Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology -Hybridization With Nucleic Acid Probes Part 1, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, N.Y.
  • hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these.
  • a "recombinant expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell.
  • An expression vector may be part of a plasmid, viral genome, or nucleic acid fragment.
  • an expression vector includes a polynucleotide to be transcribed, operably linked to a promoter.
  • "Operably linked” in this context means two or more genetic elements, such as a polynucleotide coding sequence and a promoter, placed in relative positions that permit the proper biological functioning of the elements, such as the promoter directing transcription of the coding sequence.
  • promoter is used herein to refer to an array of nucleic acid control sequences that direct transcription of a nucleic acid.
  • a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • Other elements that may be present in an expression vector include those that enhance transcription (e.g., enhancers) and terminate transcription (e.g., terminators), as well as those that confer certain binding affinity or antigenicity to the recombinant protein produced from the expression vector.
  • Recombinant refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism.
  • a recombinant polynucleotide or a copy or complement of a recombinant polynucleotide is one that has been manipulated using well known methods.
  • a recombinant expression cassette comprising a promoter operably linked to a second polynucleotide can include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook et al, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)).
  • a recombinant expression cassette (or expression vector) typically comprises polynucleotides in combinations that are not found in nature.
  • recombinant protein is one that is expressed from a recombinant polynucleotide
  • recombinant cells, tissues, and organisms are those that comprise recombinant sequences (polynucleotide and/or polypeptide).
  • single nucleotide polymorphism refers to a change of a single nucleotide with a polynucleotide, including within an allele. This can include the replacement of one nucleotide by another, as well as deletion or insertion of a single nucleotide. Most typically, SNPs are biallelic markers although tri- and tetra-allelic markers can also exist. By way of non-limiting example, a nucleic acid molecule comprising SNP A ⁇ C may include a C or A at the polymorphic position.
  • culture when referring to cell culture itself or the process of culturing, can be used interchangeably to mean that a cell (e.g., primary cell) is maintained outside its normal environment under controlled conditions, e.g., under conditions suitable for survival.
  • Cultured cells are allowed to survive, and culturing can result in cell growth, stasis, differentiation or division. The term does not imply that all cells in the culture survive, grow, or divide, as some may naturally die or senesce.
  • Cells are typically cultured in media, which can be changed during the course of the culture.
  • the terms "subject,” “patient,” and “individual” are used herein interchangeably to include a human or animal.
  • the animal subject may be a mammal, a primate (e.g., a monkey), a livestock animal (e.g., a horse, a cow, a sheep, a pig, or a goat), a companion animal (e.g., a dog, a cat), a laboratory test animal (e.g., a mouse, a rat, a guinea pig, a bird), an animal of veterinary significance, or an animal of economic significance.
  • a primate e.g., a monkey
  • livestock animal e.g., a horse, a cow, a sheep, a pig, or a goat
  • a companion animal e.g., a dog, a cat
  • a laboratory test animal e.g., a mouse, a rat, a guinea pig, a bird
  • administering includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.
  • compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though the disease, condition, or symptom may not have yet been manifested.
  • the term "effective amount” or “sufficient amount” refers to the amount of an agent (e.g., Cas nuclease, modified single guide RNA, etc.) that is sufficient to effect beneficial or desired results.
  • the therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the specific amount may vary depending on one or more of: the particular agent chosen, the target cell type, the location of the target cell in the subject, the dosing regimen to be followed, whether it is administered in combination with other agents, timing of administration, and the physical delivery system in which it is carried.
  • pharmaceutically acceptable carrier refers to a substance that aids the administration of an agent (e.g., Cas nuclease, modified single guide RNA, etc.) to a cell, an organism, or a subject.
  • agent e.g., Cas nuclease, modified single guide RNA, etc.
  • “Pharmaceutically acceptable carrier” refers to a carrier or excipient that can be included in a composition or formulation and that causes no significant adverse toxicological effect on the patient.
  • Non-limiting examples of pharmaceutically acceptable carrier include water, NaCl, normal saline solutions, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors, and the like.
  • increasing stability refers to modifications that stabilize the structure of any molecular component of the CRISPR system.
  • the term includes modifications that decrease, inhibit, diminish, or reduce the degradation of any molecular component of the CRISPR system.
  • increasing specificity refers to modifications that increase the specific activity (e.g., the on-target activity) of any molecular component of the CRISPR system.
  • the term includes modifications that decrease, inhibit, diminish, or reduce the non-specific activity (e.g., the off-target activity) of any molecular component of the CRISPR system.
  • decreasing toxicity refers to modifications that decrease, inhibit, diminish, or reduce the toxic effect of any molecular component of the CRISPR system on a cell, organism, subject, and the like.
  • enhanced activity refers to an increase or improvement in the efficiency and/or the frequency of inducing, modulating, regulating, or controlling genome editing and/or gene expression.
  • the term "about” in relation to a reference numerical value can include a range of values plus or minus 10% from that value.
  • the amount “about 10” includes amounts from 9 to 11, including the reference numbers of 9, 10, and 11.
  • the term “about” in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.
  • the present invention provides methods for inducing gene regulation of a target nucleic acid in a cell.
  • the invention includes using modified single guide RNAs (sgRNAs) that enhance genome editing and/or inhibition or activation of gene expression of a target nucleic acid in a primary cell (e.g., cultured in vitro for use in ex vivo therapy) or in a cell in a subject such as a human (e.g., for use in in vivo therapy).
  • the present invention also provides methods for preventing or treating a disease in a subject by enhancing precise genome editing to correct a mutation in a target gene associated with the disease.
  • the present invention can be used with any cell type and at any gene locus that is amenable to nuclease-mediated genome editing technology.
  • the present invention provides a method for inducing (e.g., initiating, modulating, enhancing, etc.) gene regulation of a target nucleic acid in a primary cell, the method comprising:
  • a modified single guide RNA comprising a first nucleotide sequence that is complementary to the target nucleic acid and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, wherein one or more of the nucleotides in the first nucleotide sequence and/or the second nucleotide sequence are modified nucleotides; and
  • modified sgRNA guides the Cas polypeptide to the target nucleic acid
  • modified sgRNA induces gene regulation of the target nucleic acid with an enhanced activity relative to a corresponding unmodified sgRNA.
  • the enhanced activity comprises increased stability of the modified sgRNA and/or increased specificity of the modified sgRNA for the target nucleic acid.
  • the target nucleic acid comprises a target DNA or a target RNA.
  • Gene regulation of a target nucleic acid encompasses any mechanism used by cells to increase or decrease the production of a specific gene product (e.g., protein or RNA) by the target nucleic acid and includes genome editing of the target nucleic acid or modulation (e.g., inhibition or activation) of gene expression of the target nucleic acid.
  • the gene regulation comprises genome editing of the target DNA.
  • the genome editing can be homologous-directed repair (HDR) or nonhomologous end joining (HEJ) of the target DNA.
  • the gene regulation comprises modulating (e.g., inhibiting or activating) gene expression of the target DNA or the target RNA using an endonuclease- deficient Cas polypeptide.
  • the method further comprises introducing a recombinant donor repair template into the primary cell.
  • the recombinant donor repair template comprises two nucleotide sequences comprising two non-overlapping, homologous portions of the target DNA, wherein the nucleotide sequences are located at the 5' and 3 ' ends of a nucleotide sequence corresponding to the target DNA to undergo genome editing.
  • the recombinant donor repair template comprises a synthetic single- stranded oligodeoxynucleotide (ssODN) template comprising a nucleotide sequence encoding a mutation to correct a single nucleotide polymorphism (SNP) and two nucleotide sequences comprising two non-overlapping, homologous portions of the target DNA, wherein the nucleotide sequences are located at the 5' and 3 ' ends of the nucleotide sequence encoding the mutation.
  • ssODN synthetic single- stranded oligodeoxynucleotide
  • the primary cell is isolated from a multicellular organism prior to introducing the modified sgRNA and the Cas polypeptide into the primary cell.
  • the multicellular organism can be a plant, a multicellular protist, a multicellular fungus, or an animal such as a mammal (e.g., human).
  • the primary cell is selected from the group consisting of a stem cell, an immune cell, and a combination thereof.
  • stem cells include hematopoietic stem and progenitor cells (HSPCs) such as CD34+ HSPCs, mesenchymal stem cells, neural stem cells, organ stem cells, and combinations thereof.
  • Non-limiting examples of immune cells include T cells (e.g., CD3+ T cells, CD4+ T cells, CD8+ T cells, tumor infiltrating cells (TILs), memory T cells, memory stem T cells, effector T cells), natural killer cells, monocytes, peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocytes (PBLs), and combinations thereof.
  • TILs tumor infiltrating cells
  • PBMCs peripheral blood mononuclear cells
  • PBLs peripheral blood lymphocytes
  • the primary cell or a progeny thereof e.g., a cell derived from the primary cell
  • the multicellular organism e.g., human
  • the primary cell comprises a population of primary cells.
  • the modified sgRNA induces gene regulation of the target nucleic acid in at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%), 80%), 85%), 90%), or 95% of the population of primary cells.
  • the population of primary cells comprises at least about 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , or 10 9 primary cells.
  • the gene regulation comprises genome editing (e.g., HDR or HEJ) of the target DNA in the population of primary cells.
  • the gene regulation comprises modulating (e.g., inhibiting or activating) gene expression of the target DNA or the target RNA in the population of primary cells using an endonuclease-deficient Cas polypeptide.
  • the modified sgRNA can induce HDR (e.g., in/del frequencies) in at least about 30%, 35%, 40%, 45%, 50%, 55%, or 60%) of a population of primary T cells after introducing a modified sgRNA into the primary T cells with a Cas polypeptide (e.g., as a ribonucleoprotein (RNP) complex) or an mRNA encoding a Cas polypeptide.
  • a Cas polypeptide e.g., as a ribonucleoprotein (RNP) complex
  • RNP ribonucleoprotein
  • the modified sgRNA can induce HDR (e.g., in/del frequencies) in at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%), 45%), 50%), 55%), or 60%> of a population of primary hematopoietic stem and progenitor cells (HSPCs) after introducing a modified sgRNA into the primary HSPCs with a Cas polypeptide (e.g., as an RNP complex) or an mRNA encoding a Cas polypeptide.
  • HSPCs primary hematopoietic stem and progenitor cells
  • the one or more modified nucleotides in the first nucleotide sequence and/or the second nucleotide sequence of the modified sgRNA comprise a modification in the ribose group, phosphate group, nucleobase, or combinations thereof.
  • the modification in the ribose group can be a modification at the 2' position of the ribose group. In some instances, the modification at the 2' position of the ribose group is selected from the group consisting of 2'-0-methyl, 2'-fluoro, 2'-deoxy, and 2'-0-(2-methoxyethyl).
  • the modification in the phosphate group can be a phosphorothioate modification.
  • the one or more modified nucleotides in the first nucleotide sequence and/or the second nucleotide sequence of the modified sgRNA comprise 2'-0-methyl (M) nucleotides, 2'-0-methyl 3 '-phosphorothioate (MS) nucleotides, 2'-0- methyl 3 '-thioPACE (MSP) nucleotides, or combinations thereof.
  • the modified sgRNA includes one or more MS nucleotides in the first nucleotide sequence and/or the second nucleotide sequence.
  • the modified sgRNA includes one or more MSP nucleotides in the first nucleotide sequence and/or the second nucleotide sequence. In some cases, the modified sgRNA includes one or more MS nucleotides and one or more MSP nucleotides in the first nucleotide sequence and/or the second nucleotide sequence. In some cases, the modified sgRNA does not include M nucleotides in the first nucleotide sequence and/or the second nucleotide sequence. In some cases, the modified sgRNA includes only MS nucleotides and/or MSP nucleotides as the modified nucleotides in the first nucleotide sequence and/or the second nucleotide sequence.
  • the modified sgRNA includes one or more MS nucleotides and/or one or more MSP nucleotides in the first nucleotide sequence and/or the second nucleotide sequence, and may further include one or more M nucleotides in the first nucleotide sequence and/or the second nucleotide sequence.
  • the first nucleotide sequence of the modified sgRNA is about 20 nucleotides in length. In some instances, at least two, three, four, five, six, seven, eight, nine, ten, or more of the nucleotides in the first nucleotide sequence are modified nucleotides. In certain instances, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the nucleotides in the first nucleotide sequence (e.g., a first nucleotide sequence of about 20 nucleotdies in length) are modified nucleotides.
  • nucleotides in the first nucleotide sequence are modified nucleotides.
  • the modified nucleotides are located at the 5'-end (e.g., the terminal nucleotide at the 5 '-end) or near the 5'-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5 '-end) of the first nucleotide sequence and/or at internal positions within the first nucleotide sequence.
  • from about 10% to about 30% of the nucleotides in the first nucleotide sequence are modified nucleotides.
  • the second nucleotide sequence of the modified sgRNA is about 80 nucleotides in length. In some instances, at least two, three, four, five, six, seven, eight, nine, ten, or more of the nucleotides in the second nucleotide sequence are modified nucleotides.
  • nucleotides in the second nucleotide sequence are modified nucleotides.
  • nucleotides in the second nucleotide sequence are modified nucleotides.
  • the modified nucleotides are located at the 3 '-end (e.g., the terminal nucleotide at the 3 '-end) or near the 3 '-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the 3 '- end) of the second nucleotide sequence and/or at internal positions within the second nucleotide sequence.
  • from about 1% to about 10% of the nucleotides in the second nucleotide sequence are modified nucleotides.
  • the modified sgRNA comprises one, two, or three consecutive or non-consecutive modified nucleotides starting at the 5 '-end (e.g., the terminal nucleotide at the 5 '-end) or near the 5 '-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5 '-end) of the first nucleotide sequence and one, two, or three consecutive or non-consecutive modified nucleotides starting at the 3 '-end (e.g., the terminal nucleotide at the 3 '-end) or near the 3 '-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the 3 '- end) of the second nucleotide sequence.
  • the 5 '-end e.g., the terminal nucleotide at the 5 '-end
  • near the 5 '-end e.g., within 1, 2, 3, 4, or 5 nucleo
  • the modified sgRNA comprises one modified nucleotide at the 5'-end (e.g., the terminal nucleotide at the 5'-end) or near the 5'-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5 '-end) of the first nucleotide sequence and one modified nucleotide at the 3 '-end (e.g., the terminal nucleotide at the 3 '-end) or near the 3 '-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the 3 '-end) of the second nucleotide sequence.
  • the 5'-end e.g., the terminal nucleotide at the 5'-end
  • near the 5'-end e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5 '-end
  • the 3 '-end e.g., the terminal nu
  • the modified sgRNA comprises two consecutive or non- consecutive modified nucleotides starting at the 5 '-end (e.g., the terminal nucleotide at the 5'- end) or near the 5'-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5 '-end) of the first nucleotide sequence and two consecutive or non-consecutive modified nucleotides starting at the 3 '-end (e.g., the terminal nucleotide at the 3 '-end) or near the 3 '- end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the 3 '-end) of the second nucleotide sequence.
  • the modified sgRNA comprises three consecutive or non- consecutive modified nucleotides starting at the 5 '-end (e.g., the terminal nucleotide at the 5'- end) or near the 5'-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the terminal nucleotide at the 5 '-end) of the first nucleotide sequence and three consecutive or non-consecutive modified nucleotides starting at the 3 '-end (e.g., the terminal nucleotide at the 3 '-end) or near the 3 '-end (e.g., within 1, 2, 3, 4, or 5 nucleotides of the 3 '-end) of the second nucleotide sequence.
  • the modified sgRNA comprises three consecutive modified nucleotides at the 5 '-end of the first nucleotide sequence and three consecutive modified nucleotides at the 3 '-end of the second nucleotide sequence.
  • the modified sgRNA is chemically synthesized.
  • the method for inducing gene regulation comprises multiplexed gene regulation (e.g., genome editing or modulating gene expression) of a single target nucleic acid sequence or different target nucleic acid sequences using a plurality of modified sgRNAs.
  • the multiplexed gene regulation is more efficient and/or consistent relative to the use of corresponding unmodified sgRNAs.
  • the plurality of modified sgRNAs comprises at least two, three, four, five, ten, fifteen, twenty, or more different modified sgRNAs, wherein each modified sgRNA is directed to a different target nucleic acid. In other instances, the plurality of modified sgRNAs comprises at least two, three, four, five, ten, fifteen, twenty, or more different modified sgRNAs, wherein each modified sgRNA is directed to the same target nucleic acid.
  • the Cas polypeptide is a Cas polypeptide variant or a Cas polypeptide fragment.
  • the Cas polypeptide is a Cas9 polypeptide, a variant thereof, or a fragment thereof.
  • the step of introducing into the primary cell comprises electroporating (e.g., via nucleofection) the primary cell.
  • the present invention provides a method for preventing or treating a genetic disease in a subject, the method comprising:
  • modified single guide RNA in a sufficient amount to correct a mutation in a target gene associated with the genetic disease
  • the modified sgRNA comprises a first nucleotide sequence that is complementary to the target gene and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, and wherein one or more of the nucleotides in the first nucleotide sequence and/or the second nucleotide sequence are modified nucleotides.
  • Cas CRISPR-associated protein
  • the genetic disease is selected from the group consisting of X-linked severe combined immune deficiency, sickle cell anemia, thalassemia, hemophilia, neoplasia, cancer, age-related macular degeneration, schizophrenia, trinucleotide repeat disorders, fragile X syndrome, prion-related disorders, amyotrophic lateral sclerosis, drug addiction, autism, Alzheimer' s disease, Parkinson' s disease, cystic fibrosis, blood and coagulation disease or disorders, inflammation, immune-related diseases or disorders, metabolic diseases, liver diseases and disorders, kidney diseases and disorders, muscular/skeletal diseases and disorders, neurological and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, ocular diseases and disorders, and viral infections (e.g., HIV infection).
  • X-linked severe combined immune deficiency e.g., HIV infection
  • sickle cell anemia thalassemia
  • hemophilia neoplasia
  • cancer age-related macular degeneration
  • schizophrenia tri
  • the method further comprises administering to the subject a Cas polypeptide, an mRNA encoding a Cas polypeptide, and/or a recombinant expression vector comprising a nucleotide sequence encoding a Cas polypeptide.
  • the method further comprises administering to the subject a recombinant donor repair template.
  • the recombinant donor repair template comprises two nucleotide sequences comprising two non-overlapping, homologous portions of the target gene, wherein the nucleotide sequences are located at the 5' and 3 ' ends of a nucleotide sequence corresponding to the target gene to undergo genome editing.
  • the recombinant donor repair template comprises a synthetic single-stranded oligodeoxynucleotide (ssODN) template comprising a nucleotide sequence encoding a mutation to correct a single nucleotide polymorphism (S P) in the target gene, and two nucleotide sequences comprising two non-overlapping, homologous portions of the target gene, wherein the nucleotide sequences are located at the 5' and 3 ' ends of the nucleotide sequence encoding the mutation.
  • ssODN synthetic single-stranded oligodeoxynucleotide
  • administering the modified sgRNA enhances the effect of the Cas polypeptide to correct the mutation in the target gene compared to administering the corresponding unmodified sgRNA.
  • Non-limiting embodiments related to the modified sgRNA used in the method for preventing or treating a genetic disease in a subject are described above.
  • the modified sgRNA, Cas polypeptide, and/or recombinant donor repair template is administered to the subject with a pharmaceutically acceptable carrier.
  • the modified sgRNA, Cas polypeptide, and/or recombinant donor repair template is administered to the subject via a delivery system selected from the group consisting of a nanoparticle, a liposome, a micelle, a virosome, a nucleic acid complex, and a combination thereof.
  • the nucleic acid complex comprises the modified sgRNA complexed with the Cas polypeptide.
  • the modified sgRNA, Cas polypeptide, and/or recombinant donor repair template is administered to the subject via a delivery route selected from the group consisting of oral, intravenous, intraperitoneal, intramuscular, intradermal, subcutaneous, intra-arteriole, intraventricular, intracranial, intralesional, intrathecal, topical, transmucosal, intranasal, and a combination thereof.
  • the CRISPR/Cas system of genome modification includes a Cas nuclease ⁇ e.g., Cas9 nuclease) or a variant or fragment thereof, a DNA-targeting RNA ⁇ e.g., modified sgRNA) containing a guide sequence that targets the Cas nuclease to the target genomic DNA and a scaffold sequence that interacts with the Cas nuclease ⁇ e.g., tracrRNA), and optionally, a donor repair template.
  • a variant of a Cas nuclease such as a Cas9 mutant containing one or more of the following mutations: DIOA, H840A, D839A, and H863A, or a Cas9 nickase can be used.
  • a fragment of a Cas nuclease or a variant thereof with desired properties ⁇ e.g., capable of generating single- or double-strand breaks and/or modulating gene expression) can be used.
  • the donor repair template can include a nucleotide sequence encoding a reporter polypeptide such as a fluorescent protein or an antibiotic resistance marker, and homology arms that are homologous to the target DNA and flank the site of gene modification.
  • the donor repair template can be a single- stranded oligodeoxynucleotide (ssODN).
  • the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated protein) nuclease system is an engineered nuclease system based on a bacterial system that can be used for genome engineering. It is based on part of the adaptive immune response of many bacteria and archea. When a virus or plasmid invades a bacterium, segments of the invader's DNA are converted into CRISPR RNAs (crRNA) by the "immune" response.
  • crRNA CRISPR RNAs
  • the crRNA then associates, through a region of partial complementarity, with another type of RNA called tracrRNA to guide the Cas ⁇ e.g., Cas9) nuclease to a region homologous to the crRNA in the target DNA called a "protospacer.”
  • the Cas e.g., Cas9 nuclease cleaves the DNA to generate blunt ends at the double-strand break at sites specified by a 20-nucleotide guide sequence contained within the crRNA transcript.
  • the Cas (e.g., Cas9) nuclease requires both the crRNA and the tracrRNA for site- specific DNA recognition and cleavage.
  • This system has now been engineered such that the crRNA and tracrRNA can be combined into one molecule (the "single guide RNA” or “sgRNA”), and the crRNA equivalent portion of the single guide RNA can be engineered to guide the Cas (e.g., Cas9) nuclease to target any desired sequence (see, e.g., Jinek et al. (2012) Science, 337:816-821; Jinek et al. (2013) eLife, 2:e00471; Segal (2013) eLife, 2:e00563).
  • the Cas e.g., Cas9 nuclease
  • the CRISPR/Cas system can be engineered to create a double-strand break at a desired target in a genome of a cell, and harness the cell's endogenous mechanisms to repair the induced break by homology-directed repair (HDR) or nonhomologous end-joining (NHEJ).
  • HDR homology-directed repair
  • NHEJ nonhomologous end-joining
  • the Cas nuclease has DNA cleavage activity.
  • the Cas nuclease can direct cleavage of one or both strands at a location in a target DNA sequence.
  • the Cas nuclease can be a nickase having one or more inactivated catalytic domains that cleaves a single strand of a target DNA sequence.
  • Non-limiting examples of Cas nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologs thereof, variants thereof, fragments thereof, mutants thereof, and derivatives thereof.
  • Type II Cas nucleases There are three main types of Cas nucleases (type I, type II, and type III), and 10 subtypes including 5 type I, 3 type II, and 2 type III proteins (see, e.g., Hochstrasser and Doudna, Trends Biochem Sci, 2015:40(l):58-66).
  • Type II Cas nucleases include Casl, Cas2, Csn2, and Cas9. These Cas nucleases are known to those skilled in the art.
  • the amino acid sequence of the Streptococcus pyogenes wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No.
  • NP_269215 and the amino acid sequence of Streptococcus thermophilus wild-type Cas9 polypeptide is set forth, e.g., in NBCI Ref. Seq. No. WP 011681470.
  • CRISPR-related endonucleases that are useful in the present invention are disclosed, e.g., in U.S. Application Publication Nos. 2014/0068797, 2014/0302563, and 2014/0356959.
  • Cas nucleases can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Myco
  • Torquens Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Corynebacterium diphtheria, Elusimicrobium minutum, Nitratifractor salsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp.
  • Jejuni Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perjringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteobacterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.
  • Cas9 refers to an RNA-guided double-stranded DNA-binding nuclease protein or nickase protein. Wild-type Cas9 nuclease has two functional domains, e.g., RuvC and HNH, that cut different DNA strands. Cas9 can induce double-strand breaks in genomic DNA (target DNA) when both functional domains are active.
  • the Cas9 enzyme can comprise one or more catalytic domains of a Cas9 protein derived from bacteria belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter , Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, and Campylobacter.
  • the two catalytic domains are derived from different bacteria species.
  • Useful variants of the Cas9 nuclease can include a single inactive catalytic domain, such as a RuvC " or HNH " enzyme or a nickase.
  • a Cas9 nickase has only one active functional domain and can cut only one strand of the target DNA, thereby creating a single- strand break or nick.
  • the mutant Cas9 nuclease having at least a DIOA mutation is a Cas9 nickase.
  • the mutant Cas9 nuclease having at least a H840A mutation is a Cas9 nickase.
  • a double-strand break can be introduced using a Cas9 nickase if at least two DNA-targeting RNAs that target opposite DNA strands are used.
  • a double-nicked induced double-strand break can be repaired by NHEJ or HDR (Ran et al, 2013, Cell, 154: 1380-1389). This gene editing strategy favors HDR and decreases the frequency of indel mutations at off-target DNA sites.
  • Non-limiting examples of Cas9 nucleases or nickases are described in, for example, U.S. Patent Nos.
  • the Cas9 nuclease or nickase can be codon-optimized for the target cell or target organism.
  • the Cas nuclease can be a Cas9 polypeptide that contains two silencing mutations of the RuvCl and HNH nuclease domains (DIOA and H840A), which is referred to as dCas9 (Jinek et al, Science, 2012, 337:816-821; Qi et al, Cell, 152(5): 1173- 1183).
  • the dCas9 polypeptide from Streptococcus pyogenes comprises at least one mutation at position D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, A987 or any combination thereof.
  • the dCas9 enzyme can contain a mutation at D10, E762, H983 or D986, as well as a mutation at H840 or N863. In some instances, the dCas9 enzyme contains a DIOA or DION mutation. Also, the dCas9 enzyme can include a H840A, H840Y, or H840N. In some embodiments, the dCas9 enzyme of the present invention comprises DIOA and H840A; DIOA and H840Y; DIOA and H840N; DION and H840A; DION and H840Y; or DION and H840N substitutions.
  • the substitutions can be conservative or non-conservative substitutions to render the Cas9 polypeptide catalytically inactive and able to bind to target DNA.
  • the dCas9 polypeptide is catalytically inactive such as defective in nuclease activity.
  • the dCas9 enzyme or a variant or fragment thereof can block transcription of a target sequence, and in some cases, block RNA polymerase. In other instances, the dCas9 enzyme or a variant or fragment thereof can activate transcription of a target sequence.
  • the Cas nuclease can be a Cas9 fusion protein such as a polypeptide comprising the catalytic domain of the type IIS restriction enzyme, Fokl, linked to dCas9.
  • the FokI-dCas9 fusion protein fCas9 can use two guide RNAs to bind to a single strand of target DNA to generate a double-strand break.
  • a nuclease- deficient Cas protein such as but not limited to dCas9
  • Methods of inactivating gene expression using a nuclease-null Cas protein are described, for example, in Larson et al, Nat. Protoc, 2013, 8(11):2180-2196.
  • a nucleotide sequence encoding the Cas nuclease is present in a recombinant expression vector.
  • the recombinant expression vector is a viral construct, e.g., a recombinant adeno-associated virus construct, a recombinant adenoviral construct, a recombinant lentiviral construct, etc.
  • viral vectors can be based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, human immunodeficiency virus, and the like.
  • a retroviral vector can be based on Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, mammary tumor virus, and the like.
  • Useful expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example for eukaryotic host cells: pXTl, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40.
  • any other vector may be used if it is compatible with the host cell.
  • useful expression vectors containing a nucleotide sequence encoding a Cas9 enzyme are commercially available from, e.g., Addgene, Life Technologies, Sigma- Aldrich, and Origene.
  • any of a number of transcription and translation control elements including promoter, transcription enhancers, transcription terminators, and the like, may be used in the expression vector.
  • Useful promoters can be derived from viruses, or any organism, e.g., prokaryotic or eukaryotic organisms.
  • Suitable promoters include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6), an enhanced U6 promoter, a human HI promoter (HI), etc.
  • LTR mouse mammary tumor virus long terminal repeat
  • Ad MLP adenovirus major late promoter
  • HSV herpes simplex virus
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter region
  • RSV rous sarcoma virus
  • U6 small nuclear promoter U6 small nuclear promoter
  • HI human HI promoter
  • the Cas nuclease and variants or fragments thereof can be introduced into a cell ⁇ e.g., an in vitro cell such as a primary cell for ex vivo therapy, or an in vivo cell such as in a patient) as a Cas polypeptide or a variant or fragment thereof, an mRNA encoding a Cas polypeptide or a variant or fragment thereof, or a recombinant expression vector comprising a nucleotide sequence encoding a Cas polypeptide or a variant or fragment thereof.
  • sgRNA Modified single guide RNA
  • the modified sgRNAs for use in the CRISPR/Cas system of genome modification typically include a guide sequence ⁇ e.g., crRNA) that is complementary to a target nucleic acid sequence and a scaffold sequence ⁇ e.g., tracrRNA) that interacts with a Cas nuclease ⁇ e.g., Cas9 polypeptide) or a variant or fragment thereof.
  • modified sgRNAs containing one or more chemical modifications can increase the activity, stability, and specificity, and/or decrease the toxicity of the modified sgRNAs compared to corresponding unmodified sgRNAs when used for CRISPR-based gene regulation ⁇ e.g., genome editing or modulating gene expression) in primary cells ⁇ e.g., T cells or hematopoietic stem and progenitor cells).
  • CRISPR-based gene regulation e.g., genome editing or modulating gene expression
  • primary cells e.g., T cells or hematopoietic stem and progenitor cells.
  • the advantages of the modified sgRNAs over the prior art include, but are not limited to, greater ease of delivery into target cells such as primary cells, as well as increased stability, increased duration of activity, and reduced toxicity in the target cells.
  • the modified sgRNAs as part of the CRISPR/Cas system provide higher frequencies of on-target gene regulation compared to other systems. In other cases, the modified sgRNAs provide improved activity and/or specificity compared to their unmodified sequence equivalents.
  • the modified sgRNA is complexed with a Cas nuclease ⁇ e.g., Cas9 polypeptide) or a variant or fragment thereof to form a ribonucleoprotein (RNP)-based delivery system for introduction into a cell ⁇ e.g., an in vitro cell such as a primary cell for ex vivo therapy, or an in vivo cell such as in a patient).
  • a Cas nuclease e.g., Cas9 polypeptide
  • RNP ribonucleoprotein
  • the modified sgRNA is introduced into a cell ⁇ e.g., an in vitro cell such as a primary cell for ex vivo therapy, or an in vivo cell such as in a patient) with an mRNA encoding a Cas nuclease ⁇ e.g., Cas9 polypeptide) or a variant or fragment thereof.
  • the modified sgRNA is introduced into a cell (e.g., an in vitro cell such as a primary cell for ex vivo therapy, or an in vivo cell such as in a patient) with a recombinant expression vector comprising a nucleotide sequence encoding a Cas nuclease (e.g., Cas9 polypeptide) or a variant or fragment thereof.
  • a cell e.g., an in vitro cell such as a primary cell for ex vivo therapy, or an in vivo cell such as in a patient
  • a recombinant expression vector comprising a nucleotide sequence encoding a Cas nuclease (e.g., Cas9 polypeptide) or a variant or fragment thereof.
  • a plurality of modified sgRNAs can be used for efficient multiplexed CRISPR-based gene regulation (e.g., genome editing or modulating gene expression) in target cells such as primary cells.
  • the plurality of modified sgRNAs can include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more modified sgRNAs that hybridize to the same target nucleic acid sequence or to different target nucleic acid sequences.
  • the plurality of modified sgRNAs can be introduced into a cell (e.g., an in vitro cell such as a primary cell for ex vivo therapy, or an in vivo cell such as in a patient) in a complex with a Cas nuclease (e.g., Cas9 polypeptide) or a variant or fragment thereof, or as a nucleotide sequence (e.g., mRNA or recombinant expression vector) encoding a Cas nuclease (e.g., Cas9 polypeptide) or a variant or fragment thereof.
  • a cell e.g., an in vitro cell such as a primary cell for ex vivo therapy, or an in vivo cell such as in a patient
  • a Cas nuclease e.g., Cas9 polypeptide
  • a nucleotide sequence e.g., mRNA or recombinant expression vector
  • the nucleic acid sequence of the modified sgRNA can be any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence (e.g., target DNA sequence) to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence.
  • a target polynucleotide sequence e.g., target DNA sequence
  • the degree of complementarity between a guide sequence of the modified sgRNA and its corresponding 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, 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, ELAND (Illumina, San Diego, Calif), SOAP (available at soap.genomics.org.cn), and Maq (available at ma
  • a guide sequence is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 instances, a guide sequence is about 20 nucleotides in length. In other instances, a guide sequence is about 15 nucleotides in length. In other instances, a guide sequence is about 25 nucleotides in length. 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.
  • 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 nucleotide sequence of a modified sgRNA can be selected using any of the web-based software described above. Considerations for selecting a DNA-targeting RNA include the PAM sequence for the Cas nuclease (e.g., Cas9 polypeptide) to be used, and strategies for minimizing off-target modifications. Tools, such as the CRISPR Design Tool, can provide sequences for preparing the modified sgRNA, for assessing target modification efficiency, and/or assessing cleavage at off-target sites. Another consideration for selecting the sequence of a modified sgRNA includes reducing the degree of secondary structure within the guide sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.
  • One or more nucleotides of the guide sequence and/or one or more nucleotides of the scaffold sequence of the modified sgRNA can be a modified nucleotide.
  • a guide sequence that is about 20 nucleotides in length may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more modified nucleotides.
  • the guide sequence includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides.
  • the guide sequence includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, or more modified nucleotides.
  • the modified nucleotides can be located at any nucleic acid position of the guide sequence. In other words, the modified nucleotides can be at or near the first and/or last nucleotide of the guide sequence, and/or at any position in between.
  • the one or more modified nucleotides can be located at nucleic acid position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, and/or position 20 of the guide sequence.
  • from about 10% to about 30%, e.g., about 10% to about 25%, about 10% to about 20%, about 10% to about 15%, about 15% to about 30%, about 20% to about 30%, or about 25% to about 30% of the guide sequence can comprise modified nucleotides.
  • the guide sequence can comprise modified nucleotides.
  • the scaffold sequence of the modified sgRNA contains one or more modified nucleotides.
  • a scaffold sequence that is about 80 nucleotides in length may have 1 or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 55, 60, 65, 70, 75, 76, 77, 78, 79, 80, or more modified nucleotides.
  • the scaffold sequence includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modified nucleotides.
  • the scaffold sequence includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, or more modified nucleotides.
  • the modified nucleotides can be located at any nucleic acid position of the scaffold sequence.
  • the modified nucleotides can be at or near the first and/or last nucleotide of the scaffold sequence, and/or at any position in between.
  • the one or more modified nucleotides can be located at nucleic acid position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, position 11, position 12, position 13, position 14, position 15, position 16, position 17, position 18, position 19, position 20, position 21, position 22, position 23, position 24, position 25, position 26, position 27, position 28, position 29, position 30, position 31, position 32, position 33, position 34, position 35, position 36, position 37, position 38, position 39, position 40, position 41, position 42, position 43, position 44, position 45, position 46, position 47, position 48, position 49, position 50, position 51, position 52, position 53, position 54, position 55, position 56, position 57, position 58, position 59, position 60, position 61, position 62, position 63, position 64, position 65, position 66, position 67, position 68, position 69, position 70, position 71, position 72, position 73, position 74, position 75, position 76, position 77, position
  • from about 1% to about 10%, e.g., about 1% to about 8%, about 1% to about 5%, about 5% to about 10%, or about 3% to about 7% of the scaffold sequence can comprise modified nucleotides.
  • from about 1% to about 10%, e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of the scaffold sequence can comprise modified nucleotides.
  • the modified nucleotides of the sgRNA can include a modification in the ribose (e.g., sugar) group, phosphate group, nucleobase, or any combination thereof.
  • the modification in the ribose group comprises a modification at the 2' position of the ribose.
  • the modified nucleotide includes a 2'fluoro-arabino nucleic acid, tricycle-DNA (tc-DNA), peptide nucleic acid, cyclohexene nucleic acid (CeNA), locked nucleic acid (LNA), ethylene-bridged nucleic acid (ENA), a phosphodiamidate morpholino, or a combination thereof.
  • Modified nucleotides or nucleotide analogues can include sugar- and/or backbone- modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone).
  • the phosphodiester linkages of a native or natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom.
  • the phosphoester group connecting to adjacent ribonucleotides may be replaced by a modified group, e.g., of phosphothioate group.
  • the 2' moiety is a group selected from H, OR, R, halo, SH, SR, H 2 , HR, R 2 or ON, wherein R is Ci-C 6 alkyl, alkenyl or alkynyl and halo is F, CI, Br or I.
  • the modified nucleotide contains a sugar modification.
  • sugar modifications include 2'-deoxy-2'-fluoro-oligoribonucleotide (2'- fluoro-2'-deoxycytidine-5 '-triphosphate, 2'-fluoro-2'-deoxyuridine-5 '-triphosphate), 2'-deoxy- 2'-deamine oligoribonucleotide (2'-amino-2'-deoxycytidine-5'-triphosphate, 2'-amino-2'- deoxyuridine-5 '-triphosphate), 2'-0-alkyl oligoribonucleotide, 2'-deoxy-2'-C-alkyl oligoribonucleotide (2 '-O-methylcytidine-5 '-triphosphate, 2'-methyluridine-5 '-triphosphate), 2'-C-alkyl oligoribonucleotide, and isomers thereof (2'-deoxy-2'-fluoro-oligori
  • the modified sgRNA contains one or more 2'-fluro, 2'-amino and/or 2'-thio modifications.
  • the modification is a 2'-fluoro-cytidine, 2'- fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino-adenosine, 2'-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine, 5-amino-allyl- uridine, 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and/or 5-fluoro-uridine.
  • nucleoside modifications found on mammalian RNA. See, e.g., Limbach et al., Nucleic Acids Research, 22(12):2183-2196 (1994).
  • the preparation of nucleotides and modified nucleotides and nucleosides are well- known in the art and described in, e.g., U.S. Patent Nos. 4,373,071, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5, 132,418, 5, 153,319, 5,262,530, and 5,700,642.
  • the nucleoside can be an analogue of a naturally occurring nucleoside.
  • the analogue is dihydrouridine, methyladenosine, methylcytidine, methyluridine, methylpseudouridine, thiouridine, deoxycytodine, and deoxyuridine.
  • the modified sgRNA described herein includes a nucleobase- modified ribonucleotide, i.e., a ribonucleotide containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase.
  • Non-limiting examples of modified nucleobases which can be incorporated into modified nucleosides and modified nucleotides include m5C (5-methylcytidine), m5U (5 -methyluridine), m6A (N6-methyladenosine), s2U (2-thiouridine), Um (2'-0-methyluridine), mlA (1-methyl adenosine), m2A (2- methyladenosine), Am (2-1-O-methyladenosine), ms2m6A (2-methylthio-N6- methyladenosine), i6A (N6-isopentenyl adenosine), ms2i6A (2-methylthio- N6isopentenyladenosine), io6A (N6-(cis-hydroxyisopentenyl) adenosine), ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl)a
  • the phosphate backbone of the modified sgRNA is altered.
  • the modified sgRNA can include one or more phosphorothioate, phosphoramidate (e.g., N3'- P5'-phosphoramidate (NP)), 2'-0-methoxy-ethyl (2'MOE), 2'-0-methyl-ethyl (2'ME), and/or methylphosphonate linkages.
  • one or more of the modified nucleotides of the guide sequence and/or one or more of the modified nucleotides of the scaffold sequence of the modified sgRNA include a 2'-0-methyl (M) nucleotide, a 2'-0-methyl 3 '-phosphorothioate (MS) nucleotide, a 2'-0-methyl 3'thioPACE (MSP) nucleotide, or a combination thereof.
  • M 2'-0-methyl
  • MS 2'-0-methyl 3 '-phosphorothioate
  • MSP 2'-0-methyl 3'thioPACE
  • the modified sgRNA includes one or more MS nucleotides.
  • the modified sgRNA includes one or more MSP nucleotides.
  • the modified sgRNA includes one or more MS nucleotides and one or more MSP nucleotides. In further instances, the modified sgRNA does not include M nucleotides. In certain instances, the modified sgRNA includes one or more MS nucleotides and/or one or more MSP nucleotides, and further includes one or more M nucleotides. In certain other instances, MS nucleotides and/or MSP nucleotides are the only modified nucleotides present in the modified sgRNA.
  • the modified sgRNA also includes a structural modification such as a stem loop, e.g., M2 stem loop or tetraloop.
  • the modified sgRNA can be synthesized by any method known to one of ordinary skill in the art.
  • the modified sgRNA is chemically synthesized.
  • Modified sgRNAs can be synthesized using 2'-0-thionocarbamate-protected nucleoside phosphoramidites. Methods are described in, e.g., Dellinger et al., J.American Chemical Society 133, 11540-11556 (2011); Threlfall et al., Organic & Biomolecular Chemistry 10, 746-754 (2012); and Dellinger et al, J. American Chemical Society 125, 940-950 (2003).
  • the present invention provides a recombinant donor repair template comprising two homology arms that are homologous to portions of a target DNA sequence (e.g., target gene or locus) at either side of a Cas nuclease (e.g., Cas9 nuclease) cleavage site.
  • the recombinant donor repair template comprises a reporter cassette that includes a nucleotide sequence encoding a reporter polypeptide (e.g., a detectable polypeptide, fluorescent polypeptide, or a selectable marker), and two homology arms that flank the reporter cassette and are homologous to portions of the target DNA at either side of the Cas nuclease cleavage site.
  • the reporter cassette can further comprise a sequence encoding a self-cleavage peptide, one or more nuclear localization signals, and/or a fluorescent polypeptide, e.g. superfolder GFP (sfGFP).
  • the homology arms can be about 10 bp to about 4 kb, e.g., about 10 bp to about 20 bp, about 10 bp to about 50 bp, about 10 bp to about 100 bp, about 10 bp to about 200 bp, about 10 bp to about 500 bp, about 10 bp to about 1 kb, about 10 bp to about 2 kb, about 10 bp to about 4 kb, about 100 bp to about 200 bp, about 100 bp to about 500 bp, about 100 bp to about 1 kb, about 100 bp to about 2 kb, about 100 bp to about 4 kb, about 500 bp to about 1 kb, about 500 bp to about 2 kb, about 500 bp to about 4 kb, about 1 kb to about 2 kb, about 1 kb to about 2 kb, about 1 kb to about 4 kb, or about
  • the donor repair template can be cloned into an expression vector.
  • Conventional viral and non-viral based expression vectors known to those of ordinary skill in the art can be used.
  • a single-stranded oligodeoxynucleotide (ssODN) donor template can be used for homologous recombination- mediated repair.
  • An ssODN is useful for introducing short modifications within a target DNA. For instance, ssODN are suited for precisely correcting genetic mutations such as SNPs.
  • ssODNs can contain two flanking, homologous sequences on each side of the target site of Cas nuclease cleavage and can be oriented in the sense or antisense direction relative to the target DNA.
  • Each flanking sequence can be at least about 10 base pairs (bp), e.g., at least about 10 bp, 15 bp, 20 bp, 25 bp, 30 bp, 35 bp, 40 bp, 45 bp, 50 bp, 55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, 550 bp, 600 bp, 650 bp, 700 bp, 750 bp, 800 bp, 850
  • each homology arm is about 10 bp to about 4 kb, e.g., about 10 bp to about 20 bp, about 10 bp to about 50 bp, about 10 bp to about 100 bp, about 10 bp to about 200 bp, about 10 bp to about 500 bp, about 10 bp to about 1 kb, about 10 bp to about 2 kb, about 10 bp to about 4 kb, about 100 bp to about 200 bp, about 100 bp to about 500 bp, about 100 bp to about 1 kb, about 100 bp to about 2 kb, about 100 bp to about 4 kb, about 500 bp to about 1 kb, about 500 bp to about 2 kb, about 500 bp to about 4 kb, about 1 kb to about 2 kb, about 1 kb to about 2 kb, about 1 kb to about 4 kb, about 1
  • the ssODN can be at least about 25 nucleotides (nt) in length, e.g., at least about 25 nt, 30 nt, 35 nt, 40 nt, 45 nt, 50 nt, 55 nt, 60 nt, 65 nt, 70 nt, 75 nt, 80 nt, 85 nt, 90 nt, 95 nt, 100 nt, 150 nt, 200 nt, 250 nt, 300 nt, or longer.
  • the ssODN is about 25 to about 50; about 50 to about 100; about 100 to about 150; about 150 to about 200; about 200 to about 250; about 250 to about 300; or about 25 nt to about 300 nt in length.
  • the modified nucleotides can be at the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth terminal nucleotide, or any combination thereof.
  • the modified nucleotides can be at the three terminal nucleotides at both ends of the ssODN template. Additionally, the modified nucleotides can be located internal to the terminal ends. 4. Target DNA
  • the target DNA sequence can be complementary to a fragment of a DNA-targeting RNA (e.g., modified sgRNA) and can be immediately followed by a protospacer adjacent motif (PAM) sequence.
  • the target DNA site may lie immediately 5' of a PAM sequence, which is specific to the bacterial species of the Cas9 used.
  • the PAM sequence of Streptococcus pyogenes-denwed Cas9 is NGG; the PAM sequence of Neisseria meningitidis-deri ' wed Cas9 is NNNNGATT; the PAM sequence of Streptococcus thermophilus-deri ' wed Cas9 is NNAGAA; and the PAM sequence of Treponema denticola-deri ' wed Cas9 is NAAAAC.
  • the PAM sequence can be 5' -NGG, wherein N is any nucleotide; 5' -NRG, wherein N is any nucleotide and R is a purine; or 5'-NNGRR, wherein N is any nucleotide and R is a purine.
  • the selected target DNA sequence should immediately precede (e.g. , be located 5') a 5 'NGG PAM, wherein N is any nucleotide, such that the guide sequence of the DNA- targeting RNA (e.g., modified sgRNA) base pairs with the opposite strand to mediate cleavage at about 3 base pairs upstream of the PAM sequence.
  • the degree of complementarity between a guide sequence of the DNA-targeting RNA (e.g., modified sgRNA) and its corresponding target DNA sequence, when optimally aligned using a suitable alignment algorithm is about or more than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%), 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, Selangor, Malaysia), and ELAND (Illumina, San Diego, CA).
  • the target DNA site can be selected in a predefined genomic sequence (gene) using web-based software such as ZiFiT Targeter software (Sander et al., 2007, Nucleic Acids Res, 35 :599-605; Sander et al., 2010, Nucleic Acids Res, 38:462-468), E-CRISP (Heigwer et al., 2014, Nat Methods, 1 1 : 122-123), RGEN Tools (Bae et al., 2014, Bioinformatics, 30(10): 1473-1475), CasFinder (Aach et al., 2014, bioRxiv), DNA2.0 gNRA Design Tool (DNA2.0, Menlo Park, CA), and the CRISPR Design Tool (Broad Institute, Cambridge, MA).
  • web-based software such as ZiFiT Targeter software (Sander et al., 2007, Nucleic Acids Res, 35 :599-605; Sander et al., 2010, Nucleic Acids Res, 38:462-468),
  • Such tools analyze a genomic sequence (e.g. , gene or locus of interest) and identify suitable target site for gene editing.
  • genomic sequence e.g. , gene or locus of interest
  • the CRISPR/Cas system of regulating gene expression can include a variant or fragment of the wild-type or native Cas nuclease (e.g., Cas9 polypeptide variant or fragment) and either a DNA- targeting sgRNA or an RNA-targeting sgRNA.
  • a complex comprising a Cas9 variant or fragment and an sgRNA that can bind to a target DNA sequence complementary to a portion of the sgRNA can block or hinder transcription initiation and/ elongation by RNA polymerase. This, in turn, can inhibit or repress gene expression of the target DNA.
  • a complex comprising a different Cas9 variant or fragment and an sgRNA that can bind to a target DNA sequence complementary to a portion of the sgRNA can induce or activate gene expression of the target DNA.
  • CRISPRi CRISPR interference
  • Larson et al. Nature Protocols, 2013, 8(11):2180-2196, and Qi et al, Cell, 152, 2013, 1173-1183.
  • the sgRNA-Cas9 variant complex can bind to a nontemplate DNA strand of a protein coding region and block transcription elongation.
  • the complex prevents or hinders transcription initiation.
  • a catalytically inactive variant of the Cas nuclease e.g., Cas9 polypeptide
  • the Cas nuclease is a Cas9 variant that contains at least two point mutations in the RuvC-like and HNH nuclease domains.
  • the Cas9 variant has DIOA and H840A amino acid substitutions, which is referred to as dCas9 (Jinek et al, Science, 2012, 337:816-821; Qi et al, Cell, 152(5): 1173-1183).
  • the dCas9 polypeptide from Streptococcus pyogenes comprises at least one mutation at position D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, A987 or any combination thereof.
  • Descriptions of such dCas9 polypeptides and variants thereof are provided in, for example, International Patent Publication No. WO 2013/176772.
  • the dCas9 enzyme can contain a mutation at D10, E762, H983 or D986, as well as a mutation at H840 or N863.
  • the dCas9 enzyme contains a DIOA or DION mutation.
  • the dCas9 polypeptide is catalytically inactive such as defective in nuclease activity.
  • the dCas9 enzyme or a variant or fragment thereof can block transcription of a target sequence, and in some cases, block RNA polymerase. In other instances, the dCas9 enzyme or a variant or fragment thereof can activate transcription of a target sequence.
  • the Cas9 variant lacking endonucleolytic activity ⁇ e.g., dCas9) can be fused to a transcriptional repression domain, e.g., a Kruppel associated box (KRAB) domain, or a transcriptional activation domain, e.g., a VP 16 transactivation domain.
  • the Cas9 variant is a fusion polypeptide comprising dCas9 and a transcription factor, e.g., RNA polymerase omega factor, heat shock factor 1, or a fragment thereof.
  • the Cas9 variant is a fusion polypeptide comprising dCas9 and a DNA methylase, histone acetylase, or a fragment thereof.
  • a suitable Cas nuclease ⁇ e.g., Cas9 polypeptide) variant having endoribonuclease activity as described in, e.g., O'Connell et al., Nature, 2014, 516:263-266, can be used.
  • Other useful Cas nuclease ⁇ e.g., Cas9) variants are described in, e.g., U.S. Patent Application Publication No. 2014/0302563.
  • a DNA oligonucleotide containing a PAM sequence (e.g., PAMmer) is used with the modified sgRNA and Cas nuclease (e.g., Cas9) variant described herein to bind to and cleave a single-stranded RNA transcript.
  • PAMmer e.g., PAMmer
  • Cas nuclease e.g., Cas9
  • the Cas nuclease e.g., Cas9 polypeptide
  • the Cas nuclease can be provided as a polypeptide, an mRNA encoding the polypeptide or a recombinant expression vector comprising a nucleotide sequence encoding the polypeptide. Additional details can be found above.
  • a plurality of modified sgRNAs is used to target different regions of a target gene to regulate gene expression of that target gene.
  • the plurality of modified sgRNAs can provide synergistic modulation (e.g., inhibition or activation) of gene expression of a single target gene compared to each modified sgRNA alone.
  • a plurality of modified sgRNAs is used to regulate gene expression of at least two target genes.
  • the present invention can be used to induce gene regulation of a target nucleic acid in any primary cell of interest.
  • the primary cell can be a cell isolated from any multicellular organism, e.g., a plant cell (e.g., a rice cell, a wheat cell, a tomato cell, an Arabidopsis thaliana cell, a Zea mays cell, and the like), a cell from a multicellular protist, a cell from a multicellular fungus, an animal cell such as a cell from an invertebrate animal (e.g., fruit fly, cnidarian, echinoderm, nematode, etc.) or a cell from a vertebrate animal (e.g., fish, amphibian, reptile, bird, mammal, etc.), a cell from a human, a cell from a healthy human, a cell from a human patient, a cell from a cancer patient, etc.
  • the primary cell with induced gene regulation can be
  • Any type of primary cell may be of interest, such as a stem cell, e.g., embryonic stem cell, induced pluripotent stem cell, adult stem cell (e.g., mesenchymal stem cell, neural stem cell, hematopoietic stem cell, organ stem cell), a progenitor cell, a somatic cell (e.g., fibroblast, hepatocyte, heart cell, liver cell, pancreatic cell, muscle cell, skin cell, blood cell, neural cell, immune cell), and any other cell of the body, e.g., human body.
  • a stem cell e.g., embryonic stem cell, induced pluripotent stem cell
  • adult stem cell e.g., mesenchymal stem cell, neural stem cell, hematopoietic stem cell, organ stem cell
  • a progenitor cell e.g., fibroblast, hepatocyte, heart cell, liver cell, pancreatic cell, muscle cell, skin cell, blood cell, neural cell, immune cell
  • the cells can be primary cells or primary cell cultures derived from a subject, e.g., an animal subject or a human subject, and allowed to grow in vitro for a limited number of passages.
  • the cells are disease cells or derived from a subject with a disease.
  • the cells can be cancer or tumor cells.
  • Primary cells can be harvested from a subject by any standard method. For instance, cells from tissues, such as skin, muscle, bone marrow, spleen, liver, kidney, pancreas, lung, intestine, stomach, etc., can be harvested by a tissue biopsy or a fine needle aspirate. Blood cells and/or immune cells can be isolated from whole blood, plasma or serum.
  • tissues such as skin, muscle, bone marrow, spleen, liver, kidney, pancreas, lung, intestine, stomach, etc.
  • Blood cells and/or immune cells can be isolated from whole blood, plasma or serum.
  • suitable primary cells include peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), and other blood cell subsets such as, but not limited to, T cell, a natural killer cell, a monocyte, a natural killer T cell, a monocyte- precursor cell, a hematopoietic stem and progenitor cell (HSPC) such as CD34+ HSPCs, or a non-pluripotent stem cell.
  • the cell can be any immune cell including, but not limited to, any T cell such as tumor infiltrating cells (TILs), CD3+ T cells, CD4+ T cells, CD 8+ T cells, or any other type of T cell.
  • TILs tumor infiltrating cells
  • CD3+ T cells CD3+ T cells
  • CD4+ T cells CD 8+ T cells
  • Induced pluripotent stem cells can be generated from differentiated cells according to standard protocols described in, for example, U.S. Patent Nos. 7,682,828, 8,058,065, 8,530,238, 8,871,504, 8,900,871 and 8,791,248.
  • the primary cell is in vitro. In other embodiments, the primary cell is ex vivo.
  • Ex vivo therapy can comprise administering a composition ⁇ e.g., a cell) generated or modified outside of an organism to a subject ⁇ e.g., patient).
  • a composition ⁇ e.g., a cell
  • the composition can be generated or modified by the methods disclosed herein.
  • ex vivo therapy can comprise administering a primary cell generated or modified outside of an organism to a subject (e.g., patient), wherein the primary cell has been cultured in vitro in accordance with the methods of the present invention that includes contacting the target nucleic acid in the primary cell with one or more modified sgRNAs described herein and a Cas nuclease ⁇ e.g., Cas9 polypeptide) or variant or fragment thereof, an mRNA encoding a Cas nuclease ⁇ e.g., Cas9 polypeptide) or variant or fragment thereof, or a recombinant expression vector comprising a nucleotide sequence encoding a Cas nuclease ⁇ e.g., Cas9 polypeptide) or variant or fragment thereof.
  • a primary cell generated or modified outside of an organism to a subject (e.g., patient), wherein the primary cell has been cultured in vitro in accordance with the methods of the present invention that includes contacting the target nucle
  • the composition ⁇ e.g., a cell
  • ex vivo therapy can include cell-based therapy, such as adoptive immunotherapy.
  • the composition used in ex vivo therapy can be a cell.
  • the cell can be a primary cell, including but not limited to, peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocytes (PBLs), and other blood cell subsets.
  • the primary cell can be an immune cell.
  • the primary cell can be a T cell ⁇ e.g., CD3+ T cells, CD4+ T cells, and/or CD8+ T cells), a natural killer cell, a monocyte, a natural killer T cell, a monocyte-precursor cell, a hematopoietic stem cell or a non-pluripotent stem cell, a stem cell, or a progenitor cell.
  • the primary cell can be a hematopoietic stem or progenitor cell (HSPC) such as CD34+ HSPCs.
  • the primary cell can be a human cell.
  • the primary cell can be isolated, selected, and/or cultured.
  • the primary cell can be expanded ex vivo.
  • the primary cell can be expanded in vivo.
  • the primary cell can be CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), and/or IL-7Ra(+).
  • the primary cell can be autologous to a subject in need thereof.
  • the primary cell can be non-autologous to a subject in need thereof.
  • the primary cell can be a good manufacturing practices (GMP) compatible reagent.
  • the primary cell can be a part of a combination therapy to treat diseases, including cancer, infections, autoimmune disorders, or graft-versus-host disease (GVHD), in a subject in need thereof.
  • a primary cell can be isolated from a multicellular organism ⁇ e.g., a plant, multicellular protist, multicellular fungus, invertebrate animal, vertebrate animal, etc.) prior to contacting a target nucleic acid within the primary cell with a Cas nuclease and a modified sgRNA.
  • the primary cell or its progeny e.g., a cell derived from the primary cell
  • the primary cell or its progeny can be returned to the multicellular organism.
  • Methods for introducing polypeptides and nucleic acids into a target cell are known in the art, and any known method can be used to introduce a nuclease or a nucleic acid (e.g., a nucleotide sequence encoding the nuclease, a DNA-targeting RNA (e.g., a modified single guide RNA), a donor repair template for homology-directed repair (HDR), etc.) into a cell, e.g., a primary cell such as a stem cell, a progenitor cell, or a differentiated cell.
  • a nuclease or a nucleic acid e.g., a nucleotide sequence encoding the nuclease, a DNA-targeting RNA (e.g., a modified single guide RNA), a donor repair template for homology-directed repair (HDR), etc.
  • a primary cell such as a stem cell, a progenitor cell, or a differentiated
  • Non-limiting examples of suitable methods include electroporation, viral or bacteriophage infection, transfection, conjugation, protoplast fusion, lipofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery, and the like.
  • the components of CRISPR/Cas-mediated gene regulation can be introduced into a cell using a delivery system.
  • the delivery system comprises a nanoparticle, a microparticle (e.g., a polymer micropolymer), a liposome, a micelle, a virosome, a viral particle, a nucleic acid complex, a transfection agent, an electroporation agent (e.g., using a NEON transfection system), a nucleofection agent, a lipofection agent, and/or a buffer system that includes a nuclease component (as a polypeptide or encoded by an expression construct) and one or more nucleic acid components such as a DNA-targeting RNA (e.g., a modified single guide RNA) and/or a donor repair template.
  • a nuclease component as a polypeptide or encoded by an expression construct
  • a DNA-targeting RNA e.g., a modified single guide RNA
  • the components can be mixed with a lipofection agent such that they are encapsulated or packaged into cationic submicron oil-in-water emulsions.
  • the components can be delivered without a delivery system, e.g., as an aqueous solution.
  • Methods of preparing liposomes and encapsulating polypeptides and nucleic acids in liposomes are described in, e.g., Methods and Protocols, Volume 1 : Pharmaceutical Nanocarriers: Methods and Protocols, (ed. Weissig). Humana Press, 2009 and Heyes et al. (2005) J Controlled Release 107:276-87.
  • Methods of preparing microparticles and encapsulating polypeptides and nucleic acids are described in, e.g., Functional Polymer Colloids and Microparticles volume 4 (Microspheres, microcapsules & liposomes), (eds. Arshady & Guyot). Citus Books, 2002 and Microparticulate Systems for the Delivery of Proteins and Vaccines, (eds. Cohen & Bernstein). CRC Press, 1996.
  • the target DNA can be analyzed by standard methods known to those in the art.
  • indel mutations can be identified by sequencing using the SURVEYOR ® mutation detection kit (Integrated DNA Technologies, Coralville, IA) or the Guide-it TM Indel Identification Kit (Clontech, Mountain View, CA).
  • Homology-directed repair (HDR) can be detected by PCR- based methods, and in combination with sequencing or RFLP analysis.
  • Non-limiting examples of PCR-based kits include the Guide-it Mutation Detection Kit (Clontech) and the GeneArt ® Genomic Cleavage Detection Kit (Life Technologies, Carlsbad, CA). Deep sequencing can also be used, particularly for a large number of samples or potential target/off-target sites.
  • the efficiency ⁇ e.g., specificity) of genome editing corresponds to the number or percentage of on-target genome cleavage events relative to the number or percentage of all genome cleavage events, including on-target and off-target events.
  • the modified sgRNAs described herein are capable of enhancing genome editing of a target DNA sequence in a cell such as a primary cell relative to the corresponding unmodified sgRNAs.
  • the genome editing can comprise homology- directed repair (HDR) ⁇ e.g., insertions, deletions, or point mutations) or nonhomologous end joining (NHE J).
  • HDR homology- directed repair
  • NHE J nonhomologous end joining
  • a method for inducing gene regulation e.g., genome editing and/or modulating (e.g., inhibiting or activating) gene expression, of a target nucleic acid in a cell.
  • the cell can be in vitro (e.g., a primary cell for use in ex vivo therapy) or in vivo (e.g., a cell in an organ or tissue of a subject such as a human).
  • the method for inducing genome editing includes introducing into a cell the modified sgRNA described herein and either a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof, an mRNA encoding a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof, or a recombinant expression vector comprising a nucleotide sequence encoding a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof.
  • a Cas nuclease e.g., Cas9 polypeptide
  • an mRNA encoding a Cas nuclease e.g., Cas9 polypeptide
  • a recombinant expression vector comprising a nucleotide sequence encoding a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof.
  • the modified sgRNA guides the Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof to the target nucleic acid (e.g., target DNA).
  • the modified sgRNA has enhanced activity, stability, and/or specificity for the target DNA compared to a corresponding unmodified sgRNA sequence.
  • the genome editing is nonhomologous end joining (NHEJ) of the target DNA.
  • the genome editing is homologous- directed repair (HDR) of the target DNA.
  • HDR homologous- directed repair
  • a recombinant donor repair template is added to the cell.
  • the recombinant donor repair template can include two nucleotide sequences comprising two non-overlapping, homologous portions of the target DNA, wherein the nucleotide sequences are located at the 5' and 3' ends of a nucleotide sequence corresponding to the target DNA.
  • the recombinant donor repair template comprises a synthetic single stranded oligodeoxynucleotide (ssODN) template comprising a nucleotide sequence encoding a mutation to correct a single nucleotide polymorphism (SNP) and two nucleotide sequences comprising two non-overlapping, homologous portions of the target DNA, wherein the nucleotide sequences are located at the 5' and 3' ends of nucleotide sequence encoding the mutation.
  • ssODN synthetic single stranded oligodeoxynucleotide
  • the modified sgRNA and/or either a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof, an mRNA encoding a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof, or a recombinant expression vector comprising a nucleotide sequence encoding a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof are introduced into the cell using any suitable method such as by electroporation.
  • the method for modulating (e.g., inhibiting or activating) gene expression of a target nucleic acid, e.g., a target DNA, in a cell includes introducing (e.g., electroporating) into the cell the modified sgRNA described herein and either a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof, an mRNA encoding a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof, or a recombinant expression vector comprising a nucleotide sequence encoding a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof.
  • a Cas nuclease e.g., Cas9 polypeptide
  • an mRNA encoding a Cas nuclease e.g., Cas9 polypeptide
  • a recombinant expression vector comprising a nu
  • the Cas nuclease (e.g., Cas9) variant is an endonuclease- deficient Cas (e.g., dCas9) polypeptide.
  • the Cas9 variant can have two or more amino acid substitutions compared to the wild-type Cas9 polypeptide. In other instances, the Cas9 variant cannot cleave double-stranded DNA.
  • the Cas nuclease variant can be a Cas (e.g., dCas9) fusion polypeptide.
  • the fusion polypeptide includes a transcriptional repression domain, a transcriptional activation domain, transcription factor, histone modifying enzyme (e.g., histone deacetylase, histone methyltransferase, histone acetyltransferase), a DNA modifying enzyme (e.g., DNA methyltransferase), and the like.
  • histone modifying enzyme e.g., histone deacetylase, histone methyltransferase, histone acetyltransferase
  • DNA modifying enzyme e.g., DNA methyltransferase
  • the method for modulating (e.g., inhibiting or activating) gene expression of a target nucleic acid, e.g., a target RNA, in a cell includes introducing (e.g., electroporating) into the cell the modified sgRNA described herein and either a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof, an mRNA encoding a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof, or a recombinant expression vector comprising a nucleotide sequence encoding a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof.
  • a Cas nuclease e.g., Cas9 polypeptide
  • an mRNA encoding a Cas nuclease e.g., Cas9 polypeptide
  • a recombinant expression vector comprising a
  • the Cas nuclease (e.g., Cas9) variant has reduced or lacks endonucleolytic activity.
  • the Cas nuclease variant can contain two or more amino acid substitutions such that the polypeptide is unable to cleave double-stranded DNA.
  • the Cas nuclease (e.g., Cas9) variant can have endoribonuclease activity and can cleave target RNA.
  • the modified sgRNAs described herein can be used to modulate the efficiency of gene regulation.
  • the modified sgRNA can induce gene regulation with an enhanced activity relative to a corresponding unmodified sgRNA.
  • the enhanced activity comprises increased stability of the modified sgRNA and/or increased specificity of the modified sgRNA for a target nucleic acid.
  • the modified sgRNA can induce gene regulation with a decrease in cellular toxicity relative to a corresponding unmodified sgRNA.
  • the modified sgRNAs can be applied to targeted nuclease-based therapeutics of genetic diseases.
  • the modified sgRNAs provided herein can enhance the activity of genome editing and increase the efficacy of genome editing-based therapies.
  • the modified sgRNAs may be used for in vivo gene editing of genes in subjects with a genetic disease.
  • the modified sgRNAs can be administered to a subject via any suitable route of administration and at doses or amounts sufficient to enhance the effect ⁇ e.g., improve the genome editing efficiency) of the nuclease-based therapy.
  • Provided herein is a method for preventing or treating a genetic disease in a subject in need thereof by correcting a genetic mutation associated with the disease.
  • the method includes administering to the subject a modified sgRNA described herein in an amount that is sufficient to correct the mutation. Also provided herein is the use of a modified sgRNA described herein in the manufacture of a medicament for preventing or treating a genetic disease in a subject in need thereof by correcting a genetic mutation associated with the disease.
  • the modified sgRNA can be contained in a composition that also includes a Cas nuclease ⁇ e.g., Cas9 polypeptide) or variant or fragment thereof, an mRNA encoding a Cas nuclease ⁇ e.g., Cas9 polypeptide) or variant or fragment thereof, or a recombinant expression vector comprising a nucleotide sequence encoding a Cas nuclease ⁇ e.g., Cas9 polypeptide) or variant or fragment thereof.
  • the modified sgRNA is included in a delivery system described above.
  • the genetic diseases that may be corrected by the method include, but are not limited to, X-linked severe combined immune deficiency, sickle cell anemia, thalassemia, hemophilia, neoplasia, cancer, age-related macular degeneration, schizophrenia, trinucleotide repeat disorders, fragile X syndrome, prion-related disorders, amyotrophic lateral sclerosis, drug addiction, autism, Alzheimer's disease, Parkinson's disease, cystic fibrosis, blood and coagulation disease or disorders, inflammation, immune-related diseases or disorders, metabolic diseases, liver diseases and disorders, kidney diseases and disorders, muscular/skeletal diseases and disorders ⁇ e.g., muscular dystrophy, Duchenne muscular dystrophy), neurological and neuronal diseases and disorders, cardiovascular diseases and disorders, pulmonary diseases and disorders, ocular diseases and disorders, viral infections ⁇ e.g., HIV infection), and the like. V. Examples
  • Example 1 Chemically modified guide RNAs enhance CRISPR/Cas genome editing in human primary cells.
  • CRISPR/Cas-mediated genome editing relies on guide RNAs that direct site- specific DNA cleavage facilitated by the Cas endonuclease.
  • sgRNAs chemically synthesized single guide RNAs
  • This approach is a simple and highly effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR/Cas technology.
  • Genome editing with engineered nucleases is a breakthrough technology for modifying essentially any genomic sequence of interest (Porteus, M.H. & Carroll, D., Nature biotechnology 23, 967-973 (2005)). This technology exploits engineered nucleases to generate site-specific double-strand breaks (DSBs) followed by resolution of DSBs by endogenous cellular repair mechanisms.
  • DSBs site-specific double-strand breaks
  • the outcome can be either mutation of a specific site through mutagenic nonhomologous end-joining (NHEJ), creating insertions or deletions (in/dels) at the site of the break, or precise change of a genomic sequence through homologous recombination (HR) using an exogenously introduced donor template (Hendel et al, Trends in Biotechnology 33, 132-140 (2015).
  • NHEJ nonhomologous end-joining
  • HR homologous recombination
  • CRISPR clustered regularly interspaced palindromic repeat
  • Cas RNA-guided nuclease
  • sgRNA short guide RNA
  • the guide RNA is composed of two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), which for gene editing purposes are typically fused in a chimeric single guide RNA (sgRNA).
  • the sgRNAs consist of 100 nucleotides (nt) of which 20 nt at the 5' end can hybridize to a target DNA sequence by means of Watson-Crick base pairing and guide the Cas endonuclease to cleave the target genomic DNA (FIG. 1 A).
  • sgRNAs can induce high levels of genome editing and further show that chemical alterations of the sgRNAs can dramatically enhance genome editing in both human primary T cells and CD34 + hematopoietic stem and progenitor cells (HSPCs).
  • HSPCs hematopoietic stem and progenitor cells
  • the increase in genome editing in these cell types using chemically modified sgRNAs is further improved by delivering Cas9 as mRNA or protein rather than through a DNA expression plasmid, thus generating a simple and complete RNA or ribonucleoprotein (RNP)-based delivery system for the CRISPR/Cas system.
  • RNP ribonucleoprotein
  • the highest off-target frequencies measured were with the IL2RG sgRNAs at 'off-target 2' yielding in/del frequencies of 1.0% and 7.8% when using 1 ⁇ g and 20 ⁇ g MS sgRNA, respectively.
  • the off-target activity of the IL2RG MSP sgRNA at the same site was 2.7-fold and 2.8-fold lower, respectively, compared to the MS sgRNA, despite having higher on-target activity.
  • the off-target activity of the MSP sgRNA was better compared to the MS sgRNA at the HBB off-target- 1 site, at which the HBB sgRNA has previously demonstrated significant off-target activity.
  • the MSP sgRNA had higher off-target activity compared to the MS sgRNA.
  • these results suggest that typically the chemically modified sgRNAs retain high specificity.
  • the differences observed in on:off target ratios suggest the possibility that chemical alterations to the sgRNA may have the potential to modulate the on:off-target activities however, the impact of a given chemical alteration appears to be sequence-dependent and may also depend on other factors such as cell type and delivery conditions. Whether these observations are generalizable to other sgRNAs targeting different loci in different species will require further studies.
  • FIG. 19 provides additional experimental data showing that MS-modified sgRNAs perform better than the corresponding unmodified sgRNAs in CD34+ HSPCs.
  • FIG. 20 shows that modified sgRNAs can be used for efficient multiplexed genome editing.
  • RNA oligomers were synthesized on an ABI 394 Synthesizer (Life Technologies, Carlsbad, CA, USA) using 2'-0-thionocarbamate-protected nucleoside phosphoramidites (Sigma-Aldrich, St. Louis, MO, USA or Thermo Fisher, Waltham, MA, USA) according to previously described procedures (Dellinger et al., Journal of the American Chemical Society 133, 11540-11556 (2011)). 2'-0-methyl phosphoramidites were purchased from Thermo Scientific, Grand Island, NY, and incorporated into RNA oligomers under the same conditions as the 2'-0-thionocarbamate protected phosphoramidites.
  • iodine oxidation step after the coupling reaction was replaced by a sulfurization step using a 0.05 M solution of 3-((TST,N-dimethylaminomethylidene)amino)-3H-l,2,4-dithiazole-5-thione in a pyridine-acetonitrile (3 :2) mixture for 6 minutes.
  • reagents for solid phase RNA synthesis were purchased from Glen Research (Sterling, VA, USA).
  • oligonucleotides were purified using reverse phase HPLC and analyzed by LC- MS using an Agilent 1290 Infinity series LC system coupled to an Agilent 6520 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Table 1 shows the sequences of all sgRNAs used and the masses obtained from deconvolution of the charge state series of peaks found. The deconvolution was done using Mass Hunter Qualitative Analysis (version B.06.00) software (Agilent).
  • SEQ ID NO: 18 AGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAA
  • sgRNA sequences as well as calculated and observed molecular weights are indicated. Nucleotides with 2'-0-methyl modifications are underlined. Modifications in the phosphate backbone are indicated with ⁇ (MS) and ⁇ (MSP). 2. In vitro cleavage assays
  • 4kb PAM-addressable targets were prepared by preparative PCR amplification of plasmid-borne human sequences.
  • a 20 ⁇ _, reaction volume 50 fmoles of linearized DNA target in the presence of 50 nM sgRNA, 39 nM recombinant purified Cas9 protein (Agilent) and 10 mM MgCl 2 at pH 7.6 was incubated at 37 °C for 30 min.
  • 0.5 ⁇ _, of RNace It (Agilent) was added, and incubation was continued at 37 °C for 5 min and then at 70 °C for 15 min.
  • 0.5 ⁇ _, of Proteinase K (Mol. Bio.
  • sgRNA expression vectors were constructed by cloning of 20bp oligonucleotide target sequences into px330 (Addgene plasmid #42230) containing a human codon-optimized SpCas9 expression cassette and a human U6 promoter driving the expression of the chimeric sgRNA (see Table 1 for sgRNA sequences).
  • All three plasmid targeting vectors carry approximately 2 x 800bp arms of homology, which were generated by PCR amplification of the corresponding loci using genomic DNA isolated from K562 cells. The homology arms were then cloned into a ⁇ 2,900 base pair vector based on pBluescript SK+ using standard cloning methods. Between the homology arms, both the HBB and CCR5 donors contain the EFla promoter driving expression of GFP. The IL2RG donor lacks a promoter and relies on endogenous activity of the IL2RG gene to drive GFP expression.
  • the nucleic acid sequence of the IL2RG targeting vector is set forth as SEQ ID NO: 1.
  • the nucleic acid sequence of the HBB targeting vector is set forth as SEQ ID NO:2.
  • the nucleic acid sequence of the CCR5 targeting vector is set forth as SEQ ID NO:3.
  • K562 and T cells were cultured at 37 °C, 5% C0 2 , and ambient oxygen levels.
  • CD34+ hematopoietic stem/progenitor cells were cultured at 37°C, 5% C0 2 , and 5% 0 2 .
  • K562 cells were maintained in RPMI 1640 (HyClone) supplemented with 10% bovine growth serum, 100 mg/ml streptomycin, 100 units/ml penicillin, and 2 mM L- glutamine.
  • K562 cells were nucleofected using the Lonza Nucleofector 2b (program T-016) and a nucleofection buffer containing 100 mM KH 2 P0 4 , 15 mM NaHC0 3 , 12 mM MgCl 2 x 6H 2 0, 8 mM ATP, 2 mM glucose (pH 7.4).
  • Nucleofection conditions 100 ⁇ L nucleofection solution, 10 6 cells, 1 to 20 ⁇ g chemically modified sgRNA, 1 to 15 ⁇ g Cas9 mRNA (Cas9 mRNA, 5meC, ⁇ , Product Code: L-6125, TriLink BioTechnologies, San Diego, CA, USA), 2 ⁇ g sgRNA/Cas9-encoding plasmid, or 5 ⁇ g HR donor plasmid.
  • CD3+ T cells were isolated from buffy coats obtained from the Stanford School of Medicine Blood Center using a human Pan T Cell Isolation Kit (Miltenyi Biotec, San Diego, CA, USA).
  • CD3+ cells were maintained in X-VIVO 15 (Lonza, Walkersville, MD, USA) supplemented with 5% human serum (Sigma-Aldrich, St. Louis, MO, USA), 100 IU/mL human recombinant IL-2 (Peprotech, Rocky Hill, NJ, USA), and 10 ng/mL human recombinant IL-7 (BD Biosciences, San Jose, CA, USA). Before nucleofection, T cells were activated for three days with immobilized anti-CD3 antibodies (clone: OKT3, eBioscience, San Diego, CA, USA) and soluble anti-CD28 antibodies (clone: CD28.2, eBioscience).
  • T cells were nucleofected immediately after isolation. T cells were nucleofected using the Lonza Nucleofector 2b (program U-014) and the Human T Cell Nucleofector Kit (VPA- 1002, Lonza). Nucleofection conditions: 100 ⁇ L ⁇ nucleofection solution, 10 6 cells, 10 to 20 ⁇ g chemically modified sgRNA, 15 to 30 ⁇ g Cas9 (or 15 ⁇ g eGFP mRNA, TriLink BioTechnologies, San Diego, CA, USA), 1 ⁇ g sgRNA/Cas9-encoding plasmid. Mobilized human peripheral blood CD34+ HSPCs were purchased from AllCells and thawed according to manufacturer's instructions.
  • CD34+ HSPCs were maintained in X-VIVO 15 (Lonza) supplemented with SCF (100 ng/ml), TPO (100 ng/ml), Flt3-Ligand (100 ng/ml), IL-6 (100 ng/ml), and StemRegeninl (0.75 mM).
  • CD34+ HSPCs were nucleofected using the Lonza 4D-Nucleofector (program EO-100) and the P3 Primary Cell 4D-Nucleofector Kit (V4XP- 3024).
  • Nucleofection conditions 100 ⁇ L nucleofection solution, 5xl0 5 cells, 10 ⁇ g chemically modified sgRNA, 15 ⁇ g Cas9 mRNA, 1 ⁇ g plasmid.
  • Cas9 protein was purchased from PNA Bio (Thousand Oaks, CA, USA) or Life Technologies (Carlsbad, CA, USA). For all RNP experiments except for FIG. 8, Cas9 protein from PNA Bio was used. Cas9 protein was complexed with sgRNAs in a Cas9: sgRNA molar ratio of 1 :2.5 for 10 min at 25°C.
  • RNPs were nucleofected into K562 cells or T cells as described above with 10 6 cells in 100 ⁇ L of the respective nucleofection solutions.
  • the total sgRNA amount was 10 ⁇ g (10 ⁇ g when used individually and 2x5 ⁇ g when used together).
  • sgRNAs were nucleofected with 15 ⁇ g Cas9 mRNA into 10 6 cells.
  • T cell nucleofections were performed as above whereas nucleofection of CD34+ HSPCs were similar to the T cells nucleofections using the Lonza Nucleofector 2b (program U-014) and the Human T Cell Nucleofector Kit (VPA-1002, Lonza). Directly after nucleofection CD34+ HSPCs were incubated at 30°C for 24 hrs after which they were transferred to 37 °C until harvest of genomic DNA.
  • eGFP expression was measured on an Accuri C6 flow cytometer (BD Biosciences, San Jose, CA, USA). Cell death was measured with the LIVE/DEAD Fixable Red Dead Cell Stain Kit (Life Technologies, Carlsbad, CA, USA) according to manufacturer's instructions and cells were analyzed on the Accuri C6 flow cytometer.
  • gDNA was extracted from cells three days after nucleofection (if not otherwise indicated) using QuickExtract DNA Extraction Solution (Epicentre, Madison, WI, USA) following manufacturer's instructions.
  • PCR amplicons spanning the sgRNA genomic target sites were generated using the iProof High-Fidelity Master Mix (Bio-Rad, Hercules, CA, USA) with the following primer pairs: IL2RG_fw (SEQ ID NO: 84): 5 ' -TC AC AC AGC AC AT ATTTGCC AC ACCCTCTG-3 ' , IL2RG RV (SEQ ID NO: 85): 5 ' -TGCCC AC ATGATTGTAATGGCC AGTGG-3 ' , HBB fw (SEQ ID NO: 86): 5 ' -CC AACTCCT AAGCC AGTGCC AGAAGAG-3 ' , HBB rv (SEQ ID NO: 87) 5 ' -AG
  • PCR amplicons were purified and 200 ng was denatured and re- annealed in a thermocycler and digested with T7 Endonuclease I (New England Biolabs, Waltham, MA, USA) according to manufacturer's protocol. Digested DNA was run on a 4- 20% TBE polyacrylamide gel, stained with Diamond Nucleic Acid Dye (Promega, Madison, WI, USA), and visualized on a ChemiDoc XRS+ (Bio-Rad).
  • T cells were transferred to multiple 96-well U-bottom 96-well plates at 3xl0 4 cells/well.
  • cells were transferred to white 96-well plates in 100 ⁇ L medium and adding 100 ⁇ L CellTiter-Glo 2.0 per manufacturer's guidelines.
  • Luminescence was read on a Tecan Infinite 200 PRO (Tecan, Mannedorf, Switzerland) using a 1 sec integration time.
  • Table 2 List of on- and off-target loci interrogated by deep sequencing of PCR amplicons.
  • Table 3 provides a list of oligonucleotide primers used for generation of on- and off- target amplicons to quantify in/del frequencies by deep sequencing.
  • the gene-specific hybridization sequences of the gene-specific amplicon primers and barcodes of the Illumina barcoding primers are indicated with underlined and bolded text, respectively.
  • Table 3 List of oligonucleotide primers used for generation of on- and off-target amplicons to quantify in/del frequencies by deep sequencing.
  • Table 4 provides a list of CCR5, HBB, and IL2RG on- and off-target amplicons generated for deep sequencing analysis of in/del frequencies.
  • Amplicon sizes (of unedited genomic DNA) range from 183-220 bp, with a minimum of 50 bp from the target site to the hybridization sequence of the gene-specific primer.
  • the hybridization sequences used for amplicon generation from genomic DNA and putative CRISPR-target sites are indicated in underlined and bolded text, respectively.
  • Table 4. List of CCR5, HBB, and IL2RG on- and off-target amplicons generated for deep sequencing analysis of in/del frequencies.
  • Amplicon sizes range from 183-220 bp, with a minimum of 50 bp from the target site to the hybridization sequence of the gene-specific primer.
  • the hybridization sequences used for amplicon generation from genomic DNA and putative CRISPR-target sites are indicated in underlined and bolded text, respectively.
  • Example 2 CRISPR/Cas9-based homologous recombination using chemically modified guide RNAs and a synthetic single strand oligodeoxynucleotide (ssODN) template.
  • ssODN synthetic single strand oligodeoxynucleotide
  • Stimulated human primary T cells from three different donors were nucleofected with 10 ⁇ g CCR5 sgRNA (unmodified or MS), 15 ⁇ g Cas9 mRNA, and 2.81 ⁇ g of a 183nt CCR5 ssODN (with or without phosphorothioate ('PS') linkages between the three terminal nucleotides at both ends).
  • the ssODN contained a central Hindlll restriction site not present in the WT CCR5 sequence.
  • Genomic DNA (gDNA) was extracted three days after nucleofections and PCR products spanning the target site (outside the sequence homologous to the ssODN) were generated and digested with Hindlll. Restriction fragments were analyzed on a 2% TBE agarose gel and HDR frequencies were calculated.
  • FIG. 21 depicts the agarose gels from the HDR experiment.
  • HDR frequency was increased when modified sgRNAs were used.
  • HDR frequencies were further increased when the ssODN contained phosphothioate linkages between the three terminal nucleotides at both ends.
  • the HBB homology arms are bold and italicized.
  • the EF 1 a promoter is underlined.
  • the GFP sequence is capitalized and italicized.
  • the WPRE sequence is dashed underlined.
  • the BGH poly(A) sequence is double-underlined. SEQIDNOS: 4-19
  • IL2RG_fw 5 ' -TC AC AC AGC AC AT ATTTGCC AC ACCCTCTG-3 ' , SEQIDNO: 85
  • IL2RG RV 5 ' -TGCCC AC ATGATTGT AATGGCC AGTGG-3 ' SEQIDNO: 86
  • HBB fw 5 ' -C C A ACTCC T A AGC C AGTGC C AGA AGAG-3 ' SEQIDNO: 87
  • HBB rv 5'-AGTCAGTGCCTATCAGAAACCCAAGAG-3'

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