Review

Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases

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Abstract

CRISPR–Cas9 RNA-guided nucleases are a transformative technology for biology, genetics and medicine owing to the simplicity with which they can be programmed to cleave specific DNA target sites in living cells and organisms. However, to translate these powerful molecular tools into safe, effective clinical applications, it is of crucial importance to carefully define and improve their genome-wide specificities. Here, we outline our state-of-the-art understanding of target DNA recognition and cleavage by CRISPR–Cas9 nucleases, methods to determine and improve their specificities, and key considerations for how to evaluate and reduce off-target effects for research and therapeutic applications.

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References

  1. 1.

    et al. Targeted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330, 576–578 (1987).

  2. 2.

    & Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503–512 (1987).

  3. 3.

    & Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156 (1981).

  4. 4.

    The 2007 Nobel Prize in Physiology or Medicine - Press Release. Nobelprize.org

  5. 5.

    , & Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Mol. Cell. Biol. 14, 8096–8106 (1994). This study first demonstrated that nuclease-induced DSBs could stimulate homologous recombination.

  6. 6.

    et al. Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy. Curr. Gene Ther. 11, 11–27 (2011).

  7. 7.

    , , , & Genome editing with engineered zinc finger nucleases. Nature 11, 636–646 (2010).

  8. 8.

    & CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347–355 (2014).

  9. 9.

    , & Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

  10. 10.

    & Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346, 1258096 (2014).

  11. 11.

    , & Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl Acad. Sci. USA 93, 1156–1160 (1996).

  12. 12.

    et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).

  13. 13.

    et al. Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes. Nucleic Acids Res. 39, 6315–6325 (2011).

  14. 14.

    , , & FokI dimerization is required for DNA cleavage. Proc. Natl Acad. Sci. USA 95, 10570–10575 (1998).

  15. 15.

    , , & Structure of FokI has implications for DNA cleavage. Proc. Natl Acad. Sci. USA 95, 10564–10569 (1998).

  16. 16.

    et al. megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering. Nucleic Acids Res. 42, 2591–2601 (2014).

  17. 17.

    et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012). This was the first published study to demonstrate that the target specificity of Cas9 could be programmed using an engineered single gRNA (a fusion of naturally occurring crRNA and tracrRNA).

  18. 18.

    et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

  19. 19.

    et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

  20. 20.

    et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).

  21. 21.

    et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat. Biotechnol. 31, 227–229 (2013).

  22. 22.

    et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

  23. 23.

    et al. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc. Natl Acad. Sci. USA 110, 15644–15649 (2013).

  24. 24.

    , , , & Off-target effects in CRISPR/Cas9-mediated genome engineering. Mol. Ther. Nucleic Acids 4, e264 (2015).

  25. 25.

    , & Determining the specificities of TALENs, Cas9, and other genome-editing enzymes. Methods Enzymol. 546, 47–78 (2014).

  26. 26.

    , & How specific is CRISPR/Cas9 really? Curr. Opin. Chem. Biol. 29, 72–78 (2015).

  27. 27.

    , & Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9. Int. J. Pharm. 494, 180–194 (2015).

  28. 28.

    et al. Keeping CRISPR/Cas on-target. Curr. Issues Mol. Biol. 20, 1–20 (2015).

  29. 29.

    , & Minimizing off-target mutagenesis risks caused by programmable nucleases. Int. J. Mol. Sci. 16, 24751–24771 (2015).

  30. 30.

    , & Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery. Nat. Methods 13, 41–50 (2015).

  31. 31.

    et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154, 1370–1379 (2013).

  32. 32.

    et al. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res. 24, 132–141 (2014).

  33. 33.

    et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J. Biotechnol. 208, 44–53 (2015).

  34. 34.

    et al. High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat. Biotechnol. 31, 839–843 (2013).

  35. 35.

    et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822–826 (2013). This was the first study to report high-frequency off-target CRISPR–Cas9 mutagenesis.

  36. 36.

    et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).

  37. 37.

    , , & CRISPR/Cas9 systems targeting β-globin and CCR5 genes have substantial off-target activity. Nucleic Acids Res. 41, 9584–9592 (2013).

  38. 38.

    et al. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res. 42, 7473–7485 (2014).

  39. 39.

    et al. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol. J. 12, 797–807 (2014).

  40. 40.

    , , & Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579–E2586 (2012).

  41. 41.

    , , & Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).

  42. 42.

    , & E-CRISP: fast CRISPR target site identification. Nat. Methods 11, 122–123 (2014).

  43. 43.

    , , & Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat. Methods 8, 765–770 (2011).

  44. 44.

    et al. Broad specificity profiling of TALENs results in engineered nucleases with improved DNA-cleavage specificity. Nat. Methods 11, 429–435 (2014).

  45. 45.

    et al. In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites. Nucleic Acids Res. 41, e181 (2013).

  46. 46.

    et al. Whole-genome sequencing analysis reveals high specificity of CRISPR/Cas9 and TALEN-based genome editing in human iPSCs. Cell Stem Cell 15, 12–13 (2014).

  47. 47.

    et al. Low incidence of off-target mutations in individual CRISPR-Cas9 and TALEN targeted human stem cell clones detected by whole-genome sequencing. Cell Stem Cell 15, 27–30 (2014).

  48. 48.

    et al. Off-target mutations are rare in Cas9-modified mice. Nat. Methods 12, 479–479 (2015).

  49. 49.

    & What's changed with genome editing? Cell Stem Cell 15, 3–4 (2014).

  50. 50.

    et al. An unbiased genome-wide analysis of zinc-finger nuclease specificity. Nat. Biotechnol. 29, 816–823 (2011). This is the first report of an unbiased genome-wide analysis of nuclease specificity, by analysing IDLV capture into ZFN-induced DSBs.

  51. 51.

    et al. Evaluation of TCR gene editing achieved by TALENs, CRISPR/Cas9 and megaTAL nucleases. Mol. Ther. 24, 570–581 (2016).

  52. 52.

    et al. Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat. Biotechnol. 33, 175–178 (2015).

  53. 53.

    et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015). This study describes the GUIDE-seq method for identifying Cas9 off-target cleavage sites in living cells.

  54. 54.

    , , & Open-source software guideseq for analysis of GUIDE-seq data. Nat. Biotechnol. (in the press).

  55. 55.

    et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell 147, 107–119 (2011).

  56. 56.

    et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. 33, 179–186 (2015). This study describes the HTGTS method for identifying Cas9 off-target cleavage by translocation sequencing.

  57. 57.

    et al. Nucleotide-resolution DNA double-strand break mapping by next-generation sequencing. Nat. Methods 10, 361–365 (2013). This is the original study describing the BLESS method, which was later adapted to analyse CRISPR–Cas9 off-target cleavage.

  58. 58.

    et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat. Methods 12, 237–243 (2015). This study describes Digenome-seq, an in vitro genome-wide method for identifying CRISPR–Cas9 off-target cleavage sites.

  59. 59.

    , , , & Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res. (2016).

  60. 60.

    , , , & Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat. Biotechnol. 32, 279–284 (2014).

  61. 61.

    et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

  62. 62.

    et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).

  63. 63.

    et al. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat. Biotechnol. 32, 569–576 (2014).

  64. 64.

    , & Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32, 577–582 (2014).

  65. 65.

    , , , & Dimeric CRISPR RNA-guided FokI-dCas9 nucleases directed by truncated gRNAs for highly specific genome editing. Hum. Gene Ther. 26, 425–431 (2015).

  66. 66.

    et al. Generation of mutant mice via the CRISPR/Cas9 system using FokI-dCas9. Sci. Rep. 5, 11221 (2015).

  67. 67.

    et al. Production of knockout mice by DNA microinjection of various CRISPR/Cas9 vectors into freeze-thawed fertilized oocytes. BMC Biotechnol. 15, 33 (2015).

  68. 68.

    et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (2016).

  69. 69.

    et al. Rationally engineered Cas9 nucleases with improved specificity. Science 351, 84–88 (2016).

  70. 70.

    , , & Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569–573 (2014).

  71. 71.

    et al. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343, 1247997 (2014).

  72. 72.

    et al. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949 (2014).

  73. 73.

    , , , & Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).

  74. 74.

    et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat. Biotechnol. 33, 73–80 (2015).

  75. 75.

    , , , & Targeted gene knockout by direct delivery of zinc-finger nuclease proteins. Nat. Methods 9, 805–807 (2012).

  76. 76.

    et al. Rational design of a split-Cas9 enzyme complex. Proc. Natl Acad. Sci. USA 112, 2984–2989 (2015).

  77. 77.

    , & A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).

  78. 78.

    , , & Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015).

  79. 79.

    , , , & Small molecule-triggered Cas9 protein with improved genome-editing specificity. Nat. Chem. Biol. 11, 316–318 (2015).

  80. 80.

    , , , & A Cas9-guide RNA complex preorganized for target DNA recognition. Science 348, 1477–1481 (2015).

  81. 81.

    et al. Dynamics of CRISPR-Cas9 genome interrogation in living cells. Science 350, 823–826 (2015).

  82. 82.

    , , , & DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014).

  83. 83.

    et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

  84. 84.

    et al. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat. Biotechnol. 33, 1293–1298 (2015).

  85. 85.

    et al. Structure and engineering of Francisella novicida Cas9. Cell 164, 950–961 (2016).

  86. 86.

    et al. DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat. Methods 12, 1150–1156 (2015).

  87. 87.

    , , & Conformational control of DNA target cleavage by CRISPR-Cas9. Nature 527, 110–113 (2015).

  88. 88.

    et al. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage. Science 351, 867–871 (2016).

  89. 89.

    et al. Cas9 gRNA engineering for genome editing, activation and repression. Nat. Methods 12, 1051–1054 (2015).

  90. 90.

    et al. Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat. Biotechnol. 33, 1159–1161 (2015).

  91. 91.

    et al. Targeted and genome-wide sequencing reveal single nucleotide variations impacting specificity of Cas9 in human stem cells. Nat. Commun. 5, 5507 (2014).

  92. 92.

    et al. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011).

  93. 93.

    et al. Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Nat. Biotechnol. 32, 670–676 (2014).

  94. 94.

    et al. Protospacer adjacent motif (PAM)-distal sequences engage CRISPR Cas9 DNA target cleavage. PLoS ONE 9, e109213 (2014).

  95. 95.

    , , , & A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture. Nucleic Acids Res. 43, 3389–3404 (2015).

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Acknowledgements

J.K.J. is supported by the US National Institutes of Health (NIH) Director's Pioneer Award (DP1GM105378) and by the Jim and Ann Orr Massachusetts General Hospital (MGH) Research Scholar Award. SQT is supported by an MGH Tosteson Award.

Author information

Affiliations

  1. Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, 149 13th Street, Charlestown, Massachusetts 02129, USA; and the Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Shengdar Q. Tsai
    •  & J. Keith Joung

Authors

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  2. Search for J. Keith Joung in:

Competing interests

J.K.J. is a consultant for Horizon Discovery. J.K.J. has financial interests in Editas Medicine, Hera Testing Laboratories, Poseida Therapeutics and Transposagen Biopharmaceuticals. J.K.J.'s interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. S.Q.T. and J.K.J. are co-founders of Beacon Genomics, a company that is commercializing methods for determining nuclease specificity.

Corresponding authors

Correspondence to Shengdar Q. Tsai or J. Keith Joung.

Glossary

Homology-directed repair

(HDR). A DNA repair pathway that depends on sequence homology to effect repair. A user-supplied 'donor' template can be used to introduce precise alterations of choice with this repair pathway.

Non-homologous end-joining

(NHEJ). A DNA repair pathway in which the double-stranded break (DSB) ends are directly ligated together without a requirement for homology. Variable length insertion or deletion mutations can frequently occur as a consequence of NHEJ-mediated DSB repair.

Point mutations

Genetic changes of a single DNA base pair.

CRISPR

Components of an adaptive immunity system found in bacteria.

CRISPR RNA

(crRNA). Small RNA that contains sequence complementarity to the protospacer and a short repetitive sequence with complementarity to trans-activating crRNA.

Trans-activating crRNA

(tracrRNA). A small trans-encoded RNA that has a portion of sequence complementarity with the CRISPR RNA (crRNA) and is required for Cas9 nuclease activity.

Protospacer

Target sequence for CRISPR interference, flanked by CRISPR repeats.

Protospacer adjacent motif

(PAM). Sequence required to licence Cas9 for cleavage, it is adjacent to the target sequence or protospacer.

Bulges

Gaps in base pairing between target DNA or guide RNA at an RNA-guided nuclease target≈site.

Rolling circle amplification

A method for generating many concatemerized copies of a circular template using a strand-displacing polymerase.

High-throughput sequencing

A method for sequencing populations of DNA molecules, typically with short (<300 bp) reads that have error rates an order of magnitude or more higher than standard long-read Sanger sequencing.

GUIDE-seq

(Genome-wide unbiased identification of DSBs enabled by sequencing). A cell-based method for genome-wide discovery of nuclease-induced double-stranded breaks (DSBs) based on efficient tag integration, tag-specific amplification and high-throughput sequencing.

Double-stranded oligodeoxynucleotide

(dsODN). Used as an integrated genetic tag in genome-wide unbiased identification of double-stranded breaks enabled by sequencing (GUIDE-seq).

HTGTS

(High-throughput genome-wide translocation sequencing). A method to detect nuclease-induced off-target double-stranded breaks by observation of translocation junctions.

BLESS

(Breaks labelling, enrichment on streptavidin and next-generation sequencing). A cell-based method for genome-wide discovery of nuclease-induced double-stranded breaks based on cell fixing, nuclei isolation, in situ ligation, enrichment and high-throughput sequencing.

Digested genome sequencing

(Digenome-seq). An in vitro method for detecting Cas9 cleavage of genomic DNA by whole-genome sequencing.

Cas9 nickases

(Cas9n). Engineered variants of Cas9 in which one of the two nuclease domains has been catalytically inactivated, which results in the nicking of only one DNA strand and leaving the other strand intact.

DNA curtains assay

A single-molecule assay for the visualization of protein interactions with individual DNA strands or 'curtains'.