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Genome engineering using the CRISPR-Cas9 system

Nature Protocols volume 8, pages 2281–2308 (2013)Cite this article

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Abstract

Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1–2 weeks, and modified clonal cell lines can be derived within 2–3 weeks.

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Figure 1: Schematic of the RNA-guided Cas9 nuclease.
Figure 2: DSB repair promotes gene editing.
Figure 3: Timeline and overview of experiments.
Figure 4: Target selection and reagent preparation.
Figure 5: Anticipated results for multiplex-sgRNA-targeted NHEJ.
Figure 6: Anticipated results for HDR in HEK and HUES9 cells.

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References

  1. Ding, Q. et al. A TALEN genome-editing system for generating human stem cell-based disease models. Cell Stem Cell 12, 238–251 (2013).

    Article  CAS  Google Scholar 

  2. Soldner, F. et al. Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations. Cell 146, 318–331 (2011).

    Article  CAS  Google Scholar 

  3. Carlson, D.F. et al. Efficient TALEN-mediated gene knockout in livestock. Proc. Natl. Acad. Sci. USA 109, 17382–17387 (2012).

    Article  CAS  Google Scholar 

  4. Geurts, A.M. et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 325, 433–433 (2009).

    Article  CAS  Google Scholar 

  5. Takasu, Y. et al. Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection. Insect Biochem. Molec. 40, 759–765 (2010).

    Article  CAS  Google Scholar 

  6. Watanabe, T. et al. Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases. Nat. Commun. 3, 1017 (2012).

    Article  Google Scholar 

  7. Porteus, M.H. & Baltimore, D. Chimeric nucleases stimulate gene targeting in human cells. Science 300, 763 (2003).

    Article  Google Scholar 

  8. Miller, J.C. et al. An improved zinc-finger nuclease architecture for highly specific genome editing. Nat. Biotechnol. 25, 778–785 (2007).

    Article  CAS  Google Scholar 

  9. Sander, J.D. et al. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat. Methods 8, 67–69 (2011).

    Article  CAS  Google Scholar 

  10. Wood, A.J. et al. Targeted genome editing across species using ZFNs and TALENs. Science 333, 307 (2011).

    Article  CAS  Google Scholar 

  11. Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757–761 (2010).

    Article  CAS  Google Scholar 

  12. Zhang, F. et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat. Biotechnol. 29, 149–153 (2011).

    Article  Google Scholar 

  13. Hockemeyer, D. et al. Genetic engineering of human pluripotent cells using TALE nucleases. Nat. Biotechnol. 29, 731–734 (2011).

    Article  CAS  Google Scholar 

  14. Reyon, D. et al. FLASH assembly of TALENs for high-throughput genome editing. Nat. Biotechnol. 30, 460–465 (2012).

    Article  CAS  Google Scholar 

  15. Boch, J. et al. Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326, 1509–1512 (2009).

    Article  CAS  Google Scholar 

  16. Moscou, M.J. & Bogdanove, A.J. A simple cipher governs DNA recognition by TAL effectors. Science 326, 1501 (2009).

    Article  CAS  Google Scholar 

  17. Sanjana, N.E. et al. A transcription activator-like effector toolbox for genome engineering. Nat. Protoc. 7, 171–192 (2012).

    Article  CAS  Google Scholar 

  18. Deveau, H., Garneau, J.E. & Moineau, S. CRISPR/Cas system and its role in phage-bacteria interactions. Annu. Rev. Microbiol. 64, 475–493 (2010).

    Article  CAS  Google Scholar 

  19. Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of bacteria and archaea. Science 327, 167–170 (2010).

    Article  CAS  Google Scholar 

  20. Makarova, K.S. et al. Evolution and classification of the CRISPR-Cas systems. Nat. Rev. Microbiol. 9, 467–477 (2011).

    Article  CAS  Google Scholar 

  21. Bhaya, D., Davison, M. & Barrangou, R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu. Rev. Genet. 45, 273–297 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  25. Cho, S.W., Kim, S., Kim, J.M. & Kim, J.S. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat. Biotechnol. 31, 230–232 (2013).

    Article  CAS  Google Scholar 

  26. Garneau, J.E. et al. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468, 67–71 (2010).

    Article  CAS  Google Scholar 

  27. Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    Article  CAS  Google Scholar 

  28. Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl. Acad. Sci. USA 109, E2579–E2586 (2012).

    Article  CAS  Google Scholar 

  29. Urnov, F.D., Rebar, E.J., Holmes, M.C., Zhang, H.S. & Gregory, P.D. Genome editing with engineered zinc-finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).

    Article  CAS  Google Scholar 

  30. Hsu, P.D. & Zhang, F. Dissecting neural function using targeted genome engineering technologies. ACS Chem. Neurosci. 3, 603–610 (2012).

    Article  CAS  Google Scholar 

  31. Perez, E.E. et al. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases. Nat. Biotechnol. 26, 808–816 (2008).

    Article  CAS  Google Scholar 

  32. Chen, F. et al. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat. Methods 8, 753–755 (2011).

    Article  CAS  Google Scholar 

  33. Saleh-Gohari, N. & Helleday, T. Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells. Nucleic Acids Res. 32, 3683–3688 (2004).

    Article  CAS  Google Scholar 

  34. Marraffini, L.A. & Sontheimer, E.J. CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA. Science 322, 1843–1845 (2008).

    Article  CAS  Google Scholar 

  35. Brouns, S.J. et al. Small CRISPR RNAs guide antiviral defense in prokaryotes. Science 321, 960–964 (2008).

    Article  CAS  Google Scholar 

  36. Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).

    Article  CAS  Google Scholar 

  37. Sapranauskas, R. et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 39, 9275–9282 (2011).

    Article  CAS  Google Scholar 

  38. Magadan, A.H., Dupuis, M.E., Villion, M. & Moineau, S. Cleavage of phage DNA by the Streptococcus thermophilus CRISPR3-Cas system. PLoS ONE 7, e40913 (2012).

    Article  CAS  Google Scholar 

  39. Zhang, Y. et al. Processing-Independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol. Cell 50, 488–503 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  41. Wang, H. et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910–918 (2013).

    Article  CAS  Google Scholar 

  42. Shen, B. et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res. 23, 720–723 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  44. Qi, L.S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    Article  CAS  Google Scholar 

  45. Chang, N. et al. Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos. Cell Res. 23, 465–472 (2013).

    Article  CAS  Google Scholar 

  46. Gratz, S.J. et al. Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics 4, 1029–1035 (2013).

    Article  Google Scholar 

  47. Friedland, A.E. et al. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat. Methods 10, 741–743 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  49. Schmid-Burgk, J.L., Schmidt, T., Kaiser, V., Honing, K. & Hornung, V. A ligation-independent cloning technique for high-throughput assembly of transcription activator-like effector genes. Nat. Biotechnol. 31, 76–81 (2013).

    Article  CAS  Google Scholar 

  50. Miller, J.C. et al. A TALE nuclease architecture for efficient genome editing. Nat. Biotechnol. 29, 143–148 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  52. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnol. 31, 822–826 (2013).

    Article  CAS  Google Scholar 

  53. Tuschl, T. Expanding small RNA interference. Nat. Biotechnol. 20, 446–448 (2002).

    Article  CAS  Google Scholar 

  54. Smithies, O., Gregg, R.G., Boggs, S.S., Koralewski, M.A. & Kucherlapati, R.S. Insertion of DNA sequences into the human chromosomal -globin locus by homologous recombination. Nature 317, 230–234 (1985).

    Article  CAS  Google Scholar 

  55. Thomas, K.R., Folger, K.R. & Capecchi, M.R. High frequency targeting of genes to specific sites in the mammalian genome. Cell 44, 419–428 (1986).

    Article  CAS  Google Scholar 

  56. Hasty, P., Rivera-Perez, J. & Bradley, A. The length of homology required for gene targeting in embryonic stem cells. Mol. Cell Biol. 11, 5586–5591 (1991).

    Article  CAS  Google Scholar 

  57. Wu, S., Ying, G.X., Wu, Q. & Capecchi, M.R. A protocol for constructing gene targeting vectors: generating knockout mice for the cadherin family and beyond. Nat. Protoc. 3, 1056–1076 (2008).

    Article  CAS  Google Scholar 

  58. Elliott, B., Richardson, C., Winderbaum, J., Nickoloff, J.A. & Jasin, M. Gene conversion tracts from double-strand break repair in mammalian cells. Mol. Cellular Biol. 18, 93–101 (1998).

    Article  CAS  Google Scholar 

  59. Guschin, D.Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol. Biol. 649, 247–256 (2010).

    Article  CAS  Google Scholar 

  60. Loman, N.J. et al. Performance comparison of benchtop high-throughput sequencing platforms. Nat. Biotechnol. 30, 434–439 (2012).

    Article  CAS  Google Scholar 

  61. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).

    Article  CAS  Google Scholar 

  62. Oliveira, T.Y. et al. Translocation capture sequencing: a method for high throughput mapping of chromosomal rearrangements. J. Immunol. Methods 375, 176–181 (2012).

    Article  CAS  Google Scholar 

  63. Gray, S.J. et al. Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Human Gene Ther. 22, 1143–1153 (2011).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank B. Holmes for help with computational tools. P.D.H. is a James Mills Pierce Fellow and D.A.S. is a National Science Foundation (NSF) pre-doctoral fellow. V.A. is supported by NIH Training Grants T32GM007753 and T32GM008313. This work was supported by an NIH Director's Pioneer Award (1DP1-MH100706); an NIH Transformative R01 grant (1R01-DK097768); the Keck, McKnight, Damon Runyon, Searle Scholars, Vallee, Merkin, Klingenstein and Simons Foundations; Bob Metcalfe; and Jane Pauley. Reagents are available to the academic community through Addgene and associated protocols; support forums and computational tools are available via the Zhang lab website (http://www.genome-engineering.org/).

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Author notes

  1. F Ann Ran and Patrick D Hsu: These authors contributed equally to this work.

Authors and Affiliations

  1. Broad Institute of Massachusetts Institute of Technology (MIT) and Harvard, Cambridge, Massachusetts, USA

    F Ann Ran, Patrick D Hsu, Jason Wright, Vineeta Agarwala, David A Scott & Feng Zhang

  2. McGovern Institute for Brain Research, Cambridge, Massachusetts, USA

    F Ann Ran, Patrick D Hsu, David A Scott & Feng Zhang

  3. Department of Brain and Cognitive Sciences, MIT, Cambridge, Massachusetts, USA.,

    F Ann Ran, Patrick D Hsu, David A Scott & Feng Zhang

  4. Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA

    F Ann Ran, Patrick D Hsu, David A Scott & Feng Zhang

  5. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA

    F Ann Ran & Patrick D Hsu

  6. Program in Biophysics, Harvard University, MIT, Cambridge, Massachusetts, USA

    Vineeta Agarwala

  7. Harvard-MIT Division of Health Sciences and Technology, MIT, Cambridge, Massachusetts, USA

    Vineeta Agarwala

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Contributions

F.A.R., P.D.H., J.W., D.A.S. and F.Z. designed and performed the experiments. V.A. contributed to the online tool. F.A.R., P.D.H. and F.Z. wrote the manuscript with help from all authors.

Corresponding author

Correspondence to Feng Zhang.

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Supplementary information

Supplementary Data 1

Supplementary sequences (PDF 97 kb)

Supplementary Data 2

pSpCas9(BB) plasmid sequence (TXT 12 kb)

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Ran, F., Hsu, P., Wright, J. et al. Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8, 2281–2308 (2013). https://doi.org/10.1038/nprot.2013.143

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