Genome Engineering Using CRISPR-Cas9 System

Part of the Methods in Molecular Biology book series (MIMB, volume 1239)

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is an adaptive immune system that exists in a variety of microbes. It could be engineered to function in eukaryotic cells as a fast, low-cost, efficient, and scalable tool for manipulating genomic sequences. In this chapter, detailed protocols are described for harnessing the CRISPR-Cas9 system from Streptococcus pyogenes to enable RNA-guided genome engineering applications in mammalian cells. We present all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs (gRNAs) and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination. These tools provide researchers with new instruments that accelerate both forward and reverse genetics efforts.

Key words

Genome engineering Cas9 CRISPR CRISPR-Cas9 PAM Guide RNA sgRNA DNA cleavage Mutagenesis Homologous recombination Streptococcus pyogenes 

References

  1. 1.
    Porteus MH, Baltimore D (2003) Chimeric nucleases stimulate gene targeting in human cells. Science 300(5620):763. doi:10.1126/science.1078395, 300/5620/763 [pii]PubMedCrossRefGoogle Scholar
  2. 2.
    Miller JC, Holmes MC, Wang J, Guschin DY, Lee YL, Rupniewski I, Beausejour CM, Waite AJ, Wang NS, Kim KA, Gregory PD, Pabo CO, Rebar EJ (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25(7):778–785. doi:10.1038/nbt1319 PubMedCrossRefGoogle Scholar
  3. 3.
    Sander JD, Dahlborg EJ, Goodwin MJ, Cade L, Zhang F, Cifuentes D, Curtin SJ, Blackburn JS, Thibodeau-Beganny S, Qi Y, Pierick CJ, Hoffman E, Maeder ML, Khayter C, Reyon D, Dobbs D, Langenau DM, Stupar RM, Giraldez AJ, Voytas DF, Peterson RT, Yeh JR, Joung JK (2011) Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat Methods 8(1):67–69. doi:10.1038/nmeth.1542 PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Wood AJ, Lo TW, Zeitler B, Pickle CS, Ralston EJ, Lee AH, Amora R, Miller JC, Leung E, Meng X, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Meyer BJ (2011) Targeted genome editing across species using ZFNs and TALENs. Science 333(6040):307. doi:10.1126/science.1207773 PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Stoddard BL (2005) Homing endonuclease structure and function. Q Rev Biophys 38(1):49–95. doi:10.1017/S0033583505004063 PubMedCrossRefGoogle Scholar
  6. 6.
    Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S, Lahaye T, Nickstadt A, Bonas U (2009) Breaking the code of DNA binding specificity of TAL-type III effectors. Science 326(5959):1509–1512. doi:10.1126/science.1178811, 1178811 [pii]PubMedCrossRefGoogle Scholar
  7. 7.
    Moscou MJ, Bogdanove AJ (2009) A simple cipher governs DNA recognition by TAL effectors. Science 326(5959):1501. doi:10.1126/science.1178817, 1178817 [pii]PubMedCrossRefGoogle Scholar
  8. 8.
    Zhang F, Cong L, Lodato S, Kosuri S, Church GM, Arlotta P (2011) Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol 29(2):149–153. doi:10.1038/nbt.1775 PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29(2):143–148. doi:10.1038/nbt.1755 PubMedCrossRefGoogle Scholar
  10. 10.
    Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF (2010) Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186(2):757–761. doi:10.1534/genetics.110.120717, genetics.110.120717 [pii]PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Reyon D, Tsai SQ, Khayter C, Foden JA, Sander JD, Joung JK (2012) FLASH assembly of TALENs for high-throughput genome editing. Nat Biotechnol 30(5):460–465. doi:10.1038/nbt.2170 PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Deveau H, Garneau JE, Moineau S (2010) CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol 64:475–493. doi:10.1146/annurev.micro.112408.134123 PubMedCrossRefGoogle Scholar
  13. 13.
    Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327(5962):167–170. doi:10.1126/science.1179555 PubMedCrossRefGoogle Scholar
  14. 14.
    Makarova KS, Haft DH, Barrangou R, Brouns SJ, Charpentier E, Horvath P, Moineau S, Mojica FJ, Wolf YI, Yakunin AF, van der Oost J, Koonin EV (2011) Evolution and classification of the CRISPR-Cas systems. Nat Rev Microbiol 9(6):467–477. doi:10.1038/nrmicro2577 PubMedCrossRefGoogle Scholar
  15. 15.
    Bhaya D, Davison M, Barrangou R (2011) CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 45:273–297. doi:10.1146/annurev-genet-110410-132430 PubMedCrossRefGoogle Scholar
  16. 16.
    Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E (2011) CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471(7340):602–607. doi:10.1038/nature09886 PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821. doi:10.1126/science.1225829 PubMedCrossRefGoogle Scholar
  18. 18.
    Garneau JE, Dupuis ME, Villion M, Romero DA, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadan AH, Moineau S (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468(7320):67–71. doi:10.1038/nature09523 PubMedCrossRefGoogle Scholar
  19. 19.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339(6121):819–823. doi:10.1126/science.1231143 PubMedCentralPubMedCrossRefGoogle Scholar
  20. 20.
    Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 339(6121):823–826. doi:10.1126/science.1232033 PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J (2013) RNA-programmed genome editing in human cells. Elife 2:e00471. doi:10.7554/eLife.00471 PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31(3):227–229. doi:10.1038/nbt.2501 PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153(4):910–918. doi:10.1016/j.cell.2013.04.025 PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R (2013) One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell 154(6):1370–1379. doi:10.1016/j.cell.2013.08.022 PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41(7):4336–4343. doi:10.1093/nar/gkt135 PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31(3):233–239. doi:10.1038/nbt.2508 PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Cho SW, Kim S, Kim JM, Kim JS (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31(3):230–232. doi:10.1038/nbt.2507 PubMedCrossRefGoogle Scholar
  28. 28.
    Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X, Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol 31(9):827–832. doi:10.1038/nbt.2647 PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD (2013) High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 31(9):822–826. doi:10.1038/nbt.2623 PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31(9):839–843. doi:10.1038/nbt.2673 PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM (2013) CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31(9):833–838. doi:10.1038/nbt.2675 PubMedCrossRefGoogle Scholar
  32. 32.
    Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F (2013) Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154(6):1380–1389. doi:10.1016/j.cell.2013.08.021 PubMedCrossRefGoogle Scholar
  33. 33.
    Cheng AW, Wang H, Yang H, Shi L, Katz Y, Theunissen TW, Rangarajan S, Shivalila CS, Dadon DB, Jaenisch R (2013) Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system. Cell Res 23(10):1163–1171. doi:10.1038/cr.2013.122 PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Maeder ML, Linder SJ, Cascio VM, Fu Y, Ho QH, Joung JK (2013) CRISPR RNA-guided activation of endogenous human genes. Nat Methods 10(10):977–979. doi:10.1038/nmeth.2598 PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS (2013) CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154(2):442–451. doi:10.1016/j.cell.2013.06.044 PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152(5):1173–1183. doi:10.1016/j.cell.2013.02.022 PubMedCentralPubMedCrossRefGoogle Scholar
  37. 37.
    Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA (2013) RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat Methods 10(10):973–976. doi:10.1038/nmeth.2600 PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Fu Y, Sander JD, Reyon D, Cascio VM, Joung JK (2014) Improving CRISPR-Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 32(3):279–284. doi:10.1038/nbt.2808 PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Guilinger JP, Thompson DB, Liu DR (2014) Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat Biotechnol 32:577. doi:10.1038/nbt.2909 PubMedCrossRefGoogle Scholar
  40. 40.
    Tsai SQ, Wyvekens N, Khayter C, Foden JA, Thapar V, Reyon D, Goodwin MJ, Aryee MJ, Joung JK (2014) Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing. Nat Biotechnol 32:569. doi:10.1038/nbt.2908 PubMedCrossRefGoogle Scholar
  41. 41.
    Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308. doi:10.1038/nprot.2013.143PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Broad Institute of MIT and HarvardCambridgeUSA
  2. 2.McGovern Institute for Brain Research, Department of Brain and Cognitive SciencesDepartment of Biological Engineering, Massachusetts Institute of TechnologyCambridgeUSA

Personalised recommendations