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RNA-Guided Genome Editing of Mammalian Cells

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Gene Correction

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1114))

Abstract

The microbial CRISPR-Cas adaptive immune system can be harnessed to facilitate genome editing in eukaryotic cells (Cong L et al., Science 339, 819–823, 2013; Mali P et al., Science 339, 823–826, 2013). Here we describe a protocol for the use of the RNA-guided Cas9 nuclease from the Streptococcus pyogenes type II CRISPR system to achieve specific, scalable, and cost-efficient genome editing in mammalian cells.

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References

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Urnov FD et al (2005) Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435:646–651

    Article  CAS  PubMed  Google Scholar 

  4. Porteus MH, Baltimore D (2003) Chimeric nucleases stimulate gene targeting in human cells. Science 300:763

    Article  PubMed  Google Scholar 

  5. Miller JC et al (2007) An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25:778–785

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  7. Miller JC et al (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148

    Article  CAS  PubMed  Google Scholar 

  8. Sanjana NE et al (2012) A transcription activator-like effector toolbox for genome engineering. Nat Protoc 7:171–192

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  9. Stoddard BL (2005) Homing endonuclease structure and function. Q Rev Biophys 38:49–95

    Article  CAS  PubMed  Google Scholar 

  10. Rouet P, Smih F, Jasin M (1994) Expression of a site-specific endonuclease stimulates homologous recombination in mammalian cells. Proc Natl Acad Sci U S A 91:6064–6068

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Cui X et al (2011) Targeted integration in rat and mouse embryos with zinc-finger nucleases. Nat Biotechnol 29:64–67

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Maier D et al (2013) Efficient clinical scale gene modification via zinc finger nuclease targeted disruption of the HIV co-receptor CCR5. Hum Gene Ther 24:245–258

    Article  CAS  PubMed Central  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  16. Garneau JE et al (2010) The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature 468:67–71

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Jore MM et al (2011) Structural basis for CRISPR RNA-guided DNA recognition by Cascade. Nat Struct Mol Biol 18:529–536

    Article  CAS  PubMed  Google Scholar 

  21. Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Almendros C (2009) Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155:733–740

    Article  CAS  PubMed  Google Scholar 

  22. Qi LS et al (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Gray SJ et al (2011) Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Hum Gene Ther 22:1143–1153

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Cho SW, Kim S, Kim JM, Kim JS (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31:230–232

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

We thank Randall Platt for comments and members of the Zhang Lab for discussion, support, and advice. N.P. is supported by the National Science Foundation Graduate Research Fellowship, Primary Award #1122374. P.D.H. is a James Mills Pierce Fellow. F.Z. is supported by the NIH Transformative R01 Award (R01-NS073124); the NIH Director’s Pioneer Award (DP1-MH100706); the Keck, McKnight, Gates, Damon Runyon, Searle Scholars, Merkin, Klingenstein, and Simons Foundations; Bob Metcalfe; Mike Boylan; and Jane Pauley. Sequence and reagent information are available through http://www.genome-engineering.org.

Author information

Authors and Affiliations

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

    Neena K. Pyzocha & Patrick D. Hsu

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, USA

    Neena K. Pyzocha

  3. Broad Institute of MIT and Harvard, Massachusetts Institute of Technology, Cambridge, MA, USA

    F. Ann Ran

  4. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Feng Zhang

  5. McGovern Institute for Brain Research, MIT, Cambridge, MA, USA

    Feng Zhang

  6. Department of Brain and Cognitive Sciences, MIT, Cambridge, MA, USA

    Feng Zhang

  7. Department of Biological Engineering, MIT, Cambridge, MA, USA

    Feng Zhang

Authors
  1. Neena K. Pyzocha
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  2. F. Ann Ran
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  3. Patrick D. Hsu
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  4. Feng Zhang
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Editor information

Editors and Affiliations

  1. Georgia Institute of Technology, Atlanta, Georgia, USA

    Francesca Storici

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Pyzocha, N.K., Ran, F.A., Hsu, P.D., Zhang, F. (2014). RNA-Guided Genome Editing of Mammalian Cells. In: Storici, F. (eds) Gene Correction. Methods in Molecular Biology, vol 1114. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-761-7_17

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  • DOI: https://doi.org/10.1007/978-1-62703-761-7_17

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-760-0

  • Online ISBN: 978-1-62703-761-7

  • eBook Packages: Springer Protocols

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