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Implications of human genetic variation in CRISPR-based therapeutic genome editing

Abstract

CRISPR–Cas genome-editing methods hold immense potential as therapeutic tools to fix disease-causing mutations at the level of DNA. In contrast to typical drug development strategies aimed at targets that are highly conserved among individual patients, treatment at the genomic level must contend with substantial inter-individual natural genetic variation. Here we analyze the recently released ExAC and 1000 Genomes data sets to determine how human genetic variation impacts target choice for Cas endonucleases in the context of therapeutic genome editing. We find that this genetic variation confounds the target sites of certain Cas endonucleases more than others, and we provide a compendium of guide RNAs predicted to have high efficacy in diverse patient populations. For further analysis, we focus on 12 therapeutically relevant genes and consider how genetic variation affects off-target candidates for these loci. Our analysis suggests that, in large populations of individuals, most candidate off-target sites will be rare, underscoring the need for prescreening of patients through whole-genome sequencing to ensure safety. This information can be integrated with empirical methods for guide RNA selection into a framework for designing CRISPR-based therapeutics that maximizes efficacy and safety across patient populations.

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Figure 1: Human genetic variation substantially impacts the efficacy of RNA-guided endonucleases.
Figure 2: Selection of platinum targets maximizes population efficacy.
Figure 3: Human genetic variation substantially impacts the safety of CRISPR endonuclease therapeutics.
Figure 4: Gene- and population-specific variation inform therapeutic design.

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References

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR–Cas system. Cell 163, 759–771 (2015).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Lek, M. et al. Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285–291 (2016).

    Article  CAS  Google Scholar 

  6. 1000 Genomes Project Consortium. A map of human genome variation from population-scale sequencing. Nature 467, 1061–1073 (2010).

  7. 1000 Genomes Project Consortium. An integrated map of genetic variation from 1,092 human genomes. Nature 491, 56–65 (2012).

  8. 1000 Genomes Project Consortium. A global reference for human genetic variation. Nature 526, 68–74 (2015).

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  11. Makarova, K.S. et al. An updated evolutionary classification of CRISPR–Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).

    Article  CAS  Google Scholar 

  12. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  16. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Tsai, S.Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).

    Article  CAS  Google Scholar 

  20. Frock, R.L. et al. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat. Biotechnol. 33, 179–186 (2015).

    Article  CAS  Google Scholar 

  21. Kim, D. et al. Digenome-seq: genome-wide profiling of CRISPR–Cas9 off-target effects in human cells. Nat. Methods 12, 237–243, 1, 243 (2015).

    Article  CAS  Google Scholar 

  22. Lin, Y. 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).

    Article  CAS  Google Scholar 

  23. Kleinstiver, B.P. et al. Genome-wide specificities of CRISPR–Cas Cpf1 nucleases in human cells. Nat. Biotechnol. 34, 869–874 (2016).

    Article  CAS  Google Scholar 

  24. Kim, D. et al. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat. Biotechnol. 34, 863–868 (2016).

    Article  CAS  Google Scholar 

  25. Lu, Y.-F., Goldstein, D.B., Angrist, M. & Cavalleri, G. Personalized medicine and human genetic diversity. Cold Spring Harb. Perspect. Med. 4, a008581 (2014).

    Article  Google Scholar 

  26. Cameron, P. et al. SITE-Seq: a genome-wide method to measure Cas9 cleavage. Protocol Exchange http://dx.doi.org/10.1038/protex.2017.043 (2017).

  27. Tsai, S.Q. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR–Cas9 nuclease off-targets. Nat. Methods 14, 607–614 (2017).

    Article  CAS  Google Scholar 

  28. Yan, W.X. et al. BLISS is a versatile and quantitative method for genome-wide profiling of DNA double-strand breaks. Nat. Commun. 8, 15058 (2017).

    Article  CAS  Google Scholar 

  29. Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A. & Liu, D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    Article  CAS  Google Scholar 

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Acknowledgements

We would like to thank R. Macrae, L. Francioli, S. Jones, J. Strecker, D. Cox, I. Slaymaker, and W. Yan for helpful discussions and insights. F.Z. is a New York Stem Cell Foundation–Robertson Investigator. F.Z. is supported by the US National Institutes of Health through the National Institute of Mental Health (5DP1-MH100706 and 1R01-MH110049); the National Science Foundation; the New York Stem Cell Foundation; the Howard Hughes Medical Institute; the Simons Foundation; the Paul G. Allen Family Foundation; the Vallee Foundation; the Skoltech–MIT Next-Generation Program; James and Patricia Poitras; Robert Metcalfe; and David Cheng. The computer code and resources related to this work are available through the Zhang laboratory website (http://www.genome-engineering.org/) and GitHub (http://github.com/fengzhanglab).

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D.A.S. and F.Z. conceived the study; D.A.S. performed all experiments and analyses; D.A.S. and F.Z. wrote the manuscript.

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Correspondence to Feng Zhang.

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Competing interests

F.Z. is a founder of Editas Medicine and a scientific advisor for Editas Medicine and Horizon Discovery.

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Supplementary Figures 1–5 and Supplementary Tables 1–3. (PDF 7013 kb)

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Scott, D., Zhang, F. Implications of human genetic variation in CRISPR-based therapeutic genome editing. Nat Med 23, 1095–1101 (2017). https://doi.org/10.1038/nm.4377

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