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Enhancing gene editing specificity by attenuating DNA cleavage kinetics

Abstract

Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genome editing. However the specificity of these reagents remains a concern, especially for therapeutic applications, given the potential mutagenic consequences of off-target cleavage. Here we have developed an approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites. For three ZFN pairs, we engineered single-residue substitutions in the FokI domain that preserved full on-target activity but showed a reduction in off-target indels of up to 3,000-fold. By combining this approach with substitutions that reduced the affinity of zinc fingers, we developed ZFNs specific for the TRAC locus that mediated 98% knockout in T cells with no detectable off-target activity at an assay background of ~0.01%. We anticipate that this approach, and the FokI variants we report, will enable routine generation of nucleases for gene editing with no detectable off-target activity.

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Fig. 1: Identification of FokI substitutions that improve ZFN on-target cleavage preference.
Fig. 2: Global suppression of off-target cleavage by the I479Q and Q481A FokI domain variants.
Fig. 3: Improving ZFN cleavage specificity via removal of a nonspecific DNA contact in the zinc finger repeat.
Fig. 4: Development of ZFNs for highly efficient modification of TRAC in T cells with no detectable off-target effects.

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Data availability

Illumina sequencing data underlying all key experiments have been deposited in the NCBI Sequence Read Archive under accession code PRJNA540312.

Code availability

Custom computer scripts used to perform the standard indel analysis and increased-sensitivity indel analysis can be found in Supplementary Note 2. Custom computer scripts used to automate more standard portions of the data analysis pipeline are available upon request.

References

  1. Urnov, F. D. et al. Genome editing with engineered zinc finger nucleases. Nat. Rev. Genet. 11, 636–646 (2010).

    Article  CAS  Google Scholar 

  2. Chen, J. S. et al. Enhanced proofreading governs CRISPR–Cas9 targeting accuracy. Nature 550, 407–410 (2017).

    Article  CAS  Google Scholar 

  3. Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).

    Article  CAS  Google Scholar 

  4. Tsai, S. Q. & Joung, J. K. Defining and improving the genome-wide specificities of CRISPR–Cas9 nucleases. Nat. Rev. Genet. 17, 300–312 (2016).

    Article  CAS  Google Scholar 

  5. Hacein-Bey-Abina, S. et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419 (2003).

    Article  CAS  Google Scholar 

  6. Donsante, A. et al. AAV vector integration sites in mouse hepatocellular carcinoma. Science 317, 477 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  8. Gao, X. et al. Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents. Nature 553, 217–221 (2018).

    Article  CAS  Google Scholar 

  9. Huang, C. H., Lee, K. C. & Doudna, J. A. Applications of CRISPR–Cas enzymes in cancer therapeutics and detection. Trends Cancer 4, 499–512 (2018).

    Article  CAS  Google Scholar 

  10. DiCarlo, J. E., Mahajan, V. B. & Tsang, S. H. Gene therapy and genome surgery in the retina. J. Clin. Invest. 128, 2177–2188 (2018).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Kim, Y. G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl Acad. Sci. USA 93, 1156–1160 (1996).

    Article  CAS  Google Scholar 

  13. Grizot, S. et al. Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease. Nucleic Acids Res. 37, 5405–5419 (2009).

    Article  CAS  Google Scholar 

  14. Miller, J. C. et al. Improved specificity of TALE-based genome editing using an expanded RVD repertoire. Nat. Methods 12, 465–471 (2015).

    Article  CAS  Google Scholar 

  15. Rebar, E. J. & Pabo, C. O. Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. Science 263, 671–673 (1994).

    Article  CAS  Google Scholar 

  16. Jarjour, J. et al. High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display. Nucleic Acids Res. 37, 6871–6880 (2009).

    Article  CAS  Google Scholar 

  17. Oakes, B. L. et al. Multi-reporter selection for the design of active and more specific zinc-finger nucleases for genome editing. Nat. Commun. 7, 10194 (2016).

    Article  CAS  Google Scholar 

  18. 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 

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

    Article  CAS  Google Scholar 

  20. 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 

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

    Article  CAS  Google Scholar 

  22. Guilinger, J. P., Thompson, D. B. & Liu, D. R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol. 32, 577–582 (2014).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Gao, L. et al. Engineered Cpf1 variants with altered PAM specificities. Nat. Biotechnol. 35, 789–792 (2017).

    Article  CAS  Google Scholar 

  27. Yin, H. et al. Partial DNA-guided Cas9 enables genome editing with reduced off-target activity. Nat. Chem. Biol. 14, 311–316 (2018).

    Article  CAS  Google Scholar 

  28. Ryan, D. E. et al. Improving CRISPR–Cas specificity with chemical modifications in single-guide RNAs. Nucleic Acids Res. 46, 792–803 (2018).

    Article  CAS  Google Scholar 

  29. 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 

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

    Article  CAS  Google Scholar 

  31. Kulcsár, P. I. et al. Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage. Genome Biol. 18, 190 (2017).

    Article  Google Scholar 

  32. DeWitt, M. A. et al. Selection-free genome editing of the sickle mutation in human adult hematopoietic stem/progenitor cells. Sci. Transl. Med. 8, 360ra134 (2016).

    Article  Google Scholar 

  33. Zhang, D. et al. Perfectly matched 20-nucleotide guide RNA sequences enable robust genome editing using high-fidelity SpCas9 nucleases. Genome Biol. 18, 191 (2017).

    Article  Google Scholar 

  34. Vakulskas, C. A. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med. 24, 1216–1224 (2018).

    Article  CAS  Google Scholar 

  35. Casini, A. et al. A highly specific SpCas9 variant is identified by in vivo screening in yeast. Nat. Biotechnol. 36, 265–271 (2018).

    Article  CAS  Google Scholar 

  36. Sternberg, S. H., LaFrance, B., Kaplan, M. & Doudna, J. A. Conformational control of DNA target cleavage by CRISPR–Cas9. Nature 527, 110–113 (2015).

    Article  CAS  Google Scholar 

  37. Dagdas, Y. S., Chen, J. S., Sternberg, S. H., Doudna, J. A. & Yildiz, A. A conformational checkpoint between DNA binding and cleavage by CRISPR–Cas9. Sci. Adv. 3, eaao0027 (2017).

    Article  Google Scholar 

  38. Raper, A. T., Stephenson, A. A. & Suo, Z. Functional insights revealed by the kinetic mechanism of CRISPR/Cas9. J. Am. Chem. Soc. 140, 2971–2984 (2018).

    Article  CAS  Google Scholar 

  39. Singh, D., Sternberg, S. H., Fei, J., Doudna, J. A. & Ha, T. Real-time observation of DNA recognition and rejection by the RNA-guided endonuclease Cas9. Nat. Commun. 7, 12778 (2016).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  41. Jiang, F. & Doudna, J. A. CRISPR–Cas9 structures and mechanisms. Annu. Rev. Biophys. 46, 505–529 (2017).

    Article  CAS  Google Scholar 

  42. De Ravin, S. S. et al. Targeted gene addition in human CD34+ hematopoietic cells for correction of X-linked chronic granulomatous disease. Nat. Biotechnol. 34, 424–429 (2016).

    Article  Google Scholar 

  43. Bisaria, N., Jarmoskaite, I. & Herschlag, D. Lessons from enzyme kinetics reveal specificity principles for RNA-guided nucleases in RNA interference and CRISPR-based genome editing. Cell Syst. 4, 21–29 (2017).

    Article  CAS  Google Scholar 

  44. Kaczorowski, T., Skowron, P. & Podhajska, A. J. Purification and characterization of the FokI restriction endonuclease. Gene 80, 209–216 (1989).

    Article  CAS  Google Scholar 

  45. Li, L., Wu, L. P., Clarke, R. & Chandrasegaran, S. C-terminal deletion mutants of the FokI restriction endonuclease. Gene 133, 79–84 (1993).

    Article  CAS  Google Scholar 

  46. 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 

  47. Kim, J. G., Takeda, Y., Matthews, B. W. & Anderson, W. F. Kinetic studies on Cro repressor–operator DNA interaction. J. Mol. Biol. 196, 149–158 (1987).

    Article  CAS  Google Scholar 

  48. Beane, J. D. et al. Clinical scale zinc finger nuclease-mediated gene editing of PD-1 in tumor infiltrating lymphocytes for the treatment of metastatic melanoma. Mol. Ther. 23, 1380–1390 (2015).

    Article  CAS  Google Scholar 

  49. Jen-Jacobson, L. Protein–DNA recognition complexes: conservation of structure and binding energy in the transition state. Biopolymers 44, 153–180 (1997).

    Article  CAS  Google Scholar 

  50. Pattanayak, V., Ramirez, C. L., Joung, J. K. & Liu, D. R. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection. Nat. Methods 8, 765–770 (2011).

    Article  CAS  Google Scholar 

  51. Pavletich, N. P. & Pabo, C. O. Zinc finger–DNA recognition: crystal structure of a Zif268–DNA complex at 2.1 Å. Science 252, 809–817 (1991).

    Article  CAS  Google Scholar 

  52. Khalil, A. S. et al. A synthetic biology framework for programming eukaryotic transcription functions. Cell 150, 647–658 (2012).

    Article  CAS  Google Scholar 

  53. Bauer, D. E. et al. An erythroid enhancer of BCL11A subject to genetic variation determines fetal hemoglobin level. Science 342, 253–257 (2013).

    Article  CAS  Google Scholar 

  54. Vierstra, J. et al. Functional footprinting of regulatory DNA. Nat. Methods 12, 927–930 (2015).

    Article  CAS  Google Scholar 

  55. Paschon, D. E. et al. Diversifying the structure of zinc finger nucleases for high precision editing. Nat. Commun. 10, 1133 (2019).

    Article  Google Scholar 

  56. Pernstich, C. & Halford, S. E. Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance energy transfer. Nucleic Acids Res. 40, 1203–1213 (2012).

    Article  CAS  Google Scholar 

  57. Kim, J. S. & Pabo, C. O. Getting a handhold on DNA: design of poly-zinc finger proteins with femtomolar dissociation constants. Proc. Natl Acad. Sci. USA 95, 2812–2817 (1998).

    Article  CAS  Google Scholar 

  58. Akcakaya, P. et al. In vivo CRISPR editing with no detectable genome-wide off-target mutations. Nature 561, 416–419 (2018).

    Article  CAS  Google Scholar 

  59. Liu, P. et al. Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A. J. Biol. Chem. 276, 11323–11334 (2001).

    Article  CAS  Google Scholar 

  60. Doyon, Y. et al. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat. Methods 8, 74–79 (2011).

    Article  CAS  Google Scholar 

  61. Wah, D. A., Hirsch, J. A., Dorner, L. F., Schildkraut, I. & Aggarwal, A. K. Structure of the multimodular endonuclease FokI bound to DNA. Nature 388, 97–100 (1997).

    Article  CAS  Google Scholar 

  62. Orlando, S. et al. Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res. 38, e152 (2010).

    Article  Google Scholar 

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Acknowledgements

We thank M. Lal for performing preliminary experiments.

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Authors and Affiliations

Authors

Contributions

J.C.M. and E.J.R. designed experiments, supervised experiments, analyzed data and wrote the paper. F.F., D.P.P. and P.L. designed experiments, performed experiments and analyzed data. A.R., D.E.P. and L.Z. designed experiments, supervised experiments and analyzed data. G.L. designed experiments. D.A.S. and Y.R.B. wrote custom computer code. S.J.H. supervised experiments. C.B.P., D.F.X., H.W.R., N.A.S., S.C.L., T.W. and Y.Z. performed experiments.

Corresponding author

Correspondence to Edward J. Rebar.

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All authors are full-time employees of Sangamo Therapeutics.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–15, Supplementary Tables 1–20 and Supplementary Notes 1 and 2

Reporting Summary

Supplementary Table 21

Detailed information for high-sensitivity OT1 indel assay results for K562 cells treated with Q481A AAVS1 ZFNs or GFP

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Miller, J.C., Patil, D.P., Xia, D.F. et al. Enhancing gene editing specificity by attenuating DNA cleavage kinetics. Nat Biotechnol 37, 945–952 (2019). https://doi.org/10.1038/s41587-019-0186-z

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