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Target-dependent nickase activities of the CRISPR–Cas nucleases Cpf1 and Cas9

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) machineries are prokaryotic immune systems that have been adapted as versatile gene editing and manipulation tools. We found that CRISPR nucleases from two families, Cpf1 (also known as Cas12a) and Cas9, exhibit differential guide RNA (gRNA) sequence requirements for cleavage of the two strands of target DNA in vitro. As a consequence of the differential gRNA requirements, both Cas9 and Cpf1 enzymes can exhibit potent nickase activities on an extensive class of mismatched double-stranded DNA (dsDNA) targets. These properties allow the production of efficient nickases for a chosen dsDNA target sequence, without modification of the nuclease protein, using gRNAs with a variety of patterns of mismatch to the intended DNA target. In parallel to the nicking activities observed with purified Cas9 in vitro, we observed sequence-dependent nicking for both perfectly matched and partially mismatched target sequences in a Saccharomyces cerevisiae system. Our findings have implications for CRISPR spacer acquisition, off-target potential of CRISPR gene editing/manipulation, and tool development using homology-directed nicking.

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Fig. 1: High-throughput assay for nicking and cleavage by CRISPR–Cas nucleases.
Fig. 2: Library-based assessment of nicking and cleavage activities of LbCpf1 on single mutant target variants.
Fig. 3: Library-based assessment of nicking and cleavage activities of LbCpf1 on double consecutive transversion and deletion target variants.
Fig. 4: Gel-based assessments of nicking and cleavage by LbCpf1 and Cas9.
Fig. 5: Assays for target-match-dependent nicking of precise and imprecise targets by Cas9 in vivo.

Data availability

The raw data that support our findings are available in Sequence Read Archive under accession no. PRJNA503740 (see Supplementary Table 2 for information on the data for each corresponding figure).

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Acknowledgements

We thank the D. Herschlag, P. Fineran, G. Hess, N. Jain, and colleagues in our laboratories for their input and discussion. We are grateful to J. A. Meacham for advice on the reagents used and C. Lee for lending reagents.

Author information

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Authors

Contributions

B.X.H.F conceived and designed the study, and carried out the experiments. A.Z.F. and G.B.R. participated in the experimental design and analysis of data. J.D.S. participated in the experimental design, execution, and discussion of the in vivo yeast experiments. R.T.F., M.M., and J.C. participated in enzyme purification and early investigations of nuclease activities. All authors participated in the contextualization of the results and in the preparation of the manuscript.

Corresponding authors

Correspondence to Becky Xu Hua Fu, G. Brett Robb or Andrew Z. Fire.

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Competing interests

R.T.F., M.M., J.C., and G.B.R. are employees of New England Biolabs. A provisional patent ‘Compositions and Methods for Nicking Target DNA Sequences’ related to this work has been filed by Stanford University (inventors: B.X.H.F., A.F., and J.D.S.).

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Supplementary information

Supplementary Information

Legend for Supplementary Table 1, Supplementary Table 2 and Supplementary Figures 1–54.

Reporting Summary

Supplementary Table 1

Retention scores for individual target sequence candidates from unc-22A mixed target library incubated with Cas9.

Supplementary Table 3

List of gRNAs and targets with corresponding sequences.

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Fu, B.X.H., Smith, J.D., Fuchs, R.T. et al. Target-dependent nickase activities of the CRISPR–Cas nucleases Cpf1 and Cas9. Nat Microbiol 4, 888–897 (2019). https://doi.org/10.1038/s41564-019-0382-0

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