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An engineered ScCas9 with broad PAM range and high specificity and activity

Nature Biotechnology volume 38, pages 1154–1158 (2020)Cite this article

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A Publisher Correction to this article was published on 19 June 2020

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Abstract

CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. In the present study, we combine protein motifs from several orthologs to engineer two variants of Streptococcus canis Cas9—Sc++ and a higher-fidelity mutant HiFi-Sc++—that have simultaneously broad 5′-NNG-3′ PAM compatibility, robust DNA-cleavage activity and minimal off-target activity. Sc++ and HiFi-Sc++ extend the use of CRISPR editing for diverse applications.

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Fig. 1: Engineering and characterization of broad PAM recognition by Sc++.
Fig. 2: Broad, efficient and specific genome-editing capabilities of Sc++.

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Data availability

All data needed to evaluate the conclusions in the paper are present in the paper and supplementary tables. All source data can be found at the National Center for Biotechnology Information’s Sequence Read Archive under the accession code PRJNA623032. Nuclease plasmids are available on Addgene.

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Acknowledgements

We thank E. Boyden for access to cell culture, in addition to N. Gershenfeld and S. Zhang for shared lab equipment. We further thank A. Hennes and M. Topalli for technical assistance. This work was supported by the consortium of sponsors of the MIT Media Lab and the MIT Center for Bits and Atoms, as well as the CHDI Foundation. GUIDE-seq work was supported by a grant (no. GM115911) to E.J.S. from the US National Institutes of Health.

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Author notes

  1. These authors contributed equally: Pranam Chatterjee, Noah Jakimo.

Authors and Affiliations

  1. MIT Center for Bits and Atoms, Massachusetts Institute of Technology, Cambridge, MA, USA

    Pranam Chatterjee, Noah Jakimo, Sabrina R. T. Koseki, Emma Tysinger & Joseph Jacobson

  2. MIT Media Lab, Massachusetts Institute of Technology, Cambridge, MA, USA

    Pranam Chatterjee, Noah Jakimo, Sabrina R. T. Koseki, Emma Tysinger, Rui Qing, Shilei Hao & Joseph Jacobson

  3. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA

    Jooyoung Lee, Nadia Amrani, Tomás Rodríguez & Erik J. Sontheimer

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  1. Pranam Chatterjee
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Contributions

P.C. and N.J. conceived the study. P.C. designed constructs, carried out genome-editing experiments, conducted data analyses and wrote the manuscript. J.L., N.A. and T.R. carried out GUIDE-seq experiments and analysis. S.R.T.K. and E.T. assisted in plasmid construction, transfection and sample preparation. R.Q. and S.H. performed protein expression analysis. All authors reviewed and edited the paper. J.M.J. and E.J.S. supervised the project.

Corresponding author

Correspondence to Pranam Chatterjee.

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Competing interests

P.C., N.J. and J.M.J. are listed as inventors for US Patent Application no. 20190218532: Streptococcus canis Cas9 as a genome engineering platform with novel PAM specificity.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–3.

Reporting summary

Supplementary Table 1

SgRNA sequences used in this study, grouped by editing context. Primers used for genomic amplification are also specified.

Supplementary Table 2

Curated dataset of indel formation efficiencies at each site (annotated by target sequence, genomic locus and PAM), for Fig. 2a.

Supplementary Table 3

Information for on- and off- target sites in Fig. 2c, primers used for NGS and summary of raw data used for plotted graphs.

Source data

Source Data Fig. 1

Unprocessed western blot.

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Chatterjee, P., Jakimo, N., Lee, J. et al. An engineered ScCas9 with broad PAM range and high specificity and activity. Nat Biotechnol 38, 1154–1158 (2020). https://doi.org/10.1038/s41587-020-0517-0

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