Abstract
The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas acquired immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but it can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences, and we identify an unexpectedly versatile Cas9 protein from Neisseria meningitidis. We provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins.
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Acknowledgements
We thank P.B. Stranges for protein alignments and W.L. Chew for helpful discussions. This work was supported by US National Institutes of Health NHGRI grant P50 HG005550, US Department of Energy grant DE-FG02-02ER63445 and the Wyss Institute for Biologically Inspired Engineering.
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K.M.E. and P.M. conceived of the study; K.M.E. and P.M. designed the experiments; K.M.E., J.L.B. and S.J.Y. performed experiments in E. coli; J.L.B. wrote analysis software; P.M. and M.M. performed experiments in human cells; K.M.E. and P.M. analyzed results; and K.M.E. and P.M. wrote the manuscript with input from G.M.C.
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The authors have filed for patents concerning the use of Cas9 proteins for gene targeting and regulation.
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Supplementary Text and Figures
Supplementary Figures 1–7, Supplementary Tables 1–3, Supplementary Notes 1–4 and Supplementary Software 1 and 2 (PDF 2806 kb)
Supplementary Data
Depletion tables for candidate NM PAMs, ST1 PAMs and TD PAMs; total MiSeq reads for each library; MiSeq clustering and read summaries; and source data for supplementary figures (XLS 254 kb)
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Esvelt, K., Mali, P., Braff, J. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat Methods 10, 1116–1121 (2013). https://doi.org/10.1038/nmeth.2681
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DOI: https://doi.org/10.1038/nmeth.2681
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