The RNA-programmable DNA-endonuclease Cas9 is widely used for genome engineering, where a high degree of specificity is required. To investigate which features of Cas9 determine the sensitivity to mismatches along the target DNA, we performed in vitro biochemical assays and bacterial survival assays in Escherichia coli. We demonstrate that arginines in the Cas9 bridge helix influence guide RNA, and target DNA binding and cleavage. They cluster in two groups that either increase or decrease the Cas9 sensitivity to mismatches. We show that the bridge helix is essential for R-loop formation and that R63 and R66 reduce Cas9 specificity by stabilizing the R-loop in the presence of mismatches. Additionally, we identify Q768 that reduces sensitivity of Cas9 to protospacer adjacent motif-distal mismatches. The Cas9_R63A/Q768A variant showed increased specificity in human cells. Our results provide a firm basis for function- and structure-guided mutagenesis to increase Cas9 specificity for genome engineering.
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We thank F. Richter for plasmids (pRob12, pCDF_dCas9 and pACYC_sgRNA) and help with setting up the bacterial survival assay, K. Schmidt, S. Ressel and J. Boshart for technical help, M. Ugolini for help with flow cytometry, and members of the Charpentier laboratory for valuable discussions and critical reading of the manuscript. We thank S. Klages and N. Mages from the sequencing core facility of the Max Planck Institute for Molecular Genetics for help with amplicon sequencing. We thank the Alexander von Humboldt Foundation (AvH Professorship to E.C.), the Helmholtz Association (E.C.), the Max Planck Society (E.C.), the Max Planck Foundation (E.C.), the Göran Gustafsson Foundation (Göran Gustafsson Prize to E.C.), the Kempe Foundation (E.C.), Umeå University (E.C.) and the Swedish Research Council (E.C.) for funding this study. K.C. was a fellow of the Austrian Doctoral Program in RNA Biology.
E.C. is cofounder of ERS Genomics and CRISPR Therapeutics and is a member of the scientific advisory board of CRISPR Therapeutics. All other authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Bratovič, M., Fonfara, I., Chylinski, K. et al. Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches. Nat Chem Biol 16, 587–595 (2020). https://doi.org/10.1038/s41589-020-0490-4
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