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. 2022 May 30;13:3006. doi: 10.1038/s41467-022-30515-0

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Boxplots of indel frequencies produced by Cas9 variants in HEK293T cells at targets followed by different PAMs (X-axis). Each row represents a Cas9 variant, sorted from top to bottom by efficiency and inverse specificity. Boxplots are separated in two groups for upstream (left) and downstream (right) sliding. Boxes represent the first and third quartiles (Q1 and Q3). The median is shown as a line; whiskers extend up (or down) to the last (or first) value lower (or greater) than Q3+1.5*(Q3-Q1) (or Q1-1.5*(Q3-Q1)). One-sided t-test p values, Bonferroni-corrected by the number of Cas9 variants: from left to right, top to bottom: 1.00; 4.90E-03; 4.15E-09; 0.06; 9.60E-49; 5.53E-84; 0.83; 8.44E-03; 2.43E-07; 0.40; 1.03E-39; 1.78E-63; 1.00; 0.09; 6.87E-04; 1.00; 5.54E-06; 1.08E-12; 0.85; 0.32; 0.13; 1.00; 0.078; 1.16E-05; 0.71; 0.71; 0.32; 1.00; 0.59; 0.06. The alternative hypothesis is that gRNA efficiencies at targets with G₋₁ (left) or G₊₁ (right) have larger mean, respectively, than targets with H₋₁ or H₊₁. Number of elements in each box, from left to right, top to bottom: 265; 88; 261; 85; 267; 85; 263; 88; 260; 86; 265; 86; 265; 88; 262; 86; 267; 85; 264; 88; 260; 87; 265; 86; 265; 88; 260; 86; 267; 85; 264; 88; 259; 87; 265; 86; 265; 88; 262; 86; 267; 85; 265; 88; 260; 87; 265; 86; 265; 88; 262; 86; 266; 85; 264; 88; 260; 87; 264; 86. b Illustration of the sliding mechanism at N₋₁GNN₊₁ PAMs, and boxplots of indel frequencies in HEK293T cells produced by Cas9 variants with broadened PAM compatibility at targets with different PAMs (X-axis). Boxes are defined as in (a). One-sided t-test p values, Bonferroni-corrected by the number of Cas9 variants: from left to right, top to bottom: 2.30E-59; 8.68E-25; 2.12E-135; 1.94E-186; 7.06E-69; 1.00. The alternative hypothesis used is that gRNA efficiencies at GHHH (upstream case) or HHGH/HHHG (downstream case) targets have larger mean than at HHHH. Number of elements in each box, from left to right, top to bottom: 2322; 799; 2317; 778; 2322; 786; 772; 2317; 786; 770. Source data are provided as a Source Data file.