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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. 3 — a Cas9-gRNA binding model in which guanine stretches upstream and downstream from the on-target PAM can function as local sliding PAMs at which the gRNA forms a bulged hybrid with the target DNA. The direction of Cas9 sliding is indicated with arrows, and the upstream and downstream sliding complexes are coloured in blue and red, respectively. Bulges are indicated by yellow stars. Stretches of guanine (G), representing possible PAM binding sites, are reported in the drawing and highlighted in red if used by the complex. b Cumulative distributions of indel frequencies of gRNAs categorised by the presence of local sliding PAMs at their targets (one-way ANOVA p value = 3.59E-57). Downstream n = 2239; upstream n = 1339; none n = 7565; up and downstream n = 459. T-test p value comparing upstream-none = 1.96E-10 (one-sided), downstream-none = 1.50E-40 (one-sided), up and downstream-none = 3.69E-02 (two-sided). c Boxplots of gRNA efficiencies from two datasets binned into low, medium and high CRISPRspec or CRISPRspecExt groups before (left) and after (right) the addition of local sliding contributions. Boxes represent the first and third quartiles (Q1 and Q3). The median is shown as a line and the mean by a triangle; 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)). Number of elements: 'Xiang 2021 test', CRISPRspec low n = 204, medium n = 1788, high n = 227, CRISPRspecExt low = 477, medium = 1377, high = 365; 'Hart 2015', CRISPRspec low = 18, medium = 1019, high = 204, CRISPRspecExt low = 79, medium = 774, high = 388. One-sided Kolmogorov–Smirnov two-sample test, Bonferroni-corrected within each dataset: 'Xiang 2021 test', CRISPRspec low-high = 9.61E-08, medium-high = 0.16, low-medium = 1.04E-07, CRISPRspecExt low-high = 6.63E-27, medium-high = 2.49E-06, low-medium = 3.21E-20; 'Hart 2015', CRISPRspec low-high = 0.06, medium-high = 0.36, low-medium = 0.20, CRISPRspecExt low-high = 1.91E-09, medium-high = 1.07E-12, low-medium = 1.69E-2. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.