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. 2020 Aug 17;11:4132. doi: 10.1038/s41467-020-17952-5

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© The Author(s) 2020

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. 2 — a Bio-layer interferometry (BLI) analysis of binding kinetics for dead Cas9 ribonucleoprotein complex to an immobilised biotinylated DNA substrate. Cas9 was precomplexed with tracrRNA and (NT) non-targeting control gRNA; (gRNA) 20-nt VEGFA gRNA with 4 mismatches; (GUARD RNA) a 15-nt GUARD RNA targeting the off-target site or no guide RNA. b CRISPR GUARD design. Schematic showing GUARD RNAs designs. Variation in length (14-nt versus 15-nt) and protospacer positioning likely influence binding energy and competition with the on-target gRNA (i.e., competitive/overlapping, non-competitive/proximal). Mismatches in the gRNA are shown in red. RNA bases overlapping the gRNA PAM are shown in bold. c Competition between on-target gRNA and GUARD RNA can be reduced by proximal positioning. On-target (VEGFA) and off-target (CAVIN4) DNA abundance was measured by qPCR following an in vitro Cas9 DNA cleavage assay. The corresponding GUARD RNA was added at a molar ratio of 5:1 to the VEGFA on-target gRNA (100 nM to 20 nM). d GUARD RNAs are more effective at blocking Cas9-mediated DNA cutting with pre-incubation in vitro. On-target (VEGFA) and off-target (CAVIN4) DNA abundance was measured by qPCR following an in vitro Cas9 DNA cleavage assay, with a 30 min pre-incubation with GUARD RNAs and Cas9, before addition of the on-target gRNA and Cas9. Increasing molar ratios of GUARD RNA to the VEGFA on-target gRNA were used (10:10, 20:10, 50:10, 100:10 nM). e GUARD RNAs are effective in blocking Cas9-mediated DNA cleavage of off-target sites within a 10 bp window of the gRNA PAM. Synthetic DNA fragments containing the VEGFA gRNA off-target site CAVIN4 positioned progressively further away from the GUARD RNA binding site were quantified by qPCR following an in vitro Cas9 DNA cleavage assay (50:10 nM GUARD RNA to gRNA ratio). f CRISPR GUARD is effective at blocking off-target editing in cells. Indel rates from NGS of amplicons from Cas9-expressing HEK293 cells transfected with VEGFA gRNA and multiple GUARD RNA designs for protection of the CAVIN4 proximal off-target site (25:25 nM GUARD RNA to gRNA ratio). Data represent the mean of two (f) independent experiments, or the mean ± SD of three (c, e) or five (d) independent experiments with symbols representing each replicate. For a, data are representative of two independent experiments. NT non-targeting gRNA, UTC untransfected control. Unpaired, two-tailed student’s t-test. For c, *P = 0.0208, **P = 0.0083. For d, *P = 0.0164, **P = 0.0021 (VEGFA) and **P = 0.007 (CAVIN4), ***P = 0.0003, ***P < 0.0001. For e, **P = 0.0041 and ***P = 0.0001. Source data are provided as a Source Data file.