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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. 3 — a Increasing GUARD RNA concentration leads to complete protection of Cas9 off-target sites in cells. Indel rates from NGS of amplicons from Cas9-expressing HEK293 cells transfected with VEGFA gRNA and increasing concentrations of 14-nt GUARD RNA for protection of the CAVIN4 off-target site (10:10, 20:10, 50:10, 100:10 nM). The total concentration of delivered RNA in each case was kept constant by co-delivery of NT gRNA (first condition is 100 nM NT). b Indel rates from amplicons sequencing of Cas9-expressing HEK293 cells transfected with EMX1 or VEGFA on-target gRNAs, and a single GUARD RNA (25:25 nM ratio). GUARD RNA positioning relative to the off-target protospacer sequence is shown schematically in each panel. Data represent the mean of two independent experiments with symbols representing each replicate. Source data are provided as a Source Data file.