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sgRNA sequence motifs blocking efficient CRISPR/Cas9-mediated gene editing

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Item Type:Article
Title:sgRNA sequence motifs blocking efficient CRISPR/Cas9-mediated gene editing
Creators Name:Graf, R. and Li, X. and Chu, V.T. and Rajewsky, K.
Abstract:Cas9 nucleases can be programmed with single guide RNAs (sgRNAs) to mediate gene editing. High CRISPR/Cas9-mediated gene knockout efficiencies are essential for genetic screens and critically depend on the properties of the sgRNAs used. The specificity of an sgRNA is defined by its targeting sequence. Here, we discovered that two short sequence motifs at the 3′ end of the targeting sequence are almost exclusively present in inefficient sgRNAs of published sgRNA-activity datasets. By specific knock-in of sgRNA target sequences with or without these motifs and quantitative measurement of knockout efficiency, we show that the presence of these motifs in sgRNAs per se results in a 10-fold reduction of gene knockout frequencies. Mechanistically, the cause of the low efficiency differs between the two motifs. These sequence motifs are relevant for future sgRNA design approaches and studies of Cas9-DNA interactions.
Keywords:CRISPR/Cas9, Gene Targeting, Knockout Efficiency, sgRNA Design, sgRNA Efficiency, sgRNA Motif, scaffold RNA, CrispRGold, CRISPR Screening, Animals, Mice
Source:Cell Reports
ISSN:2211-1247
Publisher:Cell Press / Elsevier (U.S.A.)
Volume:26
Number:5
Page Range:1098-1103
Date:29 January 2019
Official Publication:https://doi.org/10.1016/j.celrep.2019.01.024
PubMed:View item in PubMed

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