sgRNA Sequence Motifs Blocking Efficient CRISPR/Cas9-Mediated Gene Editing

Summary: 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. : CRISPR/Cas9-mediated gene editing efficiency critically depends on the properties of the sgRNAs used. Graf et al. show that the presence of two position-specific sequence motifs in the targeting sequence of sgRNAs per se results in a severe reduction of gene knockout frequencies. Keywords: CRISPR/Cas9, gene targeting, knockout efficiency, sgRNA design, sgRNA efficiency, sgRNA motif, scaffold RNA, CrispRGold, CRISPR screening