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3006 barcody al5/2/2023 In the case of the most broadly used SpCas9 protein, DSBs are introduced by the HNH and RuvC nuclease domains 3 nt upstream from the PAM 3.Ī large number of in silico methods for gRNA design report that the cleavage efficiency of Cas9 can largely vary due to the sequence and structural properties of the gRNA 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. Once a stable gRNA–DNA heteroduplex is formed, double-strand breaks (DSBs) are produced on the DNA by the Cas9 nuclease domains. In this study we refer to this canonical PAM sequence as 5′-N −1GGN +1-3′, thus including both the GG binding motif and its 1-bp flanking sequence context (N −1 and N +1) in the definition of PAM. The canonical PAM of SpCas9 is 5′- 3′ “NGG”. Cleavage by the widely studied Streptococcus pyogenes Cas9 ortholog (SpCas9), hereafter referred to as Cas9, is mediated by a 20-nt segment of a guide RNA (gRNA) complementary to a target DNA sequence preceding a protospacer adjacent motif (PAM), where the Cas9 is recruited 2. The bacterial CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 endonuclease has been transformed into a powerful genome-editing tool that is nowadays broadly applied in biological, agricultural and medical research 1. Our results provide insights into the mechanisms of Cas9-PAM compatibility and cleavage activation, underlining the importance of accounting for local sliding in gRNA design. We validate the effects of local sliding on gRNA efficiency using both public data and in-house data generated by measuring SpCas9 cleavage efficiency at 1024 sites designed to cover all possible combinations of 4-nt PAM and context sequences of 4 gRNAs. Combining the effects of local sliding for a given PAM context with global off-targets allows us to better identify highly specific, and thus efficient, gRNAs. Using an energy-based model, we show that local gRNA-DNA interactions resulting from Cas9 “sliding” on overlapping protospacer adjacent motifs (PAMs) profoundly impact gRNA activities. Here, we identify a sweet spot range of binding free energy change for optimal efficiency which largely explains why gRNAs display changes in efficiency at on- and off-target sites, including why gRNAs can cleave an off-target with higher efficiency than the on-target. Although the heterogeneity of gRNA activity is well recognized, the current understanding of how CRISPR/Cas9 activity is regulated remains incomplete. A major challenge of CRISPR/Cas9-mediated genome engineering is that not all guide RNAs (gRNAs) cleave the DNA efficiently.
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