Journal
NATURE COMMUNICATIONS
Volume 13, Issue 1, Pages -Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41467-022-30515-0
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Funding
- Innovation Fund Denmark [4108-00008B, 4096-00001B]
- Independent Research Fund Denmark, FTP [9041-00317B]
- European Union [899417]
- Novo Nordisk Foundation [NNF21OC0068988]
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This study identifies a range of optimal binding free energy change for efficient CRISPR/Cas9-mediated genome engineering. The authors show that gRNA efficiency is influenced by local gRNA-DNA interactions resulting from Cas9 sliding on overlapping PAMs. Incorporating the effects of local sliding with global off-targets helps identify highly specific and efficient gRNAs. The findings highlight the significance of accounting for local sliding in gRNA design and improve our understanding of Cas9-PAM compatibility and cleavage activation.
A major challenge of CRISPR/Cas9-mediated genome engineering is that not all guide RNAs (gRNAs) cleave the DNA efficiently. Although the heterogeneity of gRNA activity is well recognized, the current understanding of how CRISPR/Cas9 activity is regulated remains incomplete. 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. 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. 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. 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. 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. CRISPR/Cas9-mediated cleavage efficiency varies at different target locations. Here the authors explain this variation with binding free energy changes and show that overlapping Cas9 binding sites influence cleavage efficiency by enabling Cas9 sliding.
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