CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context

Abstract

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.

Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43

“…Although we predicted the gRNAs with three SNPs would have a negative effect on CasRx cleavage, we found that these guides significantly improved its nuclease activity in both RdRp and N compared to the guides without mismatches ( p = 0.0278 and p = 0.0304, respectively). Recent work using an energy-based model with Cas9 has found that interactions between gRNA and the targeted DNA may influence cleavage activity, resulting in Cas9 cleaving off-target sites with higher efficiency than on-target sites [ 22 , 23 ]. It is possible there is a similar interaction between gRNAs and targeted RNA when Cas13 is used, but this should be further elucidated in future work using a robust number of gRNA sequences.…”

Massively targeted evaluation of therapeutic CRISPR off-targets in cells

“…Our data further showed that mismatches at the two upstream PAM proximal position (N19 and N20) were more tolerated than other nucleotides of the seed region. This site-dependent effect could be explained by our recent binding energy model about the effect of sliding PAMs on CRISPR-Cas9 specificity 67 . Indeed, when performing benchmarking of the different CRISPR-Cas9 off-target prediction tools with our data, our results also showed that energy-based predictors out-performed other tools in their accuracy of predicting true off-targets.…”

“…10.1101/2020.05.20.103614). Recently, we further demonstrated that this surrogate library approach can be used to evaluate PAM compatibility in human cells 67 . We anticipate that SURRO-seq could be adapted to evaluate off-targets of other DNA editing RGN systems, including prime editing 68 .…”

Expanding the scope of bacterial CRISPR activation with PAM-flexible dCas9 variants

“…We found that PAM-flexible dCas9 variants exhibited weaker CRISPRi-based gene repression compared to dCas9 (Figure 3B). One possible explanation for this behavior follows from the observation that increasing PAM promiscuity reduces affinity towards NGG PAMs (Corsi et al, 2022; Hu et al, 2018; Legut et al, 2020; Nishimasu et al, 2018; Walton et al, 2020). This reduced PAM-binding affinity could allow RNA polymerase to more readily displace the CRISPRi complex.…”