Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement

Abstract

The RNA-guided nuclease Cas9 has unlocked powerful methods for perturbing both the genome through targeted DNA cleavage and the regulome through targeted DNA binding, but limited biochemical data have hampered efforts to quantitatively model sequence perturbation of target binding and cleavage across diverse guide sequences. We present scalable, sequencing-based platforms for high-throughput filter binding and cleavage and then perform 62,444 quantitative binding and cleavage assays on 35,047 on- and off-target DNA sequences across 90 Cas9 ribonucleoproteins (RNPs) loaded with distinct guide RNAs. We observe that binding and cleavage efficacy, as well as specificity, vary substantially across RNPs; canonically studied guides often have atypically high specificity; sequence context surrounding the target modulates Cas9 on-rate; and Cas9 RNPs may sequester targets in nonproductive states that contribute to “proofreading” capability. Lastly, we distill our findings into an interpretable biophysical model that predicts changes in binding and cleavage for diverse target sequence perturbations.

Mechanism of R-loop formation and conformational activation of Cas9

“…We identify mutations that destabilise the binding intermediate states and thus increase off-target discrimination, which presents an opportunity for the development of novel high-fidelity SpCas9 variants. As most off-target sequences are only bound but not cleaved [19][20][21]42 , these variants could prove useful for applications that rely on the fidelity of Cas9 target binding [43][44][45][46] , such as transcriptional regulation or base editing 47 . Directional target DNA hybridization is associated with dynamic repositioning of the REC2/3 and HNH domains to initially assume a catalytically inactive, checkpoint conformation upon R-loop completion.…”

“…The resulting off-target activity is dependent on the number, type, and positioning of base mismatches within the guidetarget heteroduplex 15,[19][20][21] . PAM-proximal mismatches within the seed region are discriminated against through substantially increased dissociation rates 11,19,21,22 . In contrast, PAM-distal mismatches are compatible with stable binding, but trap the enzyme in a cleavage-incompetent, dead-end complex 13,23,24 .…”

“…Mutation of these residues, which would further destabilise this intermediate state and thus promote off-target dissociation, presents an opportunity to generate novel high-fidelity SpCas9 variants. As most off-targets are only bound but not cleaved [19][20][21]31 , these variants could prove to be valuable for biotechnological applications that rely on the fidelity of Cas9 target binding [32][33][34][35] , such as transcriptional regulation or base editing.…”

“…Although highly specific, SpCas9 can nevertheless cleave offtarget genomic sites with imperfect complementarity to the guide RNA [14][15][16][17][18] . The resulting off-target activity is dependent on the number, type, and positioning of base mismatches within the guidetarget heteroduplex 15,[19][20][21] . PAM-proximal mismatches within the seed region are discriminated against through substantially increased dissociation rates 11,19,21,22 .…”

Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity

“…Nevertheless, these mismatch-dependent rules only partially account for the observed off-target effects. For example, recent studies revealed a guide-intrinsic mismatch tolerance (GMT) in addition to the impact of specific mismatches 21,22 . While the observations of GMT suggest the possibility to identify highly specific gRNA regardless of genetic context, the sequence determinants underlying GMT is unclear.…”