A kinetic model improves off-target predictions and reveals the physical basis ofSpCas9 fidelity

Abstract

The SpCas9 endonuclease has become an important tool in gene-editing and basic science alike. Though easily programmed to target any sequence, SpCas9 also shows considerable activity over genomic offtargets. Many empirical facts regarding the targeting reaction have been established, but a comprehensive mechanistic description is still lacking-limiting fundamental understanding, our ability to predict off-target activity, and ultimately the safe adaptation of the SpCas9 toolkit for therapeutics. By mechanistically modelling the SpCas9 structure-function relationship, we simultaneously capture binding and cleavage dynamics for SpCas9 and Sp-dCas9 in terms of free-energies. When our model is trained on high-throughput data, we outperform state-of-the-art off-target prediction tools. Based on the biophysical parameters we extract, our model predicts the open, intermediate, and closed complex configurations described in singlemolecule FRET experiments, and indicates that R-loop progression is tightly coupled to structural changes in the targeting complex. We further show that SpCas9 targeting kinetics are tuned for extended sequence specificity while maintaining on-target efficiency. Our approach can be used to characterize any other CRISPR derived nuclease, and contrasting future studies of high-fidelity variants with the SpCas9 benchmark we here provide will help elucidate the determinants of CRISPR fidelity and the path to increased specificity and efficiency in engineered systems.

Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin

“…Once the R-loop is fully formed, the nuclease is activated and cleaves the target and non-target DNA strands to generate a DNA break 5 . R-loop extension is rate-limiting for cleavage by Cas12a and Cas9 in vitro [6][7][8][9] , providing a mechanistic explanation for Cas12a's observed higher specificity in vivo 8,[10][11][12][13][14][15] .…”

A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex

“…We show that the observed dynamics can be quantitatively described by a random walk model in a simplified one-dimensional energy landscape. The model was adapted from previous descriptions of protein-free strand displacement reactions in dynamic DNA nanotechnology (Irmisch et al, 2020;Machinek et al, 2014;Srinivas et al, 2013), which have recently also been introduced to the CRISPR-Cas field (Eslami-Mossallam et al, 2020;Klein et al, 2018). Importantly, our modelling (i) provides direct evidence that R-loop expansion follows a random-walk process (ii), shows that the single-base pair stepping of R-loop expansion occurs at a sub-millisecond time scale, (iii) returns absolute free energy penalties imposed by mismatches, (iv) explains the non-trivial dependence of R-loop formation on the proximity between multiple mismatches and (v) reveals that the length of the seed region in Cascade is a function of the applied supercoiling rather than a structural property.…”

Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

“…Upon R-loop formation, the nuclease is activated and cleaves the target and nontarget DNA strands to generate a DNA break (5). R-loop extension is rate-limiting for cleavage by Cas12a and Cas9 in vitro and more sensitive to mismatches for Cas12a (6)(7)(8)(9), providing a mechanistic explanation for Cas12a's observed higher specificity in vivo (8,(10)(11)(12)(13)(14)(15).…”