2020
DOI: 10.1101/2020.05.21.108613
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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 mechanistic… Show more

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Cited by 8 publications
(7 citation statements)
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References 75 publications
(148 reference statements)
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“…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] .…”
Section: Introductionmentioning
confidence: 89%
“…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] .…”
Section: Introductionmentioning
confidence: 89%
“…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.…”
Section: Introductionmentioning
confidence: 99%
“…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).…”
Section: Introductionmentioning
confidence: 99%