Kinetic Basis for Improved Specificity of CRISPR/Cas9 High Fidelity Variants

CRISPR/Cas9-mediated is a powerful, programmable genome editing tool that has been widely used for many applications. While engineered Cas9s have been reported to minimize the off-target cleavage efficiency compared with wild type Cas9 in vivo, a rational mechanism for enhanced specificity has not been put forward. Here, we provide kinetic characterization of the Hyper-accurate Cas9 variant (HypaCas9) to provide a basis for improved discrimination against off-target cleavage. We show that chemistry is the rate-limiting step for off-target cleavage in HypaCas9 and is much slower than on-target cleavage in WTCas9, where DNA unwinding is the rate-limiting step. In addition, the intrinsic cleavage rate and non-productive DNA binding state has also greatly been affected in engineered Cas9s. We find that HypaCas9 gains discrimination mainly through slowing down chemistry and rebalancing kinetic partitioning to favor release rather than cleavage of the bound substrate. Support or Funding Information This work was supported in part by the Welch Foundation grants (F-1604) (to K.A.J.) and (F-1938) (to D.W.T.). Free energy profile for WTCas9 and HypaCas9 for on-target and off-target cleavage (A)kobs, HNH cleavage rates for on-target and off-target cleavage by WTCas9 and HypaCas9. (B)The two-steps for R-loop formation rates for on-target and off-target cleavage of WTCas9 and HypaCas9 were estimated by stopped-flow fluorescence measurements. (C)The free energy profile was calculated using transition state theory: ΔG‡ = RT[ln(kT/h) − ln(kobs)] kcal/mol using the equilibrium constants from globally fitting. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.