Structural basis for mismatch surveillance by CRISPR/Cas9

Abstract

The widespread use of CRISPR/Cas9 as a programmable genome editing tool has been hindered by off-target DNA cleavage (Cong et al., 2013; Doudna, 2020; Fu et al., 2013; Jinek et al., 2013). While analysis of such off-target editing events have enabled the development of Cas9 variants with greater discrimination against mismatches (Chen et al., 2017; Kleinstiver et al., 2016; Slaymaker et al., 2016), the underlying molecular mechanisms by which Cas9 rejects or accepts mismatches are poorly understood (Kim et al., 2019; Liu et al., 2020; Slaymaker and Gaudelli, 2021). Here, we used kinetic analysis to guide cryo-EM structure determination of Cas9 at different stages of mismatch surveillance. We observe a distinct, previously undescribed linear conformation of the duplex formed between the guide RNA (gRNA) and DNA target strand (TS), that occurs in the presence of PAM-distal mismatches, preventing Cas9 activation. The canonical kinked gRNA:TS duplex is a prerequisite for Cas9 activation, acting as a structural scaffold to facilitate Cas9 conformational rearrangements necessary for DNA cleavage. We observe that highly tolerated PAM- distal mismatches achieve this kinked conformation through stabilization of a distorted duplex conformation via a flexible loop in the RuvC domain. Our results provide molecular insights into the underlying structural mechanisms that may facilitate off- target cleavage by Cas9 and provides a molecular blueprint for the design of next- generation high fidelity Cas9 variants that selectively reduce off-target DNA cleavage while retaining efficient cleavage of on-target DNA.

Mechanism of R-loop formation and conformational activation of Cas9

“…Together, these structural observations provide a rationale for the allosteric coupling of R-loop formation with HNH domain rearrangement and RuvC active site accessibility, in agreement with single-molecule studies showing that PAM-distal end positioning modulates HNH domain conformation 32 . The concerted mechanism of HNH domain and NTS repositioning has also been reported in a complementary structural study 36 .…”

An engineered xCas12i with high activity, high specificity and broad PAM range

“…In agreement with the hypothesis that introducing arginine at key sites could strengthen the binding between Cas and DNA, the introduction of N243R in the PI domain and E336R at REC domain significantly increased editing activity and expanded PAM recognition. Interestingly, D892R or G883R substitutions in the RuvC domain reduced off-target and retained on-target cleavage activity, whereas alanine substitutions 24, 25 , which has been used to reduce off-target activity, did not. The D892R substituted hfCas12Max was obviously more sensitive to mismatch, which suggests that D892R or G883R improved sgRNA binding specificity.…”

“…Interestingly, D892R or G883R substitutions in the RuvC domain reduced off-target and retained on-target cleavage activity, whereas alanine substitutions 24,25 , which has been used to reduce off-target activity, did not. The D892R substituted hfCas12Max was obviously more sensitive to mismatch, which suggests that D892R or G883R improved sgRNA binding specificity.…”

CRISPR-Cas9 library screening approach for anti-cancer drug discovery: overview and perspectives

“…There are many SpCas9 variants for high-fidelity experiments. SpCas9-HF1, eSpCas9, HypaCas9, and SuperFi-Cas9 are examples of structure-guided engineered proteins in which amino acid residues that contact with the DNA strands are modified 95 , 113 - 115 . Another approach for obtaining improved variants is random mutagenesis and end-point selection, examples of which include evoCas9, Sniper-Cas9, and xCas9 96 , 112 , 116 .…”