Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches

Abstract

C as9 is an RNA-guided endonuclease that specifically binds and cleaves DNA with complementarity to the spacer sequence of CRISPR RNA (crRNA) located upstream of a protospacer adjacent motif (PAM) 1,2. Sufficient base pairing of the crRNA spacer of either dual-RNA (trans-activating crRNA (tracrRNA) in complex with crRNA) or single-guide RNA (sgRNA, tracrRNA fused to crRNA) with the target DNA strand leads to displacement of the nontarget strand and R-loop formation 1-5. On completion of this process, Cas9 endonuclease domains, HNH and RuvC, cleave the target and nontarget strand, respectively, resulting in a double-strand DNA break 1,2. Due to its programmability, CRISPR-Cas9 was adapted for a wide variety of genome engineering approaches 6-9. However, a main concern for the application of the CRISPR-Cas9 technology is off-target cleavage that occurs when Cas9 cleaves mismatched targets; that is, targets with imperfect complementarity to the dual-RNA/sgRNA spacer. It is therefore crucial to identify regions and amino acids that determine Cas9 specificity. Streptococcus pyogenes Cas9 (SpCas9) requires a seed sequence of 10-12 base pairs (bp) adjacent to the PAM for successful cleavage 2. The REC3 domain senses PAM-distal mismatches and controls the transition of the HNH domain into an active conformation, which serves as a mechanism to block the cleavage in the presence of mismatches 10,11. Alongside other strategies to reduce off-target cleavage 12 , both structure-guided and random mutagenesis have been used to generate enhanced specificity Cas9 variants 10,13-19. Three of the improved variants 10,15,17 contain mutations that increase the energetic barrier for transition of the HNH domain to the cleavage-competent state 10. eSpCas9 (ref. 17) and SpCas9-HF1 (ref. 15) require one additional base pair between crRNA and target DNA for stable binding, which makes target unwinding more sensitive to mismatches and slows cleavage in the presence of mismatches 20. It was shown that mutation of two residues in the loop region of the bridge helix impairs target cleavage and improves specificity 21. The conformational checkpoint through the HNH domain before cleavage and the seed sequence are the only known specificity determinants for Cas9. In addition, stable R-loop formation is an essential prerequisite for cleavage 5 , but residues involved in this process have not been identified. Therefore, we investigated whether other Cas9 domains are involved in mismatch sensing and R-loop stabilization focusing on the PAM-adjacent region of the target. We show that arginines of the Cas9 bridge helix influence guide RNA binding, and target DNA binding and cleavage. We identify two groups of arginines with opposite effects on Cas9 mismatch sensitivity. We demonstrate that R63, R66 and R70 stabilize the R-loop in the presence of PAM-adjacent mismatches thus reducing the Cas9 specificity. Additionally, we observe that Q768 mediates Cas9 insensitivity to a mismatch in target position 15 and is involved in sensitivity to PAM-distal mism...

High concentration of Cas12a effector tolerates more mismatches on ssDNA

“…In addition, according to the study of Majda Bratovič et al, arginines in the Cas9 bridge helix influenced guide RNA, and target DNA binding and cleavage. 31 There were two residues of arginine on the 9th base of Cas12a (951 and 955), so the residue of arginine might have an impact on the binding of single-stranded DNA and crRNA and the activation of trans-cleavage activity by this base. Of course, these required in-depth study of protein structure.…”

“…According to Takashi Yamano et al, bridge helix structure of AsCas12a, LbCas12a, and FnCas12a was located 9‐10 bases from the PAM region 30 and our reverse 11th base was exactly on the position of the 9th base from the PAM region. In addition, according to the study of Majda Bratovič et al, arginines in the Cas9 bridge helix influenced guide RNA, and target DNA binding and cleavage 31 . There were two residues of arginine on the 9th base of Cas12a (951 and 955), so the residue of arginine might have an impact on the binding of single‐stranded DNA and crRNA and the activation of trans‐cleavage activity by this base.…”

CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future

“…Desensitizing the Rec3 domain increases the dependence on specificity for the DNA:RNA heteroduplex to induce DSBs, thereby reducing OTEs while maintaining editing efficacy (38,39). One of the more recent developments is the Cas9_R63A/Q768A variant, in which the R63A mutation destabilizes R-loop formation in the presence of mismatches and Q768A mutation increases sensitivity to PAM-distal mismatches (49). Despite the different strategies, the rational for generating many Cas9 variants with reduced OTEs has been to ultimately reduce general Cas9 and DNA interactions and give a stronger role for the DNA:RNA heteroduplex in facilitating the edits.…”

Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules

“…S4), a finding that has been previously observed in other proteins 17 22 . Two clear exceptions are the mechanistically essential 'bridge helix' 35 , which orders and stabilizes the R-loop 41,42 , and the 'phosphate lock loop' 43 , which interacts with the PAMproximal target strand phosphate to enable gRNA strand invasion. It should be noted that the enrichment data presented here is somewhat sparse and only a relative measurement of CRISPRi function; the larger-scale features of acceptable domain and sub-domain level deletions were therefore extensively validated with further in vivo and biochemical assays.…”