TY - JOUR
T1 - Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence
AU - Babu, Kesavan
AU - Kathiresan, Venkatesan
AU - Kumari, Pratibha
AU - Newsom, Sydney
AU - Parameshwaran, Hari Priya
AU - Chen, Xiongping
AU - Liu, Jin
AU - Qin, Peter Z.
AU - Rajan, Rakhi
N1 - Funding Information:
The work reported here was supported in part by grants from the National Science Foundation [MCB-1716423, R.R.; MCB-1716744, P.Z.Q.], and the J.L. members were supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health [R15HL147265].
Funding Information:
We thank the OU Protein Production & Purification Core (PPC Core) for protein purification services and instrument support. The OU PPC Core is supported by an IDeA grant from NIGMS [grant number P20GM103640]. We also acknowledge the use of computational resources provided by the High-Performance Computing (HPC) Center at the University of North Texas (UNT). We acknowledge the financial support provided from the Office of the Vice President for Research and Partnerships and the Office of the Provost, University of Oklahoma towards the publication cost.
Publisher Copyright:
©
PY - 2021/12/14
Y1 - 2021/12/14
N2 - CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection in bacteria and archaea against intruding genomic materials. Cas9, a type-II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA can direct Cas9 to cleave specific genomic targets. The conformational changes associated with RNA/DNA binding are being modulated to develop Cas9 variants with reduced off-Target cleavage. Previously, we showed that proline substitutions in the arginine-rich bridge helix (BH) of Streptococcus pyogenes Cas9 (SpyCas9-L64P-K65P, SpyCas92Pro) improve target DNA cleavage selectivity. In this study, we establish that kinetic analysis of the cleavage of supercoiled plasmid substrates provides a facile means to analyze the use of two parallel routes for DNA linearization by SpyCas9: (i) nicking by HNH followed by RuvC cleavage (the TS (target strand) pathway) and (ii) nicking by RuvC followed by HNH cleavage (the NTS (nontarget strand) pathway). BH substitutions and DNA mismatches alter the individual rate constants, resulting in changes in the relative use of the two pathways and the production of nicked and linear species within a given pathway. The results reveal coordinated actions between HNH and RuvC to linearize DNA, which is modulated by the integrity of the BH and the position of the mismatch in the substrate, with each condition producing distinct conformational energy landscapes as observed by molecular dynamics simulations. Overall, our results indicate that BH interactions with RNA/DNA enable target DNA discrimination through the differential use of the parallel sequential pathways driven by HNH/RuvC coordination.
AB - CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection in bacteria and archaea against intruding genomic materials. Cas9, a type-II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA can direct Cas9 to cleave specific genomic targets. The conformational changes associated with RNA/DNA binding are being modulated to develop Cas9 variants with reduced off-Target cleavage. Previously, we showed that proline substitutions in the arginine-rich bridge helix (BH) of Streptococcus pyogenes Cas9 (SpyCas9-L64P-K65P, SpyCas92Pro) improve target DNA cleavage selectivity. In this study, we establish that kinetic analysis of the cleavage of supercoiled plasmid substrates provides a facile means to analyze the use of two parallel routes for DNA linearization by SpyCas9: (i) nicking by HNH followed by RuvC cleavage (the TS (target strand) pathway) and (ii) nicking by RuvC followed by HNH cleavage (the NTS (nontarget strand) pathway). BH substitutions and DNA mismatches alter the individual rate constants, resulting in changes in the relative use of the two pathways and the production of nicked and linear species within a given pathway. The results reveal coordinated actions between HNH and RuvC to linearize DNA, which is modulated by the integrity of the BH and the position of the mismatch in the substrate, with each condition producing distinct conformational energy landscapes as observed by molecular dynamics simulations. Overall, our results indicate that BH interactions with RNA/DNA enable target DNA discrimination through the differential use of the parallel sequential pathways driven by HNH/RuvC coordination.
UR - http://www.scopus.com/inward/record.url?scp=85119340324&partnerID=8YFLogxK
U2 - 10.1021/acs.biochem.1c00354
DO - 10.1021/acs.biochem.1c00354
M3 - Article
C2 - 34757726
AN - SCOPUS:85119340324
SN - 0006-2960
VL - 60
SP - 3783
EP - 3800
JO - Biochemistry
JF - Biochemistry
IS - 49
ER -