@article{781f8fb1b8f54a65bfbc89919ebdf117,
title = "Rational Engineering of CRISPR-Cas9 Nuclease to Attenuate Position-Dependent Off-Target Effects",
abstract = "The RNA-guided Cas9 nuclease from Streptococcus pyogenes has become an important gene-editing tool. However, its intrinsic off-Target activity is a major challenge for biomedical applications. Distinct from some reported engineering strategies that specifically target a single domain, we rationally introduced multiple amino acid substitutions across multiple domains in the enzyme to create potential high-fidelity variants, considering the Cas9 specificity is synergistically determined by various domains. We also exploited our previously derived atomic model of activated Cas9 complex structure for guiding new modifications. This approach has led to the identification of the HSC1.2 Cas9 variant with enhanced specificity for DNA cleavage. While the enhanced specificity associated with the HSC1.2 variant appeared to be position-dependent in the in vitro cleavage assays, the frequency of off-Target DNA editing with this Cas9 variant is much less than that of the wild-Type Cas9 in human cells. The potential mechanisms causing the observed position-dependent effect were investigated through molecular dynamics simulation. Our discoveries establish a solid foundation for leveraging structural and dynamic information to develop Cas9-like enzymes with high specificity in gene editing.",
author = "Zhicheng Zuo and Kesavan Babu and Chhandosee Ganguly and Ashwini Zolekar and Sydney Newsom and Rakhi Rajan and Wang, {Yu Chieh} and Jin Liu",
note = "Funding Information: This work was supported by National Natural Science Foundation of China (grant no. 32000885) and Shanghai Municipal Education Commission under the program for Professor of Special Appointment at Shanghai Institutions of Higher Learning to Z.Z., National Science Foundation of USA (grant no. MCB-1716423) to R.R., University of North Texas Health Science Center (Start-up Fund and Faculty Pilot Grant) and Medical College of Wisconsin (Advancing a Healthier Wisconsin Endowment) to Y.C.W., and National Heart, Lung, and Blood Institute of the National Institutes of Health (grant no. R15HL147265) to J.L. The Rajan lab thank the OU Protein Production and Characterization Core (PPC Core) facility for protein purification services and instrument support. The OU PPC core is supported by an Institutional Development Award (IDeA) grant from the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health (grant no. P20GM103640). Publisher Copyright: {\textcopyright} Copyright 2022, Mary Ann Liebert, Inc., publishers 2022.",
year = "2022",
month = apr,
doi = "10.1089/crispr.2021.0076",
language = "English",
volume = "5",
pages = "329--340",
journal = "CRISPR Journal",
issn = "2573-1599",
publisher = "Mary Ann Liebert Inc.",
number = "2",
}