Quantifying Protein-Nucleic Acid Interactions for Engineering Useful CRISPR-Cas9 Genome-Editing Variants.

Autor:	Chu HY; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.; Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China., Peng J; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.; Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China., Mou Y; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China.; Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China., Wong ASL; Laboratory of Combinatorial Genetics and Synthetic Biology, School of Biomedical Sciences, The University of Hong Kong, Pokfulam, Hong Kong SAR, China. aslw@hku.hk.; Centre for Oncology and Immunology, Hong Kong Science Park, Hong Kong SAR, China. aslw@hku.hk.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2025; Vol. 2870, pp. 227-243.
DOI:	10.1007/978-1-0716-4213-9_12
Abstrakt:	Numerous high-specificity Cas9 variants have been engineered for precision genome editing. These variants typically harbor multiple mutations designed to alter the Cas9-single guide RNA (sgRNA)-DNA complex interactions for reduced off-target cleavage. By dissecting the contributions of individual mutations, we attempt to derive principles for designing high-specificity Cas9 variants. Here, we computationally modeled the specificity harnessing mutations of the widely used Cas9 isolated from Streptococcus pyogenes (SpCas9) and investigated their individual mutational effects. We quantified the mutational effects in terms of energy and contact changes by comparing the wild-type and mutant structures. We found that these mutations disrupt the protein-protein or protein-DNA contacts within the Cas9-sgRNA-DNA complex. We also identified additional impacted amino acid sites via energy changes that constitute the structural microenvironment encompassing the focal mutation, giving insights into how the mutations contribute to the high-specificity phenotype of SpCas9. Our method outlines a strategy to evaluate mutational effects that can facilitate rational design for Cas9 optimization. (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu