Differential Divalent Metal Binding by SpyCas9's RuvC Active Site Contributes to Nonspecific DNA Cleavage.

Autor: Newsom SN; Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, The University of Oklahoma, Norman, Oklahoma, USA., Wang DS; Department of Pharmaceutical Sciences, University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, USA., Rostami S; Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, The University of Oklahoma, Norman, Oklahoma, USA., Schuster I; Department of Chemistry, University of Southern California, Los Angeles, California, USA., Parameshwaran HP; Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, The University of Oklahoma, Norman, Oklahoma, USA., Joseph YG; Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, The University of Oklahoma, Norman, Oklahoma, USA., Qin PZ; Department of Chemistry, University of Southern California, Los Angeles, California, USA., Liu J; Department of Pharmaceutical Sciences, University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, USA., Rajan R; Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, The University of Oklahoma, Norman, Oklahoma, USA.
Jazyk: angličtina
Zdroj: The CRISPR journal [CRISPR J] 2023 Dec; Vol. 6 (6), pp. 527-542.
DOI: 10.1089/crispr.2023.0022
Abstrakt: To protect against mobile genetic elements (MGEs), some bacteria and archaea have clustered regularly interspaced short palindromic repeats-CRISPR associated (CRISPR-Cas) adaptive immune systems. CRISPR RNAs (crRNAs) bound to Cas nucleases hybridize to MGEs based on sequence complementarity to guide the nucleases to cleave the MGEs. This programmable DNA cleavage has been harnessed for gene editing. Safety concerns include off-target and guide RNA (gRNA)-free DNA cleavages, both of which are observed in the Cas nuclease commonly used for gene editing, Streptococcus pyogenes Cas9 (SpyCas9). We developed a SpyCas9 variant (SpyCas9 H982A ) devoid of gRNA-free DNA cleavage activity that is more selective for on-target cleavage. The H982A substitution in the metal-dependent RuvC active site reduces Mn 2+ -dependent gRNA-free DNA cleavage by ∼167-fold. Mechanistic molecular dynamics analysis shows that Mn 2+ , but not Mg 2+ , produces a gRNA-free DNA cleavage competent state that is disrupted by the H982A substitution. Our study demonstrates the feasibility of modulating cation:protein interactions to engineer safer gene editing tools.
Databáze: MEDLINE