SpyCas9 的 RuvC 活性位点通过差向二价金属结合有助于非特异性 DNA 切割。
Differential Divalent Metal Binding by SpyCas9's RuvC Active Site Contributes to Nonspecific DNA Cleavage.
机构信息
Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, The University of Oklahoma, Norman, Oklahoma, USA.
Department of Pharmaceutical Sciences, University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, USA.
出版信息
CRISPR J. 2023 Dec;6(6):527-542. doi: 10.1089/crispr.2023.0022.
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, Cas9 (SpyCas9). We developed a SpyCas9 variant (SpyCas9) 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-dependent gRNA-free DNA cleavage by ∼167-fold. Mechanistic molecular dynamics analysis shows that Mn, but not Mg, 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.
为了防止移动遗传元件（MGE）的侵害，一些细菌和古菌拥有成簇规律间隔短回文重复序列- CRISPR 相关（CRISPR-Cas）适应性免疫系统。CRISPR 核糖核酸（crRNA）与 Cas 核酸酶结合，根据与 MGE 序列互补的程度来指导核酸酶切割 MGE。这种可编程的 DNA 切割已被用于基因编辑。安全性问题包括脱靶和无向导 RNA（gRNA）的 DNA 切割，这两种情况都在常用于基因编辑的 Cas 核酸酶 Cas9（SpyCas9）中观察到。我们开发了一种缺乏无 gRNA DNA 切割活性的 SpyCas9 变体（SpyCas9），它对靶标切割的选择性更高。金属依赖性 RuvC 活性位点中的 H982A 取代将 Mn 依赖性无 gRNA DNA 切割降低了约 167 倍。机制分子动力学分析表明，Mn 而不是 Mg 产生 gRNA 无 DNA 切割活性状态，该状态被 H982A 取代所破坏。我们的研究证明了调节阳离子：蛋白质相互作用以设计更安全的基因编辑工具的可行性。
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