Differential Divalent Metal Binding by SpyCas9's RuvC Active Site Contributes to Nonspecific DNA Cleavage
Differential Divalent Metal Binding by SpyCas9's RuvC Active Site Contributes to Nonspecific DNA Cleavage
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SpyCas9 的 RuvC 活性位点的差异二价金属结合有助于非特异性 DNA 切割
DOI：
10.1089/crispr.2023.0022
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Rajan, Rakhi
中科院分区：
文献类型：
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作者：
Newsom, Sydney N.;Wang, Duen-Shian;Rostami, Saadi;Schuster, Isabelle;Parameshwaran, Hari Priya;Joseph, Yadin G.;Qin, Peter Z.;Liu, Jin;Rajan, Rakhi
为了保护免受移动的遗传元件（MGE）的影响，一些细菌和古细菌具有成簇的规则间隔的短回文重复序列-CRISPR相关（CRISPR-Cas）适应性免疫系统。与Cas核酸酶结合的CRISPR RNA（crRNA）基于序列互补性与MGE杂交以引导核酸酶切割MGE。这种可编程的DNA切割已被用于基因编辑。安全问题包括脱靶和无指导RNA（gRNA）的DNA切割，这两种情况都在通常用于基因编辑的Cas核酸酶化脓性链球菌Cas9（SpyCas 9）中观察到。我们开发了一种缺乏无gRNA DNA切割活性的SpyCas 9变体（SpyCas 9 H982 A），其对靶向切割更具选择性。金属依赖性RuvC活性位点中的H982 A取代使Mn 2+依赖性gRNA游离DNA切割减少了167倍。机械分子动力学分析表明，Mn 2+，而不是Mg 2+，产生一个无gRNA的DNA切割能力的状态，被破坏的H982 A取代。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。
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 pyogenesCas9 (SpyCas9). We developed a SpyCas9 variant (SpyCas9H982A) 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 Mn2+-dependent gRNA-free DNA cleavage by ∼167-fold. Mechanistic molecular dynamics analysis shows that Mn2+, but not Mg2+, 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.