Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence.
Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence.
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DOI：
10.1021/acs.biochem.1c00354
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发表时间：
2021-12-14
期刊：
影响因子：
2.9
通讯作者：
Rajan R
中科院分区：
文献类型：
--
作者：
Babu K;Kathiresan V;Kumari P;Newsom S;Parameshwaran HP;Chen X;Liu J;Qin PZ;Rajan R
CRISPR-Cas系统是RNA引导的核酸酶，其在细菌和古细菌中提供针对入侵基因组物质的适应性免疫保护。Cas9是一种II型CRISPR效应蛋白，广泛用于基因编辑应用，因为单个指导RNA可以指导Cas9切割特定的基因组靶标。与RNA/DNA结合相关的构象变化被调节以开发具有减少的脱靶切割的Cas9变体。先前，我们显示了酿脓链球菌Cas9（SpyCas 9-L 64 P-K65 P，SpyCas 92 Pro）的富含丝氨酸的桥螺旋（BH）中的脯氨酸取代改善了靶DNA切割选择性。在这项研究中，我们确定了超螺旋质粒底物切割的动力学分析提供了一种简单的方法来分析SpyCas 9对DNA线性化的两种平行途径的使用：（i）通过HNH产生切口，然后是RuvC切割（TS（靶链）途径）和（ii）通过RuvC产生切口，然后是HNH切割（NTS（非靶链）途径）。BH取代和DNA错配改变了个体速率常数，导致两种途径的相对使用以及给定途径内的切口和线性物质的产生发生变化。结果揭示了HNH和RuvC之间的协调行动，线性化DNA，这是调制的BH的完整性和在基板中的错配的位置，与每个条件产生不同的构象能量景观所观察到的分子动力学模拟。总体而言，我们的研究结果表明，BH与RNA/DNA的相互作用，使目标DNA的歧视，通过差分使用的HNH/RuvC协调驱动的平行顺序的途径。
CRISPR-Cas systems are RNA-guided nucleases that provide adaptive immune protection in bacteria and archaea against intruding genomic materials. Cas9, a type-II CRISPR effector protein, is widely used for gene editing applications since a single guide RNA can direct Cas9 to cleave specific genomic targets. The conformational changes associated with RNA/DNA binding are being modulated to develop Cas9 variants with reduced off-target cleavage. Previously, we showed that proline substitutions in the arginine-rich bridge helix (BH) of Streptococcus pyogenes Cas9 (SpyCas9-L64P-K65P, SpyCas92Pro) improve target DNA cleavage selectivity. In this study, we establish that kinetic analysis of the cleavage of supercoiled plasmid substrates provides a facile means to analyze the use of two parallel routes for DNA linearization by SpyCas9: (i) nicking by HNH followed by RuvC cleavage (the TS (target strand) pathway) and (ii) nicking by RuvC followed by HNH cleavage (the NTS (nontarget strand) pathway). BH substitutions and DNA mismatches alter the individual rate constants, resulting in changes in the relative use of the two pathways and the production of nicked and linear species within a given pathway. The results reveal coordinated actions between HNH and RuvC to linearize DNA, which is modulated by the integrity of the BH and the position of the mismatch in the substrate, with each condition producing distinct conformational energy landscapes as observed by molecular dynamics simulations. Overall, our results indicate that BH interactions with RNA/DNA enable target DNA discrimination through the differential use of the parallel sequential pathways driven by HNH/RuvC coordination.
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影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin
影响因子：
56.9
作者：
Jiang, Fuguo;Zhou, Kaihong;Doudna, Jennifer A.
通讯作者：
Doudna, Jennifer A.
影响因子：
15
作者：
East, Kyle W.;Newton, Jocelyn C.;Lisi, George P.
通讯作者：
Lisi, George P.
DOI：
10.1126/science.1159689
发表时间：
2008-08-15
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Brouns SJ;Jore MM;Lundgren M;Westra ER;Slijkhuis RJ;Snijders AP;Dickman MJ;Makarova KS;Koonin EV;van der Oost J
通讯作者：
van der Oost J
影响因子：
2.8
作者：
Bolotin, A;Ouinquis, B;Ehrlich, SD
通讯作者：
Ehrlich, SD