How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration.
How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration.
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DOI：
10.1038/nature24020
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发表时间：
2017-10-05
期刊：
影响因子：
64.8
通讯作者：
Ke A
中科院分区：
文献类型：
--
作者：
Xiao Y;Ng S;Nam KH;Ke A
CRISPR （Clustered Regularly Interspaced Short Palindromic Repeats）及其附近的cas （CRISPR相关）操纵子在原核生物中建立了基于rna的适应性免疫系统。当一个短的外源dna衍生的预间隔物作为新的间隔物被整合到CRISPR阵列中时，就会产生分子记忆。尽管rna引导的CRISPR干扰机制在CRISPR- cas系统中差异很大，但间隔器整合机制本质上是相同的。保守的Cas1和Cas2蛋白形成一个整合酶复合体，由两个远端Cas1二聚体组成，中间由一个Cas2二聚体桥接。预间隔物与Cas1-Cas2结合形成双分叉DNA，在整合过程中，每个3 ‘悬空的末端3 ’ -OH作为攻击亲核试剂。重要的是，在先导序列和CRISPR重复序列内的一对反向重复序列（IRs）的引导下，前置器优先集成到CRISPR阵列的先导-近端区域。研究最充分的大肠杆菌I-E型CRISPR系统中的间隔整合进一步依赖于细菌整合宿主因子（IHF）。然而，在II-A型CRISPR中，Cas1-Cas2单独在体外有效地整合间隔物；其他Cas蛋白（Cas9和Csn2）在预间隔生物发生中起辅助作用。以粪肠球菌II-A型系统为研究对象，本文报道了间隔整合过程中Cas1-Cas2的四个结构快照。EfaCas1-Cas2选择性地结合到带有4-nt - 3 '悬垂的30-bp的预垫层上。遇到靶标时会发生三个分子事件：Cas1-Cas2/pre - spacer首先随机搜索半位点，然后优先与前导侧CRISPR重复序列相互作用，并催化亲核攻击，将前导-近端重复序列的一条链连接到pre - spacer 3 ' -悬空。识别间隔半位点需要DNA弯曲并导致完全整合。我们推导了一个解释逐步间隔整合过程和领导-近端偏好的机制框架。
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the nearby cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical. The conserved Cas1 and Cas2 proteins form an integrase complex consisting two distal Cas1 dimers bridged by a Cas2 dimer in the middle. The prespacer is bound by Cas1-Cas2 as a dual forked DNA, and the terminal 3′-OH of each 3′-overhang serves as an attacking nucleophile during integration. Importantly, the prespacer is preferentially integrated into the leader-proximal region of the CRISPR array, guided by the leader sequence and a pair of inverted repeats (IRs) inside the CRISPR repeat. Spacer integration in the most well-studied Escherichia coli Type I-E CRISPR system further relies on the bacterial Integration Host Factor (IHF). In Type II-A CRISPR, however, Cas1-Cas2 alone integrates spacer efficiently in vitro; other Cas proteins (Cas9 and Csn2) play accessory roles in prespacer biogenesis. Focusing on the Enterococcus faecalis Type II-A system, here we report four structure snapshots of Cas1-Cas2 during spacer integration. EfaCas1-Cas2 selectively binds to a splayed 30-bp prespacer bearing 4-nt 3′-overhangs. Three molecular events take place upon encountering a target: Cas1-Cas2/prespacer first searches for half-sites stochastically, then preferentially interacts with the leader-side CRISPR repeat and catalyzes a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3′-overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework explaining the stepwise spacer integration process and the leader-proximal preference.
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DOI：
10.1126/science.1165771
发表时间：
2008-12-19
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Marraffini LA;Sontheimer EJ
通讯作者：
Sontheimer EJ
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
2.8
作者：
Pourcel, C;Salvignol, G;Vergnaud, G
通讯作者：
Vergnaud, G
影响因子：
3.9
作者：
Mojica, FJM;Díez-Villaseñor, C;Soria, E
通讯作者：
Soria, E
影响因子：
16
作者：
Nunez, James K.;Bai, Lawrence;Doudna, Jennifer A.
通讯作者：
Doudna, Jennifer A.