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How type II CRISPR-Cas establish immunity through Cas1-Cas2-mediated spacer integration

机译：II型CRISPR-Cas如何通过Cas1-Cas2介导的间隔子整合建立免疫力

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摘要

CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby Cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes(1-5). Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer(6-9). Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR-Cas systems, the spacer integration mechanism is essentially identical(7-9). The conserved Cas1 and Cas2 proteins form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer(6,10). 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(11-14). The prespacer is preferentially integrated into the leader-proximal region of the CRISPR array(1,7,10,15,) guided by the leader sequence and a pair of inverted repeats inside the CRISPR repeat(7,15-20). Spacer integration in the well-studied Escherichia coli type I-E CRISPR system also relies on the bacterial integration host factor(21,22). In type II-A CRISPR, however, Cas1-Cas2 alone integrates spacers efficiently in vitro(18); other Cas proteins (such as Cas9 and Csn2) have accessory roles in the biogenesis phase of prespacers(17,23). Here we present four structural snapshots from the type II-A system(24) of Enterococcus faecalis Cas1 and Cas2 during spacer integration. Enterococcus faecalis Cas1-Cas2 selectively binds to a splayed 30-base-pair prespacer bearing 4-nucleotide 3' overhangs. Three molecular events take place upon encountering a target: first, the Cas1-Cas2-prespacer complex searches for half-sites stochastically, then it preferentially interacts with the leader-side CRISPR repeat, and finally, it catalyses 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 to explain the stepwise spacer integration process and the leader-proximal preference.
机译：CRISPR（聚簇的规则间隔的短回文重复序列）和附近的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（1-5）。当短的外源DNA衍生的预间隔子作为新的间隔子整合到CRISPR阵列中时，就会创建分子记忆（6-9）。在CRISPR-Cas系统中，RNA引导的CRISPR干扰机制差异很大，而间隔子整合机制本质上是相同的（7-9）。保守的Cas1和Cas2蛋白形成由两个远端Cas1二聚体（由Cas2二聚体桥接）组成的整合酶复合物（6,10）。该预间隔子与Cas1-Cas2结合为双叉DNA，每个3'突出端的末端3'-OH在整合过程中充当攻击亲核试剂（11-14）。预间隔子优先整合到CRISPR阵列的前导近端区域（1,7,10,15，）中，并由前导序列和CRISPR重复序列内的一对反向重复序列（7,15-20）引导。在经过充分研究的大肠杆菌I-E CRISPR系统中的间隔子整合还依赖于细菌整合宿主因子（21,22）。然而，在II-A型CRISPR中，Cas1-Cas2单独在体外有效整合了间隔子（18）。其他Cas蛋白（例如Cas9和Csn2）在前间隔物的生物合成阶段具有辅助作用（17,23）。在这里，我们介绍间隔物整合过程中粪肠球菌Cas1和Cas2的II-A型系统（24）的四个结构快照。粪肠球菌Cas1-Cas2选择性结合带有4个核苷酸3'突出端的张开的30个碱基对的预间隔子。遇到靶标时发生三个分子事件：首先，Cas1-Cas2-prespacer复合体随机搜索半个位点，然后优先与前导侧CRISPR重复序列相互作用，最后，它催化连接一条链的亲核攻击前导-近端重复序列的“末端”到预间隔物3'突出端。识别间隔物半位需要DNA弯曲并导致完全整合。我们得出一个机制框架来解释逐步间隔集成过程和领导者-近端偏好。

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  • 来源
    《Nature》 |2017年第7674期|137-141|共5页
  • 作者

    Xiao Yibei; Ng Sherwin; Nam Ki Hyun; Ke Ailong;

  • 作者单位

    Cornell Univ, Dept Mol Biol & Genet, 253 Biotechnol Bldg, Ithaca, NY 14353 USA;

    Cornell Univ, Dept Mol Biol & Genet, 253 Biotechnol Bldg, Ithaca, NY 14353 USA;

    Pohang Univ Sci & Technol, Pohang Accelerator Lab, Pohang, South Korea;

    Cornell Univ, Dept Mol Biol & Genet, 253 Biotechnol Bldg, Ithaca, NY 14353 USA;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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