Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity

摘要

认为细菌也许具有免疫记忆的观点曾经只是一种想象，但却因下面的发现而成为被接受的事实：CRISPR-Cas基因位点会迅速演变以获得短噬菌体序列或spacers,后者然后会在CRISPR重复段之前整合，构成噬菌体感染的一个记录。这些spacers被转录成小CRISPR RNAs(crRNAs),后者被用来定位入侵病毒的DNA。本期Nature上发表的两篇论文描述了关于细菌怎样形成入侵病毒的一个DNA记忆的分子层面的详细情况。Jennifer Doudna及同事发现，纯化的大肠杆菌Cas1-Cas2复合物能以与逆转录病毒整合酶和DNA转座酶相似的方式将寡核苷酸DNA基质整合到受体DNA内。Cas1是催化亚单元，而Cas2则会提高整合活性，它们一起形成"spacer之获取"所需的最小机构。Luciano Marraffini及同事发现，在酿脓链球菌的Ⅱ-型CRISPR-Cas系统中，以crRNA为引导使入侵病毒的DNA失活的Cas9核酸酶也是新spacer序列的整合所必需的，但其机制还有待确定。%Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3' -OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1-Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1-Cas2-mediated adaptive immunity.