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CRISPR基因座中功能性间隔子整合机制
2020-03-02 12:46

荷兰代尔夫特理工大学Chirlmin Joo和Sungchul Kim课题组合作发现原核生物可选择性装载和处理预间隔子以实现精确的CRISPR适应。该项研究成果发表在2020219日出版的《自然》杂志上。

利用高时空分辨率的单分子荧光,他们表明Cas1-Cas2从各种形式DNA中(包括单链DNA和部分双链体)选择预间隔子的前体,其方式取决于DNA的链长度和原间隔子相邻基序(PAM)序列的存在。

他们还确定了DnaQ外切核酸酶是将Cas1–Cas2装载的预间隔子前体加工成适合整合的成熟预间隔物的酶。Cas1-Cas2保护PAM序列不被加工成熟,从而导致产生不对称修剪的预间隔子,并随后以正确的方向整合间隔子。

他们的结果证明了原间隔子前体选择和PAM修剪的动力学协调性,从而提供了深入了解CRISPR基因座中功能性间隔子整合基础的机制。

据介绍,CRISPR–Cas免疫保护原核生物免受入侵的遗传元件侵染。它使用高度保守的Cas1-Cas2复合体来建立可遗传记忆(间隔)。目前对于Cas1-Cas2如何从外源DNA片段(预间隔子)中获得间隔子,并以正确的方向将它们整合到CRISPR基因座中的,尚不清楚。

附:英文原文

Title: Selective loading and processing of prespacers for precise CRISPR adaptation

Author: Sungchul Kim, Luuk Loeff, Sabina Colombo, Slobodan Jergic, Stan J. J. Brouns, Chirlmin Joo

Issue&Volume: 2020-02-19

Abstract: CRISPR–Cas immunity protects prokaryotes against invading genetic elements1. It uses the highly conserved Cas1–Cas2 complex to establish inheritable memory (spacers)2,3,4,5. How Cas1–Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1–Cas2 selects precursors of prespacers from DNA in various forms—including single-stranded DNA and partial duplexes—in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1–Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1–Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.

DOI: 10.1038/s41586-020-2018-1

Source: https://www.nature.com/articles/s41586-020-2018-1

Nature:《自然》,创刊于1869年。隶属于施普林格·自然出版集团,最新IF:69.504
官方网址:http://www.nature.com/
投稿链接:http://www.nature.com/authors/submit_manuscript.html


本期文章:《自然》:Online/在线发表

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