The effect of crRNA-target mismatches on cOA-mediated interference by a type III-A CRISPR-Cas system.
The effect of crRNA-target mismatches on cOA-mediated interference by a type III-A CRISPR-Cas system.
复制标题
DOI：
10.1080/15476286.2022.2150812
复制
发表时间：
2022-01
期刊：
影响因子：
4.1
通讯作者：
Dunkle, Jack A.
中科院分区：
文献类型：
--
作者：
Nasef, Mohamed;Khweis, Sarah A.;Dunkle, Jack A.
当外源核酸通过其与crRNA碱基对的能力被检测到时，CRISPR系统会引发干扰。了解外源核酸和crRNA之间需要多大程度的互补性才能产生干扰是CRISPR系统研究中的一个核心问题。由于系统的复杂性，利用三种不同的干扰活动，对III型含cas10的CRISPR系统中哪些靶标- crrna错配消除了干扰的明确描述已被证明是难以捉摸的。我们表征了靶- crrna错配对体外环寡腺苷酸（cOA）合成的影响，以及在体内依赖于cOA合成的干扰实验。我们发现，在体外和体内，序列背景都会影响不匹配的靶标是否被crRNA识别。我们还研究了靶- crRNA双工中不匹配的位置如何影响crRNA的识别。我们的数据支持了crrna靶双工中存在cas10激活区，双工的cas10近端区域在调节cOA合成中最关键的假设。了解III型CRISPR系统控制目标识别的规则是至关重要的：作为自然界中最普遍的CRISPR系统之一，它在许多属细菌的生存中起着重要作用。最近，III型系统被重新用作敏感和准确的分子诊断工具。理解这个系统中目标识别的规则将是至关重要的，因为它是为生物技术目的而设计的。
CRISPR systems elicit interference when a foreign nucleic acid is detected by its ability to base-pair to crRNA. Understanding what degree of complementarity between a foreign nucleic acid and crRNA is required for interference is a central question in the study of CRISPR systems. A clear description of which target–crRNA mismatches abrogate interference in type III, Cas10-containing, CRISPR systems has proved elusive due to the complexity of the system which utilizes three distinct interference activities. We characterized the effect of target–crRNA mismatches on in vitro cyclic oligoadenylate (cOA) synthesis and in vivo in an interference assay that depends on cOA synthesis. We found that sequence context affected whether a mismatched target was recognized by crRNA both in vitro and in vivo. We also investigated how the position of a mismatch within the target–crRNA duplex affected recognition by crRNA. Our data provide support for the hypothesis that a Cas10-activating region exists in the crRNA–target duplex, that the Cas10-proximal region of the duplex is the most critical in regulating cOA synthesis. Understanding the rules governing target recognition by type III CRISPR systems is critical: as one of the most prevalent CRISPR systems in nature, it plays an important role in the survival of many genera of bacteria. Recently, type III systems were re-purposed as a sensitive and accurate molecular diagnostic tool. Understanding the rules of target recognition in this system will be critical as it is engineered for biotechnology purposes.
登录
查看更多内容
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
影响因子：
30.3
作者：
Pyenson NC;Gayvert K;Varble A;Elemento O;Marraffini LA
通讯作者：
Marraffini LA
影响因子：
64.5
作者：
Jiang W;Samai P;Marraffini LA
通讯作者：
Marraffini LA
影响因子：
46.9
作者：
Kleinstiver BP;Tsai SQ;Prew MS;Nguyen NT;Welch MM;Lopez JM;McCaw ZR;Aryee MJ;Joung JK
通讯作者：
Joung JK
DOI：
10.1261/rna.078739.121
发表时间：
2021-05-13
期刊：
RNA (New York, N.Y.)
影响因子：
--
作者：
Athukoralage JS;White MF
通讯作者：
White MF