Prevalence, formation, and function of “extraneous” CRISPR RNAs derived from the extra repeat in CRISPR arrays

源自 CRISPR 阵列中额外重复序列的“外来”CRISPR RNA 的普遍性、形成和功能

• 批准号: 468749960
• 负责人: Professor Dr. Chase Beisel
• 金额: --
• 依托单位: Helmholtz-Institut für RNA-basierte Infektionsforschung (HIRI)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/468749960?language=en
• 关键词: Prevalence; formation; function; extraneous; CRISPR

CRISPR-Cas systems in bacteria and archaea provide adaptive immunity against plasmids and phages. As part of adaptive immunity, small fragments of an invader’s genetic material are stored within a CRISPR array as spacers each located between fixed repeats. Each spacer-repeat pair then gives rise to a CRISPR RNA (crRNA) that directs the system’s effector nuclease to seek out and cleave nucleic-acid sequences matching the spacer. Because this spacer was derived from an invader, the crRNA helps confer resistance against this invader if it appears again.Each CRISPR array possessing the set of acquired spacers begins and ends with a repeat, leaving one repeat without a paired spacer. While the extra repeat is often necessary to acquire new spacers, it would also give rise to a crRNA with the spacer portion originating outside of the CRISPR array. This “extraneous” crRNA (ecrRNA) would be disconnected from adaptive immunity because the spacer portion was not derived from an invader, and it could interfere with crRNA biogenesis. Accordingly, we recently discovered different mechanisms by which CRISPR-Cas systems block formation of ecrRNAs. However, we observed instances in which these mechanisms were absent, potentially allowing natural ecrRNA formation. These observations raise the intriguing possibility that the produced ecrRNAs are functionally expressed and direct effector nucleases for purposes extending beyond adaptive immunity. Through this project, we will investigate the propensity of CRISPR-Cas systems to generate ecrRNAs and direct genome targeting by the effector nuclease. Our hypothesis is that functional ecrRNAs can be generated by different types of CRISPR-Cas systems. To investigate this hypothesis, we will explore three CRISPR sub-types (II-A, V-A, VI-B) in which we elucidated a distinct mechanism for blocking ecrRNA formation, with one objective devoted to each sub-type. We will apply complementary bioinformatics and experimental approaches to determine the extent ecrRNA formation and whether the resulting RNAs direct targeting by the effector nuclease. The proposed work represents an ongoing collaboration between the Weinberg and Beisel groups and combines their respective expertise in the bioinformatic prediction of functional RNAs and the experimental investigation of CRISPR biology. If successful, this project will establish the ecrRNA as a unique class of CRISPR-associated RNAs and reveal new modes by which CRISPR-Cas systems can enact functions beyond adaptive immunity.

细菌和古细菌中的CRISPR-Cas系统提供针对质粒和噬菌体的适应性免疫。作为适应性免疫的一部分，入侵者遗传物质的小片段作为间隔片段存储在CRISPR阵列中，每个间隔片段位于固定重复序列之间。然后，每个间隔-重复序列对产生一个CRISPR RNA (crRNA)，该RNA指导系统的效应核酸酶寻找并切割与间隔序列匹配的核酸序列。因为这个间隔来自于入侵者，如果入侵者再次出现，crRNA可以帮助赋予抵抗这种入侵者的能力。每个拥有获得的间隔序列的CRISPR阵列都以一个重复序列开始和结束，留下一个重复序列没有配对的间隔序列。虽然额外的重复通常是获得新的间隔片段所必需的，但它也会产生间隔片段部分来自CRISPR阵列之外的crRNA。这种“外来”crRNA （ecrRNA）将与适应性免疫分离，因为间隔部分不是来自入侵者，它可能干扰crRNA的生物发生。因此，我们最近发现了CRISPR-Cas系统阻断ecrRNAs形成的不同机制。然而，我们观察到这些机制缺失的情况，可能允许自然形成ecrRNA。这些观察结果提出了一种有趣的可能性，即所产生的ecrnas在功能上表达和直接效应核酸酶的作用超出了适应性免疫的范围。通过这个项目，我们将研究CRISPR-Cas系统产生ecrRNAs和通过效应核酸酶直接靶向基因组的倾向。我们的假设是，功能性ecrnas可以由不同类型的CRISPR-Cas系统产生。为了研究这一假设，我们将探索三种CRISPR亚型（II-A, V-A, VI-B），在这些亚型中，我们阐明了阻断ecrRNA形成的独特机制，每种亚型都有一个目标。我们将应用互补的生物信息学和实验方法来确定ecrRNA形成的程度以及所产生的rna是否被效应核酸酶直接靶向。拟议的工作代表了Weinberg和Beisel团队之间的持续合作，并结合了他们各自在功能性rna的生物信息学预测和CRISPR生物学实验研究方面的专业知识。如果成功，该项目将确立ecrRNA作为一类独特的crispr相关rna，并揭示CRISPR-Cas系统可以发挥适应性免疫以外功能的新模式。