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

细菌和古生菌中的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物存储在CRISPR阵列中，每个间隔物位于固定重复之间。然后，每个间隔子-重复对产生一个CRISPR RNA(CrRNA)，它指导系统的效应器核酸酶寻找和切割与间隔子匹配的核酸序列。由于该间隔区来自入侵者，如果再次出现，crRNA有助于增强对该入侵者的抵抗力。拥有获得的间隔区集合的每个CRISPR阵列以重复开始和结束，留下一个没有成对间隔区的重复。虽然额外的重复通常是获得新的间隔物所必需的，但它也会产生具有起源于CRISPR阵列外部的间隔物部分的crRNA。这种“外来的”crRNA(EcrRNA)将与适应性免疫断开，因为间隔区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系统阻止ecrRNAs形成的不同机制。然而，我们观察到这些机制缺失的例子，潜在地允许自然的ecrRNA形成。这些观察结果提出了一种耐人寻味的可能性，即产生的ecrRNA是功能性表达的，并为超越适应性免疫的目的而直接作用于核酸酶。通过这个项目，我们将研究CRISPR-CAS系统产生ecrRNAs的倾向，并通过效应器核酸酶直接进行基因组靶向。我们的假设是，功能ecrRNA可以由不同类型的CRISPR-Cas系统产生。为了探讨这一假设，我们将探索三个CRISPR亚型(II-A，V-A，VI-B)，在这三个亚型中，我们阐明了阻止ecrRNA形成的不同机制，每个亚型都有一个目标。我们将应用互补的生物信息学和实验方法来确定ecrRNA的形成程度，以及由此产生的RNA是否直接被效应器核酸酶靶向。这项拟议的工作代表了Weinberg和Beisel团队之间的持续合作，并结合了他们在功能RNA的生物信息学预测和CRISPR生物学的实验研究方面的各自专业知识。如果成功，该项目将建立ecrRNA作为CRISPR相关RNA的独特类别，并揭示CRISPR-CAS系统执行适应性免疫以外的功能的新模式。

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.