Mechanisms and functions of endogenous RNA-targeting by CRISPR-Cas9 in Campylobacter jejuni

II型CRISPR-Cas9核酸酶天然地利用CRISPR RNA（crRNA）和tracrRNA来沉默外源双链DNA。除了它们在适应性免疫中的作用得到充分研究外，还有新的证据表明CRISPR-Cas系统具有超越防御的作用，并对细菌毒力和群体行为等表型产生影响。例如，II型组分的缺失会影响不同病原体的毒力，如新杀弗朗西斯氏菌、脑膜炎奈瑟氏菌或空肠弯曲杆菌。虽然最近的工作表明，一些Cas9核酸酶也可以靶向RNA，但RNA识别需要核酸酶修饰或辅助因子。例如F. novicida Cas9使用相关的scaRNA和tracrRNA来抑制内源性脂蛋白mRNA以影响毒力，尽管确切的调控机制仍不清楚。 我们对食源性病原体C. jejuni揭示了其大量转录的II-C型CRISPR-Cas系统的最小crRNA生物合成途径。此外，我们未发表的RIP-seq（免疫共沉淀结合RNA-seq）研究表明，C。空肠Cas9（CjCas 9）使用其天然crRNA来结合和切割互补的内源性mRNA。大约100种转录物与CjCas 9共纯化，并且通常可以通过其碱基配对潜力细分为菌株NCTC 11168的四种crRNA。这些RNA中的几个在预测的结合位点附近进行切割。突变分析显示RNA靶向是crRNA和tracrRNA依赖性的，并且RNA切割需要CjCas 9 HNH结构域。我们进一步观察到，随着crRNA和RNA靶标之间的互补性增加，RNA切割得到改善，并且在体外是可编程的。这些发现表明，CjCas 9是一种混杂的核酸酶，可以协同靶向DNA和RNA。在这里，我们将进一步研究CjCas 9靶向RNA的生理作用和潜在的分子机制，以了解它是否介导内源性基因调控或只是免疫监视的副作用。首先，表达不同crRNA库的菌株的RIP-seq将揭示内源RNA靶向的程度，并揭示是否存在单独的crRNA依赖性和非依赖性调节子。调节突变体和各种条件下的表达分析将显示CRISPR-Cas何时受到调节，以了解其功能。CRISPR-Cas突变体的RNA-seq和核糖体分析将鉴定全局切割模式，并可鉴定转录后控制的靶标。然后，我们将使用遗传学和生物化学方法来验证CjCas 9靶点，并详细研究调控的分子机制，包括与crRNA碱基配对的决定因素，从选定的候选靶mRNA开始。最后，我们将确定CjCas 9的RNA靶向是否调节毒力。这将为CRISPR-Cas在细菌基因调控和致病性中的作用提供深入了解。

The Type II CRISPR-Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) and tracrRNA to silence foreign double-stranded DNA. Besides their well-studied role in adaptive immunity, there is emerging evidence that CRISPR-Cas systems have roles beyond defense and impact on phenotypes such as bacterial virulence and group behavior. For example, deletion of Type II components affects virulence in diverse pathogens, such as Francisella novicida, Neisseria meningitidis or Campylobacter jejuni. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. For example, F. novicida Cas9 uses an associated scaRNA and tracrRNA to repress an endogenous lipoprotein mRNA to affect virulence, although the exact mechanism of regulation remains unclear. Our RNA-seq analysis of multiple strains of the foodborne pathogen C. jejuni revealed a minimal crRNA biogenesis pathway for its abundantly transcribed Type II-C CRISPR-Cas system. Moreover, our unpublished RIP-seq (co-immunoprecipitation combined with RNA-seq) study revealed that C. jejuni Cas9 (CjCas9) uses its native crRNAs to bind and cleave complementary endogenous mRNAs. Approximately 100 transcripts co-purify with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs of strain NCTC11168. Several of these RNAs underwent cleavage close to the predicted binding site. Mutational analyses revealed RNA targeting was crRNA- and tracrRNA-dependent, and that RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage improved with greater complementarity between the crRNA and the RNA target, and was programmable in vitro. These findings suggest that CjCas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.Here, we will further investigate the physiological roles and underlying molecular mechanisms of RNA targeting by CjCas9 to understand if it mediates endogenous gene regulation or is just a side effect of immune surveillance. First, RIP-seq of strains expressing different crRNA repertoires will reveal the extent of endogenous RNA targeting and reveal if there are separate crRNA-dependent and -independent regulons. Expression analyses in regulator mutants and under various conditions will show when CRISPR-Cas is regulated to provide insight into its function. RNA-seq and ribosome profiling of CRISPR-Cas mutants will identify global cleavage patterns and may identify targets of post-transcriptional control. We will then use genetics and biochemistry approaches to validate CjCas9 targets and study in detail the molecular mechanisms underlying regulation, including determinants for base-pairing with crRNAs, starting with selected candidate target mRNAs. Finally, we will determine if RNA targeting by CjCas9 regulates virulence. This will provide insight into the roles of CRISPR-Cas in bacterial gene regulation and pathogenicity.