Processing-Independent CRISPR RNAs Limit Natural Transformation in Neisseria meningitidis

In bacteria and archaea, CRISPR loci confer adaptive, sequence-based immunity against viruses and plasmids. CRISPR interference is specified by CRISPR RNAs (crRNAs) that are transcribed and processed from CRISPR spacers and repeats. Pre-crRNA processing is essential for CRISPR interference in all systems studied thus far. Here we examine crRNA biogenesis and CRISPR interference in naturally competent Neisseria spp., including the human pathogen N. meningitidis. Our studies reveal a unique crRNA maturation pathway in which crRNA transcription is driven by promoters that are embedded within each repeat, yielding crRNA 5' ends are not formed by processing. Although crRNA 3' end formation occurs through RNase III cleavage of a pre-crRNA/tracrRNA duplex, as in other Type II CRISPR systems, this processing event is dispensable for interference. The meningococcal pathway is the most streamlined CRISPR/cas system characterized to date. Endogenous CRISPR spacers frequently target genomic sequences of other Neisseria strains and so limit natural transformation, which is the primary source of genetic variation that contributes to immune evasion, antibiotic resistance, and virulence in N. meningitidis. Overall design: dRNA-seq approach for RNA samples from cultures of N. lactamica 020-06, harvested at mid-log. Two cDNA libraries from total RNA were prepared to distinguish between transcripts with either primary orprocessed 5' ends: one library is generated from untreated RNA, whereas the other is treated with terminator exonuclease (TEX),

在细菌与古菌中，成簇规律间隔短回文重复序列（Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR）位点可赋予宿主针对病毒与质粒的序列特异性适应性免疫。CRISPR干扰由CRISPR RNA（crRNA）介导，这类RNA由CRISPR间隔序列与重复序列转录并加工获得。截至目前的所有研究均证实，前CRISPR RNA（pre-crRNA）的加工过程对于所有已表征的CRISPR系统的干扰功能均不可或缺。本研究针对具有自然感受态的奈瑟菌属（Neisseria spp.）菌株（包括人类致病菌脑膜炎奈瑟菌N. meningitidis），开展了crRNA生物发生与CRISPR干扰机制的相关研究。研究揭示了一条独特的crRNA成熟通路：crRNA的转录由嵌入于每个重复序列内部的启动子驱动，由此产生的crRNA 5'端并非通过加工过程生成。尽管与其他II型CRISPR系统类似，crRNA 3'端的形成通过核糖核酸酶III（RNase III）切割pre-crRNA/反式激活crRNA（trans-activating crRNA, tracrRNA）双链复合体完成，但该加工事件对于CRISPR干扰功能并非必需。脑膜炎奈瑟菌的这一CRISPR/Cas通路是目前已报道的最为精简的CRISPR/Cas系统。内源性CRISPR间隔序列常靶向其他奈瑟菌菌株的基因组序列，因此会限制自然转化过程；而自然转化正是脑膜炎奈瑟菌遗传变异的主要来源，这类变异可帮助致病菌逃避免疫、获得抗生素耐药性并增强毒力。实验整体设计：针对从对数生长期中期收获的乳糖奈瑟菌020-06（N. lactamica 020-06）培养物提取的RNA样本，采用dRNA-seq技术进行分析。研究人员从总RNA中构建了两种cDNA文库，以区分带有原始5'端与加工后5'端的转录本：其中一个文库由未经处理的总RNA构建，另一个文库则经末端核酸外切酶（terminator exonuclease, TEX）处理。