Key Laboratory of RNA Biology and National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
University of the Chinese Academy of Sciences, Beijing, China.
FASEB J. 2019 Jan;33(1):1496-1509. doi: 10.1096/fj.201800557RR. Epub 2018 Jul 6.
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are prokaryotic adaptive immune systems against invading nucleic acids. CRISPR locus variability has been exploited in evolutionary and epidemiological studies of Mycobacterium tuberculosis, the causative agent of tuberculosis, for over 20 yr, yet the biological function of this type III-A system is largely unexplored. Here, using cell biology and biochemical, mutagenic, and RNA-seq approaches, we show it is active in invader defense and has features atypical of type III-A systems: mature CRISPR RNA (crRNA) in its crRNA-CRISPR/Cas protein complex are of uniform length (∼71 nt) and appear not to be subject to 3'-end processing after Cas6 cleavage of repeat RNA 8 nt from its 3' end. crRNAs generated resemble mature crRNA in type I systems, having both 5' (8 nt) and 3' (28 nt) repeat tags. Cas6 cleavage of repeat RNA is ion dependent, and accurate cleavage depends on the presence of a 3' hairpin in the repeat RNA and the sequence of its stem base nucleotides. This study unveils further diversity among CRISPR/Cas systems and provides insight into the crRNA recognition mechanism in M. tuberculosis, providing a foundation for investigating the potential of a type III-A-based genome editing system.-Wei, W., Zhang, S., Fleming, J., Chen, Y., Li, Z., Fan, S., Liu, Y., Wang, W., Wang, T., Liu, Y., Ren, B., Wang, M., Jiao, J., Chen, Y., Zhou, Y., Zhou, Y., Gu, S., Zhang, X., Wan, L., Chen, T., Zhou, L., Chen, Y., Zhang, X.-E., Li, C., Zhang, H., Bi, L. Mycobacterium tuberculosis type III-A CRISPR/Cas system crRNA and its maturation have atypical features.
成簇规律间隔短回文重复序列（CRISPR）/CRISPR 相关蛋白（Cas）系统是原核生物针对入侵核酸的适应性免疫系统。20 多年来，结核分枝杆菌（导致结核病的病原体）的 CRISPR 基因座的变异性已被用于进化和流行病学研究，但该 III-A 系统的生物学功能在很大程度上仍未得到探索。在这里，我们使用细胞生物学和生化、诱变和 RNA-seq 方法，表明它在防御入侵方面具有活性，并且具有非典型的 III-A 系统特征：成熟的 CRISPR RNA（crRNA）在其 crRNA-CRISPR/Cas 蛋白复合物中的长度一致（约 71nt），并且似乎在 Cas6 从其 3'端切割重复 RNA 的 8nt 后，不经过 3'端加工。生成的 crRNA 类似于 I 型系统中的成熟 crRNA，具有 5'（8nt）和 3'（28nt）重复标签。Cas6 对重复 RNA 的切割依赖于离子，并且准确的切割取决于重复 RNA 3'发夹的存在及其茎碱基核苷酸的序列。这项研究揭示了 CRISPR/Cas 系统之间的进一步多样性，并为结核分枝杆菌中 crRNA 识别机制提供了新的见解，为研究基于 III-A 型的基因组编辑系统的潜力奠定了基础。-魏伟，张硕，弗莱明，陈阳，李哲，范胜，刘阳，王婉婷，王涛，刘阳，任波，王明，焦娇，陈阳，周颖，周阳，顾帅，张欣，万磊，陈婷，周亮，陈阳，张欣怡，李畅，张涵，毕丽。结核分枝杆菌 III-A 型 CRISPR/Cas 系统的 crRNA 及其成熟具有非典型特征。
