Stachler Aris-Edda, Turgeman-Grott Israela, Shtifman-Segal Ella, Allers Thorsten, Marchfelder Anita, Gophna Uri
Department of Biology II, Ulm University, 89069 Ulm, Germany.
Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978-01, Israel.
Nucleic Acids Res. 2017 May 19;45(9):5208-5216. doi: 10.1093/nar/gkx150.
CRISPR-Cas systems allow bacteria and archaea to acquire sequence-specific immunity against selfish genetic elements such as viruses and plasmids, by specific degradation of invader DNA or RNA. However, this involves the risk of autoimmunity if immune memory against host DNA is mistakenly acquired. Such autoimmunity has been shown to be highly toxic in several bacteria and is believed to be one of the major costs of maintaining these defense systems. Here we generated an experimental system in which a non-essential gene, required for pigment production and the reddish colony color, is targeted by the CRISPR-Cas I-B system of the halophilic archaeon Haloferax volcanii. We show that under native conditions, where both the self-targeting and native crRNAs are expressed, self-targeting by CRISPR-Cas causes no reduction in transformation efficiency of the plasmid encoding the self-targeting crRNA. Furthermore, under such conditions, no effect on organismal growth rate or loss of the reddish colony phenotype due to mutations in the targeted region could be observed. In contrast, in cells deleted for the pre-crRNA processing gene cas6, where only the self-targeting crRNA exists as mature crRNA, self-targeting leads to moderate toxicity and the emergence of deletion mutants. Sequencing of the deletions caused by CRISPR-Cas self targeting indicated DNA repair via microhomology-mediated end joining.
CRISPR-Cas系统使细菌和古生菌能够通过特异性降解入侵的DNA或RNA，获得针对病毒和质粒等自私遗传元件的序列特异性免疫。然而，如果错误地获得了针对宿主DNA的免疫记忆，这就存在自身免疫的风险。这种自身免疫在几种细菌中已被证明具有高度毒性，并且被认为是维持这些防御系统的主要代价之一。在这里，我们构建了一个实验系统，其中嗜盐古生菌沃氏嗜盐栖热菌的CRISPR-Cas I-B系统靶向一个对色素产生和红色菌落颜色必不可少的非必需基因。我们发现，在天然条件下，当自身靶向和天然crRNA都表达时，CRISPR-Cas的自身靶向不会降低编码自身靶向crRNA的质粒的转化效率。此外，在这种条件下，未观察到对生物体生长速率有影响，也未观察到由于靶向区域的突变导致红色菌落表型丧失。相反，在缺失前体crRNA加工基因cas6的细胞中，只有自身靶向crRNA以成熟crRNA的形式存在，自身靶向会导致中度毒性并出现缺失突变体。由CRISPR-Cas自身靶向引起的缺失测序表明通过微同源性介导的末端连接进行DNA修复。
