Pyne Michael E, Bruder Mark R, Moo-Young Murray, Chung Duane A, Chou C Perry
Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada.
Department of Pathology and Molecular Medicine, McMaster University, Ontario, Canada.
Sci Rep. 2016 May 9;6:25666. doi: 10.1038/srep25666.
Application of CRISPR-Cas9 systems has revolutionized genome editing across all domains of life. Here we report implementation of the heterologous Type II CRISPR-Cas9 system in Clostridium pasteurianum for markerless genome editing. Since 74% of species harbor CRISPR-Cas loci in Clostridium, we also explored the prospect of co-opting host-encoded CRISPR-Cas machinery for genome editing. Motivation for this work was bolstered from the observation that plasmids expressing heterologous cas9 result in poor transformation of Clostridium. To address this barrier and establish proof-of-concept, we focus on characterization and exploitation of the C. pasteurianum Type I-B CRISPR-Cas system. In silico spacer analysis and in vivo interference assays revealed three protospacer adjacent motif (PAM) sequences required for site-specific nucleolytic attack. Introduction of a synthetic CRISPR array and cpaAIR gene deletion template yielded an editing efficiency of 100%. In contrast, the heterologous Type II CRISPR-Cas9 system generated only 25% of the total yield of edited cells, suggesting that native machinery provides a superior foundation for genome editing by precluding expression of cas9 in trans. To broaden our approach, we also identified putative PAM sequences in three key species of Clostridium. This is the first report of genome editing through harnessing native CRISPR-Cas machinery in Clostridium.
CRISPR-Cas9系统的应用彻底改变了所有生命领域的基因组编辑。在此，我们报告了在巴氏梭菌中实现用于无标记基因组编辑的异源II型CRISPR-Cas9系统。由于梭菌属中74%的物种含有CRISPR-Cas基因座，我们还探索了利用宿主编码的CRISPR-Cas机制进行基因组编辑的前景。观察到表达异源cas9的质粒在梭菌中的转化效果不佳，这增强了我们开展这项工作的动力。为克服这一障碍并建立概念验证，我们着重于对巴氏梭菌I-B型CRISPR-Cas系统的表征和利用。通过计算机模拟间隔区分析和体内干扰试验，揭示了位点特异性核酸酶攻击所需的三个原间隔序列临近基序（PAM）序列。引入合成的CRISPR阵列和cpaAIR基因缺失模板，编辑效率达到了100%。相比之下，异源II型CRISPR-Cas9系统产生的编辑细胞总产量仅为25%，这表明天然机制通过排除反式表达cas9为基因组编辑提供了更好的基础。为拓展我们的方法，我们还在三种关键梭菌属物种中鉴定出了假定的PAM序列。这是关于利用梭菌属天然CRISPR-Cas机制进行基因组编辑的首次报道。
