Zhao Ran, Liu Yanqiang, Zhang Huan, Chai Changsheng, Wang Jin, Jiang Weihong, Gu Yang
Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences , Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences , Shanghai 200032 , China.
School of Life Sciences , Henan University , Kaifeng 475001 , China.
ACS Synth Biol. 2019 Oct 18;8(10):2270-2279. doi: 10.1021/acssynbio.9b00033. Epub 2019 Sep 26.
Uncovering the full potential of gas-fermenting Clostridia, attractive autotrophic bacteria capable of using synthesis gases (CO-CO-H) to produce a range of chemicals and fuels, for industrial applications relies on having efficient molecular tools for genetic modifications. Although the CRISPR-Cas9-mediated genome editing system has been developed in Clostridia, its use is limited owing to low GC content (approx. 30%) in these anaerobes. Therefore, the effector protein Cas12a, which recognizes T-rich instead of G-rich protospacer-adjacent motifs (PAMs), has evident advantages over Cas9 in CRISPR genome editing in Clostridia. Here, we developed the CRISPR-Cas12a system for efficient gene deletion and regulation in the gas-fermenting species. On the basis of screening for the most suitable Cas12a and significantly improved electrotransformation efficiency that bypassed poor repair efficiency of the Cas12a-caused DNA double-strand break (DSB) in , efficient deletion (80-100%) of four genes (, , , and ) was achieved by using the CRISPR-Cas12a system. Furthermore, a DNase-deactivated Cas12a (ddCas12a) was adopted to construct a CRISPRi system to downregulate targeted genes, reaching over 80% repression for most of the chosen binding sites. This CRISPRi system was also used in a butyric acid-producing strain to redirect carbon flux, leading to 20-40% reductions in ethanol titer that were accompanied by increased butyric acid titer. These results demonstrate the high efficiency of the CRISPR-Cas12a system for genome engineering in , which effectively expands the existing CRISPR-Cas toolbox in gas-fermenting species and may play important roles in genetic manipulations where CRISPR-Cas9 is incompetent.
发掘能够利用合成气（CO-CO-H）生产一系列化学品和燃料的有吸引力的自养细菌——产气梭菌在工业应用中的全部潜力，依赖于拥有用于基因改造的高效分子工具。尽管已经在梭菌中开发出了CRISPR-Cas9介导的基因组编辑系统，但由于这些厌氧菌的GC含量较低（约30%），其应用受到限制。因此，识别富含T而非富含G的原间隔序列临近基序（PAM）的效应蛋白Cas12a，在梭菌的CRISPR基因组编辑中比Cas9具有明显优势。在此，我们开发了用于产气物种中高效基因缺失和调控的CRISPR-Cas12a系统。基于筛选最合适的Cas12a以及显著提高的电转化效率（该效率绕过了Cas12a导致的DNA双链断裂（DSB）在中的低修复效率），通过使用CRISPR-Cas12a系统实现了四个基因（、、、和）的高效缺失（80-100%）。此外，采用无核酸酶活性的Cas12a（ddCas12a）构建了CRISPRi系统以下调靶向基因，对于大多数选定的结合位点，抑制率超过80%。该CRISPRi系统还用于产丁酸的菌株中以重新引导碳通量，导致乙醇滴度降低20-40%，同时丁酸滴度增加。这些结果证明了CRISPR-Cas12a系统在产气物种基因组工程中的高效性，其有效地扩展了产气物种中现有的CRISPR-Cas工具库，并且可能在CRISPR-Cas9无法胜任的基因操作中发挥重要作用。
