Metabolic Engineering Research Laboratory, Institute of Chemical and Engineering Sciences, A*STAR, Singapore, Singapore.
Molecular Engineering Laboratory, Biomedical Institutes of Sciences, A*STAR, Singapore, Singapore.
Biotechnol Bioeng. 2019 Sep;116(9):2330-2338. doi: 10.1002/bit.27021. Epub 2019 May 30.
Application of the well-characterized Streptococcus pyogenes CRISPR-Cas9 system in actinomycetes streptomycetes has enabled high-efficiency multiplex genome editing and CRISPRi-mediated transcriptional regulation in these prolific bioactive metabolite producers. Nonetheless, SpCas9 has its limitations and can be ineffective depending on the strains and target sites. Here, we built and tested alternative CRISPR-Cas constructs based on the standalone pCRISPomyces-2 editing plasmid. We showed that Streptococcus thermophilus CRISPR1 Cas9 (sth1Cas9), Staphylococcus aureus Cas9 (saCas9), and Francisella tularensis subsp. novicida U112 Cpf1 (fnCpf1) are functional in multiple streptomycetes, enabling efficient homology-directed repair-mediated knock-in and deletion. In strains where spCas9 was nonfunctional, these alternative Cas systems enabled precise genomic modifications within biosynthetic gene clusters for the discovery, production, and diversification of natural products. These additional Cas proteins provide us with the versatility to overcome the limitations of individual CRISPR-Cas systems for genome editing and transcriptional regulation of these industrially important bacteria.
在放线菌链霉菌中应用特征明确的酿脓链球菌 CRISPR-Cas9 系统，使得在这些高产生物活性代谢产物产生菌中进行高效多重基因组编辑和 CRISPRi 介导的转录调控成为可能。尽管如此，SpCas9 也有其局限性，并且根据菌株和靶位点的不同，其效果可能不佳。在这里，我们构建并测试了基于独立的 pCRISPomyces-2 编辑质粒的替代 CRISPR-Cas 构建体。我们表明，嗜热链球菌 CRISPR1 Cas9(sth1Cas9)、金黄色葡萄球菌 Cas9(saCas9) 和土拉弗朗西斯菌亚种 novicida U112 Cpf1(fnCpf1) 在多种链霉菌中均具有功能，能够有效地进行同源定向修复介导的敲入和缺失。在 spCas9 无效的菌株中，这些替代 Cas 系统能够在生物合成基因簇内进行精确的基因组修饰，从而发现、生产和多样化天然产物。这些额外的 Cas 蛋白使我们能够克服单个 CRISPR-Cas 系统在这些工业上重要的细菌的基因组编辑和转录调控方面的局限性。
