Characterization of Cas proteins for CRISPR‐Cas editing in streptomycetes

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Characterization of Cas proteins for CRISPR‐Cas editing in streptomycetes
Characterization of Cas proteins for CRISPR‐Cas editing in streptomycetes
Other Titles:
Biotechnology and Bioengineering
Publication Date:
15 May 2019
Yeo WL, Heng E, Tan LL, et al. Characterization of Cas proteins for CRISPR‐Cas editing in streptomycetes. Biotechnology and Bioengineering. 2019;116: 2330–2338.
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 repairmediated 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.
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Funding Info:
Funded by by the Agency for Science, Technology and Research (A*STAR), Singapore and National Research Foundation, Singapore (NRF2013‐THE001–094 to MMZ, FTW, YHL) and the A*STAR Visiting Investigator Program (1535j00137 to HZ).
"This is the peer reviewed version of the following article: [Wan Lin Yeo, Elena Heng, Lee Ling Tan, Yi Wee Lim, Yee Hwee Lim, Shawn Hoon, Huimin Zhao, Mingzi M Zhang, Fong Tian Wong. Characterization of Cas proteins for CRISPR-Cas editing in streptomycetes. Biotechnol Bioeng. 2019 Sep;116(9):2330-2338 ], which has been published in final form at [10.1002/bit.27021]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions."

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