Abstract: By this invention, for the first time, a method for high-efficiency site-specific genetic engineering, utilizing either native or heterologous CRISPR-Cas9 systems, in the anaerobic bacterium Clostridium pasteurianum, is provided. Application of CRISPR-Cas9 systems has revolutionized genome editing across all domains of life. Here we report implementation of the heterologous Type 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 CRISPR-Cas system.
July 4, 2017
May 16, 2019
Michael E. Pyne, Mark Bruder, Murray Moo-Young, Duane Chung, C. Perry Chou