AUTHOR=Li Mingyue , Chen Jiuzhou , Wang Yu , Liu Jiao , Huang Jingwen , Chen Ning , Zheng Ping , Sun Jibin 

TITLE=Efficient Multiplex Gene Repression by CRISPR-dCpf1 in Corynebacterium glutamicum

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=8

YEAR=2020

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2020.00357

DOI=10.3389/fbioe.2020.00357

ISSN=2296-4185

ABSTRACT=<p><italic>Corynebacterium glutamicum</italic> is an important workhorse for industrial production of diversiform bioproducts. Multiplex control of metabolic pathway genes is crucial for maximizing biosynthesis of desired products. However, few tools for simultaneously regulating multiple genes in <italic>C. glutamicum</italic> have been reported. Here, a CRISPR-dCpf1-based multiplex gene repression system was developed for <italic>C. glutamicum</italic>. This system successfully repressed two fluorescent reporter genes simultaneously by expressing a dCpf1 (E1006A, D917A) and a designed single crRNA array. To demonstrate applications of this CRISPR-dCpf1 system in metabolic engineering, we applied this system to repress four genes involved in lysine biosynthesis (<italic>gltA</italic>, <italic>pck</italic>, <italic>pgi</italic>, and <italic>hom</italic>) with a single array, which increased the lysine titer and yield for over 4.0-fold. Quantitative PCR demonstrated that transcription of all the four endogenous target genes were repressed by over 90%. Thus, the CRISPR-dCpf1 system is a simple and effective technique for multiplex gene repression in <italic>C. glutamicum</italic> and holds promise for metabolic engineering of <italic>C. glutamicum</italic> to produce valuable chemicals and fuels.</p>