CRISPR-Cpf1 Assisted Genome Editing of Corynebacterium Glutamicum

Overview

Journal Nat Commun

Specialty Biology

Date 2017 May 5

PMID 28469274

Citations 141

Authors

Yu Jiang

Fenghui Qian

Junjie Yang

Yingmiao Liu

Feng Dong

Chongmao Xu

Bingbing Sun

Biao Chen

Xiaoshu Xu

Yan Li

Renxiao Wang

Sheng Yang

Affiliations

Soon will be listed here.

Abstract

Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum. Here we report a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method for C. glutamicum. CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86-100%. Large gene deletions and insertions are also obtained using an all-in-one plasmid consisting of FnCpf1, CRISPR RNA, and homologous arms. The two CRISPR-Cpf1-assisted systems enable N iterative rounds of genome editing in 3N+4 or 3N+2 days. A proof-of-concept, codon saturation mutagenesis at G149 of γ-glutamyl kinase relieves L-proline inhibition using Cpf1-assisted ssDNA recombineering. Thus, CRISPR-Cpf1-based genome editing provides a highly efficient tool for genetic engineering of Corynebacterium and other bacteria that cannot utilize the Sp CRISPR-Cas9 system.

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