CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

Yu Jiang,Fenghui Qian,Bingbing Sun,Yingmiao Liu,Chongmao Xu,Renxiao Wang,Feng Dong,Sheng Yang,Biao Chen,Yan Li,Junjie Yang,Xiaoshu Xu

doi:10.1038/ncomms15179

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.

Highlights

Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer
We find that Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cpf[1] systems, when applied to C. glutamicum and related species, generate nucleotide substitutions, insertions and gene deletions with high efficiency (Fig. 1)
PXMJ19ts-Plcpf[1], carrying Cpf[1] from Francisella novicida (FnCpf1), transformed C. glutamicum at a high efficiency, close to that achieved by the pXMJ19ts control (Fig. 2a)

Summary

Introduction

Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. CRISPR-Cpf[1], combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86–100%. CRISPR-Cpf1-based genome editing provides a highly efficient tool for genetic engineering of Corynebacterium and other bacteria that cannot utilize the Sp CRISPR-Cas[9] system. Cpf[1], a single-strand RNA-guided endonuclease of the class 2 CRISPR-Cas system that cleaves targeted DNA with features distinct from those of Cas[9] When combine with single-stranded (ss) DNA recombineering technology, the CRISPR-Cpf[1] system enables us to achieve genomic in situ codon saturation mutagenesis without relying on laborious pre-construction of libraries. CRISPR-Cpf1assisted genome editing tools for C. glutamicum allow us to complete each round of iterative metabolic engineering in as little as 3 days, less than half the time required for sacB-based allelic exchange protocols[8]

Methods

Results

Conclusion