CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum

Nat Commun. 2017 May 4:8:15179. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Corynebacterium glutamicum / genetics*
  • DNA, Single-Stranded / genetics
  • Endonucleases / metabolism*
  • Francisella / enzymology
  • Gene Editing / methods*
  • Genetic Engineering / methods*
  • Genome, Bacterial / genetics

Substances

  • DNA, Single-Stranded
  • Endonucleases