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Gene Synthesis> | To generate pXMJ19ts-Plcpf1, pXMJ19ts-Pncas9 was used as a template to amplify a B5.7 kb fragment of pSC101-pBL1ts-Knr with primers P22/P23. A B3.9 kb fragment of PlacM-FnCpf1 was amplified using a synthetic C. glutamicum codon-optimized FnCpf1 (GenScript) as a template and primers P24/P25. | Get A Quote |
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 enabl... More