AUTHOR=Zhang Feng , Wang Jin-Yu , Li Chang-Lon , Zhang Wei-Guo 

TITLE=HyCas9-12aGEP: an efficient genome editing platform for Corynebacterium glutamicum

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=12

YEAR=2024

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2024.1327172

DOI=10.3389/fbioe.2024.1327172

ISSN=2296-4185

ABSTRACT=<p><italic>Corynebacterium glutamicum</italic> plays a crucial role as a significant industrial producer of metabolites. Despite the successful development of CRISPR-Cas9 and CRISPR-Cas12a-assisted genome editing technologies in <italic>C. glutamicum</italic>, their editing resolution and efficiency are hampered by the diverse on-target activities of guide RNAs (gRNAs). To address this problem, a hybrid CRISPR-Cas9-Cas12a genome editing platform (HyCas9-12aGEP) was developed in <italic>C</italic>. <italic>glutamicum</italic> in this study to co-express sgRNA (corresponding to <italic>Sp</italic>Cas9 guide RNA), crRNA (corresponding to <italic>Fn</italic>Cas12a guide RNA), or hfgRNA (formed by the fusion of sgRNA and crRNA). HyCas9-12aGEP improves the efficiency of mapping active gRNAs and outperforms both CRISPR-Cas9 and CRISPR-Cas12a in genome editing resolution and efficiency. In the experiment involving the deletion of the <italic>cg0697-0740</italic> gene segment, an unexpected phenotype was observed, and HyCas9-12aGEP efficiently identified the responsible genotype from more than 40 genes. Here, HyCas9-12aGEP greatly improve our capability in terms of genome reprogramming in <italic>C. glutamicum.</italic></p>