Establishment of a RecET-Assisted CRISPR-Cas12a System for Large Deoxyribonucleic Acid-Fragment Manipulation in <i>Zymomonas mobilis</i>

The lack of effective and efficient genome-editing toolkits for large DNA-fragment manipulation impedes the development of robust cell factories to meet the needs of fast-growing biomanufacturing. <i>Zymomonas mobilis</i> is an important nonmodel polyploid industrial strain with excellent industrial characteristics. Although diverse CRISPR-Cas systems have been established in <i>Z. mobilis</i> for gene deletion, replacement, and ssDNA recombination, it is still challenging to achieve large DNA-fragment manipulation due to its low recombination and repair efficiencies for double-strand DNA breaks. In this study, a RecET-assisted CRISPR-Cas12a genome editing system was developed using a chromosome-borne <i>cas12a</i> and <i>recET</i>-encoded recombinase, as well as an all-in-one editing plasmid. Different promoters were used for <i>recET</i> and <i>cas12a</i> to determine optimal expression. The combination of PB-cas12a_Pt-recET had the highest efficiency of 97.92 ± 2.95% for 9-kb DNA-fragment deletion, which also had efficiencies about 100%, 80%, and 5%, respectively, for the deletion of 9-16, 20-25, and 30 kb DNA fragments. The RecET-assisted CRISPR-Cas12a was further applied for deletions of different large gene clusters and had the potential for efficient pathway knock-in. This study highlights the importance of the Cas12a nuclease expression levels and the combination of the RecET system in improving the double-strand DNA repair capability for large DNA-fragment manipulation in <i>Z. mobilis</i>. The RecET-assisted CRISPR-Cas12a system established in this study provides a versatile and powerful tool for large DNA-fragment manipulation in <i>Z. mobilis</i>, which is beneficial for functional genomic research, strain improvement, as well as the development of synthetic microbial chassis.