CRISPR/Cas12a-Mediated Genome Editing in Thioalkalivibrio versutus

Haloalkaliphilic Thioalkalivibrio versutus, a dominant species for sulfide removal, has attracted increasing attention. However, research on T. versutus is limited by the lack of genetic manipulation tools. In this work, we developed a CRISPR/AsCas12a-mediated system in T. versutus for an efficient and implementable genome editing workflow. Compared to the CRISPR/Cas9-mediated system, the CRISPR/AsCas12a system exhibited enhanced editing efficiency. Additionally, as Cas12a is capable of processing the crRNA maturation independently, the CRISPR/AsCas12a system allowed multiplex gene editing and large-fragment DNA knockout by expressing more than one crRNA under the control of one promoter. Using the CRISPR/AsCas12a system, five key genes of the elemental sulfur oxidation pathway were knocked out. Simultaneous deletion of the rhd and tusA genes disrupted the ability of T. versutus to metabolize elemental sulfur, resulting in a 24.7% increase in elemental sulfur generation and a 15.2% reduction in sulfate production. This genome engineering strategy significantly improved our understanding of sulfur metabolism in Thioalkalivibrio spp.