Application of the SpCas9 inhibitor BRD0539 for CRISPR/Cas9-based genetic tools in <i>Lacticaseibacillus paracasei</i>

Although the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has been extensively developed since its discovery for eukaryotic and prokaryotic genome editing and other genetic manipulations, there are still areas warranting improvement, especially regarding bacteria. In this study, BRD0539, a small-molecule inhibitor of <i>Streptococcus pyogenes</i> Cas9 (SpCas9), was used to suppress the activity of the nuclease during genetic modification of <i>Lacticaseibacillus paracasei</i>, as well as to regulate CRISPR interference (CRISPRi). First, we developed and validated a CRISPR-SpCas9 system targeting the <i>sirA</i> gene of <i>L. paracasei</i>. Then BRD0539 was used for CRISPR-dependent DNA cleavage <i>in vivo</i>. Our results suggested that the inhibitor worked partially in both <i>Escherichia coli</i> and <i>L. paracasei</i>. Next, we designed a CRISPRi system in a <i>L. paracasei</i> strain by inserting an inactive SpCas9 gene into the chromosome and introducing a plasmid encoding for a single guide RNA (sgRNA) targeting the <i>sirA</i> gene. Expression of <i>sirA</i> was successfully inhibited in the recombinant strains, and CRISPRi was abolished in an inhibitor-dependent manner. Our findings may help expand the CRISPR toolbox for research on lactic acid bacteria and other microbes.