Characterization of Type IV-A1 CRISPR interference mechanism in vivo

The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans presents a unique self- targeting feature affecting the transcript levels of its native target in the genome. This study investigates the system's interference mechanism and regulatory dynamics using synthetic crRNAs in the native or recombinant system in Escherichia coli. Targeting an integrated fluorescent protein gene (sfGFP) in the genome revealed extended locational effects of CRISPR-Cas activity. Here, a complex interplay between synthetic and endogenous crRNAs was revealed, emphasizing the need for careful crRNA design considerations. Using the recombinant system in comparison with dCas9 highlighted advanced interference effects by Type IV-A1 on targeted and neighboring genes. Furthermore, evaluation of spatiotemporal organization of Type IV-A1 in presence of plasmids revealed its plasmids targeting preferences. Our results provide insights of dynamic regulation by Type IV-A1 and suggest it as potential tool as CRISPRi tool for gene regulation and biotechnology.