Analysis of Type IV-A1 CRISPR-Cas activity in Pseudomonas oleovorans in vivo

Survival is a constant battle against environmental threats in the microbial world, while bacteria as well as archaea can suffer from invaders and need adaptive strategies to evade harmful mobile genetic elements. Here, CRISPR-Cas systems, classified into two major classes and several functionally distinct types, serve as adaptive immune systems against foreign DNA in prokaryotes. Type IV CRISPR-Cas systems are classified as class 1 systems, acting through multi-subunit ribonucleoprotein complexes. Unlike many other systems, Type IV usually lacks an adaptation module and does not rely on DNA cleavage for CRISPR interference. Within recent studies a Type IV-A1 CRISPR-Cas system in the bacterium Pseudomonas oleovorans was found. Type IV-A1 is associated with the helicase CasDinG that is involved in unwinding the DNA during the interference process. The precise interference mechanism of Type IV-A1 has not yet been fully understood. Thus, this study addresses gaps in knowledge to further examine the activity and mechanism of the Type IV-A1 CRISPR-Cas system found in Pseudomonas oleovorans strain DSM 1045. We first established a Golden Gate method for development and optimization of minimal CRISPR arrays to facilitate convenient cloning of synthetic crRNAs and pave the way of Type IV-A1 as a biotechnological tool. Various plasmids expressing synthetic crRNAs were generated using the Golden Gate cloning strategy to further analyze the native or a recombinant Type IV-A1 system. Furthermore, a novel time-lapse imaging system (SMARTIS) was developed, which is specifically designed for real-time visualization of bacterial colony growth. SMARTIS provides high-resolution images that can be further analyzed. Thus, variations in colony sizes and colors of Escherichia coli colonies expressing Type IV-A1 crRNPs were examined and revealed that colony heterogeneity is potentially caused by CRISPR-mediated targeting in combination with a used expression system. We created P. oleovorans strains genomically expressing sfGFP and used various synthetic crRNAs to target different genomic regions. The range of mediated interference on genomic targets was determined and we demonstrated that the Type IV-A1 CRISPR-Cas system achieves long-range transcriptional repression. This extended interference effect is facilitated by the associated helicase CasDinG, which unwinds DNA in a 5′-3′ direction and potentially interrupts activity of the RNA polymerase. Eventually, this research advances our understanding of Type IV-A1 CRISPR-Cas systems, emphasizing their distinctive interference mechanisms and potential as native tools for non-destructive gene regulation.