Characterization of Type IV-A1 CRISPR-interference on gene expression and plasmid replication
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Philipps-Universität Marburg
Abstract
Among the extensive repertoire of bacterial defense mechanisms against mobile genetic elements (MGEs), CRISPR-Cas systems have emerged as versatile tools, primarily relying on nucleases that recognize foreign DNA or RNA. These systems are classified into two classes, seven types, and 33 subtypes based on the composition of the effector complexes involved in the interference process. Since the discovery of CRISPR-Cas systems as adaptive immune systems in bacteria, research has increasingly explored their broader functions beyond immunity against MGEs. One intriguing aspect is the presence of self-targeting spacers (STS) that target functional genes within the host genome, potentially leading to lethal effects. Bacterial cells employ various strategies to mitigate these effects, such as mutations in CRISPR elements, partial complementarity or using anti-CRISPR proteins.
Interestingly, the Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans contains an STS within its CRISPR array that targets the host pilN gene, which is involved in Type IV pili formation. Notably, this system operates without common mitigation strategies. Instead, the presence of a functional system with a tolerated STS suggests additional functions beyond adaptive immunity.
Type IV systems are typically found on large plasmids and lack adaptation modules like Cas1 and Cas2, as well as nucleases for target degradation. Despite this, they exhibit active defense mechanisms, particularly against plasmids and conjugative elements. Recent studies have highlighted the importance of the helicase-containing protein CasDinG in the interference process. However, the exact interference mechanism and the extent to which the system could be used as a genetic tool remain to be fully elucidated.
In my thesis, I address the characterization of the Type IV-A1 CRISPR ribonucleoprotein (crRNP) interference activities through two main research approaches: characterizing CRISPR interference using Illumina RNA-seq and visualizing crRNP dynamics using single-molecule microscopy (SMM). In Chapter II, we present the first publication on the characterization of the self-targeting activities of the crRNPs in P. oleovorans. My contributions to the study revealed that fluorescently tagged crRNPs displayed distinct dynamics in the presence and absence of the STS, evaluated in both wild-type and ΔCRISPR array strains. In the wild-type strain, significant numbers of confined molecules displayed lower diffusion rates over the bacterial nucleoid, suggesting active nucleoid scanning and probing in the presence of STS. Additionally, comparison of the transcriptomes from the RNA-seq data of wild-type and ΔCRISPR strains showed significant changes in the transcript abundance of the host pilN, with increased transcripts for pilN and neighboring genes in the ΔCRISPR strain. This suggests an interference mechanism that, while tolerating STS, is able to downregulate transcripts while maintaining DNA integrity.
Following these findings, we explored the scope of the interference and potential as a CRISPR interference (CRISPRi) tool in Chapter III. We compared crRNP interference at the transcript level with the well-known dCas9 system, which interferes by physically blocking RNA polymerase (RNAP) and preventing transcription elongation. Targeting different genes in the histidine operon resulted in broader transcriptomic impacts by crRNPs, effectively silencing the entire operon, compared to the local downregulation seen with dCas9 treatments.
In the final part of Chapter III, we evaluated the spatiotemporal dynamics of fluorescently tagged crRNPs in a recombinant system exposed to both genome and target-containing plasmids. Results confirm the distribution of crRNPs over the bacterial nucleoid in presence of genome targets. In contrast, introducing target-containing plasmids redistributed the crRNPs towards cell poles and showed particles with lower diffusion rates, indicating prolonged target interaction times. This supports the hypothesis of crRNP interaction with plasmids. Our findings suggest that crRNPs interfere with plasmid replication by interacting with the replication/transcription machinery, particularly the DnaX clamp loader complex, which was fluorescently tagged to evaluate interactions of crRNPs with replication forks upon plasmid targeting. Experiments showed decreased DnaX diffusion rates in the presence of target-containing plasmids, implying replication fork stalling. This effect aligns with observations in other CRISPR systems and supports the notion of crRNP-mediated plasmid replication inhibition.
Altogether, this research enhances our understanding of Type IV-A1 CRISPR-Cas systems, highlighting their unique interference mechanisms and potential as natural CRISPRi tools. The findings provide a foundation for future studies to explore their broader biological roles and applications in bacterial gene regulation.