Characterizing CRISPR-Cas systems with non-defensive functions

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CRISPR-Cas systems are widely recognized as RNA-guided, adaptive immune systems in prokaryotes that detect and eradicate foreign genetic material. However, recent work highlighting alternative roles in gene regulation and immune avoidance are challenging the singular definition of CRISPR as an immune system. Still unclear are the inherent roles of CRISPR and how they impact the overall physiology and behavior of prokaryotic life.We previously showed that the most abundant Type I CRISPR-Cas systems can be readily converted into transcriptional repressors by disrupting the system’s endonuclease Cas3. We hypothesized that Type I systems may perform a similar function naturally. By searching prokaryotic genomes for genome-targeting CRISPR arrays, we identified the plant pathogen Xanthomonas albilineans as an extremely promising case. The genome of this bacterium contains two complete Type I CRISPR-Cas systems, a I-C system and a I-F system, encoding a total of 64 genome-targeting spacers. Further analysis showed that the target sites are flanked by known protospacer-adjacent motifs (PAMs), and the I-C system appears to have a defective Cas3. These preliminary results strongly suggest that the CRISPR-Cas systems in this agriculturally important bacterium could act as transcriptional repressors and regulate a collection of cellular processes.The goal of this proposal is to characterize the properties of the two CRISPR-Cas systems in X. albilineans. We hypothesize that both systems are functionally expressed and regulate transcription of target sites through lack of Cas3 activity. To test this hypothesis, we propose the following two objectives: Objective 1: Determine the outcome of DNA targeting by each CRISPR-Cas system using cell-free transcription-translation systems.Objective 2: Determine the expression pattern and function of each CRISPR-Cas system in X. albilineans. If successful, the proposed work could reveal novel roles of CRISPR-Cas systems that extend beyond adaptive immunity and provide the basis to identify similar functions across the diversity of Type I CRISPR-Cas systems found in the prokaryotic world. The PI has extensive experience with CRISPR-Cas systems and bacterial genetics, and the project directly aligns with the goals of SPP 2141.