Extensive Self-Targeting By CRISPR-Cas Systems in the Plant Pathogen Xanthomonas Albilineans

CRISPR-Cas systems are RNA-directed adaptive immune systems in bacteria and archaea. Genetic information of invaders is collected and incorporated as spacers in CRISPR arrays. As targeting relies on spacers, self-targeting spacers are expected to lead to autoimmunity and cell death. Nevertheless, there exist examples of strains with genome-targeting spacers. Xanthomonas albilineans is a unique example with over 20 self-targeting spacers, the largest content reported to date. The bacterium also harbors two CRISPR-Cas systems, a type I-C and a type I-F system and all cas genes required for these systems are encoded within X. albilineans genome. Type I systems encode a multi‑protein effector complex called Cascade that is sufficient for target binding and, after recruiting Cas3, degrades its target. We used a cell-free transcription-translation system (TXTL) to characterize X. albilineans CRISPR-Cas systems and to address the function of these genome-targeting spacers.

We found that both systems could yield target DNA binding as well as cleavage activity. Furthermore, 5’CC and 5’TTC for I-F and I-C system, respectively, are functional PAMs. These PAMs are also associated with genome-targeting spacers, suggesting extensive self-targeting. Anti-CRISPR proteins encoded in X. albilineans genome could prevent the bacterium from lethal self-targeting. Supporting this hypothesis, we could identify ~15 putative anti-CRISPR proteins encoded within X. albilineans genome or plasmids. These proteins could serve as regulators and prevent cell-death. With this, a new alternative function for CRISPR-Cas systems as gene-regulating system could be discovered and new information about the evolution of CRISPR-Cas systems can be gathered.