Cas9 interaction with the tracrRNA nexus modulates the repression of type II-A CRISPR-cas genes.

Immune responses need to be regulated to prevent autoimmunity. CRISPR-Cas systems provide adaptive immunity in prokaryotes through the acquisition of short DNA sequences from invading viruses (bacteriophages), known as spacers. Spacers are inserted into the CRISPR locus and serve as templates for the transcription of guides used by RNA-guided nucleases to recognize complementary nucleic acids of the invaders and start the CRISPR immune response. In type II-A CRISPR systems, Cas9 uses the guide RNA to cleave target DNA sequences in the genome of infecting phages, and the tracrRNA to bind the promoter of cas genes and repress their transcription. We previously isolated a Cas9 mutant carrying the I473F substitution that increased the frequency of spacer acquisition by 2-3 orders of magnitude, leading to a fitness cost due to higher levels of autoimmunity. Here, we investigated the molecular basis underlying these findings. We found that the I473F mutation decreases the association of Cas9 to tracrRNA, limiting its repressor function, leading to high levels of expression of cas genes, which in turn increase the strength of the type II-A CRISPR-Cas immune response. We obtained similar results for a related type II-A system, and therefore our findings highlight the importance of the interaction between Cas9 and its tracrRNA cofactor in tuning the immune response to balanced levels that enable phage defense but avoid autoimmunity.