Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.09.243105v1?rss=1 Authors: Bourgeois, J., Lazinski, D. W., Camilli, A. Abstract: The prokaryotic adaptive immune system CRISPR/Cas serves as defense against bacteriophage and invasive nucleic acid. A Type I-E CRISPR/Cas system has been detected in classical biotype isolates of Vibrio cholerae, the causative agent of the disease cholera. Experimental characterization of this system revealed a functional immune system that operates using a 5'-TT-3' protospacer-adjacent motif (PAM) for interference. However, several designed spacers against the 5'-TT-3' PAM do not interfere as expected, indicating further investigation of this system is necessary. In this study, we identified additional sequence requirements of a pyrimidine in the 5' position of the spacer and purine in the complementary position of the protospacer using 873 unique spacers and 2267 protospacers mined from CRISPR arrays in deposited sequences of V. cholerae. We present bioinformatic evidence that during acquisition the protospacer purine is captured in the prespacer and that a 5'-RTT-3' PAM is necessary for spacer acquisition. Finally, we demonstrate experimentally that a 5'-RTT-3' PAM is necessary for CRISPR interference by designing and manipulating spacer and cognate PAMs in a plasmid conjugation assay and discover functional consequences of base pairing with the 5' spacer pyrimidine in spacer efficacy. Copy rights belong to original authors. Visit the link for more info