Primed acquisition and microhomology-mediated end-joining cooperate to confer specific CRISPR immunity against invasive genetic elements

Abstract CRISPR-Cas systems provide archaea and bacteria with adaptive immunity against invasive genetic elements through acquisition of invader-derived spacers. De novo spacer acquisition generally adapts spacers from both invaders and hosts with a bias towards invaders, while primed acquisition shows higher specificity to adapt spacers from invaders. Here, in Zymomonas mobilis subtype I-F system, primed acquisition showed much higher efficiency than de novo acquisition in this system. However, both routes recognised a large proportion of protospacers with the less conserved 5’-end CC PAM. Moreover, primed acquisition showed a preference towards protospacers located at the opposite strand and 3’ direction of the priming protospacer sites, differing from the canonical subtype I-F system. Further, self-spacers were adapted at a higher frequency during de novo spacer acquisition, probably leading to self-interference. Importantly, this species employed microhomology-mediated end-joining (MMEJ) for repair of host DNA breaks guided by self-targeted spacers, and overexpression of a host NAD + -dependent Ligase-A significantly increased the repair efficiency. In summary, our findings demonstrate that Z. mobilis uses primed acquisition for higher specific uptake of invader DNA and employs MMEJ to repair host DNA breaks guided by self-targeted spacers, showing specific immunity against invasive genetic elements.