Primed adaptation tolerates extensive structural and size variations of the CRISPR RNA guide in Haloarcula hispanica

Abstract

Abstract Recent studies on CRISPR adaptation revealed that priming is a major pathway of spacer acquisition, at least for the most prevalent type I systems. Priming is guided by a CRISPR RNA which fully/partially matches the invader DNA, but the plasticity of this RNA guide has not yet been characterized. In this study, we extensively modified the two conserved handles of a priming crRNA in Haloarcula hispanica, and altered the size of its central spacer part. Interestingly, priming is insusceptible to the full deletion of 3′ handle, which seriously impaired crRNA stability and interference effects. With 3′ handle deletion, further truncation of 5′ handle revealed that its spacer-proximal 6 nucleotides could provide the least conserved sequence required for priming. Subsequent scanning mutation further identified critical nucleotides within 5′ handle. Besides, priming was also shown to tolerate a wider size variation of the spacer part, compared to interference. These data collectively illustrate the high tolerance of priming to extensive structural/size variations of the crRNA guide, which highlights the structural flexibility of the crRNA-effector ribonucleoprotein complex. The observed high priming effectiveness suggests that primed adaptation promotes clearance of the fast-replicating and ever-evolving viral DNA, by rapidly and persistently multiplexing the interference pathway.