Molecular Details of DNA Integration by CRISPR-Associated Proteins During Adaptation in Bacteria and Archaea

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in bacteria and archaea, where immunological memory is retained in the CRISPR locus as short pieces of the intruding nucleic acid, termed spacers. The adaptation to new infections occurs through the integration of a new spacer into the CRISPR array. For immune protection, spacers are transcribed into CRISPR RNAs (crRNA) that are used to guide the effector nuclease of the system in sequence-dependent target cleavage. Spacers originate as a prespacer from either DNA or RNA depending on the CRISPR-Cas system being observed, and the nearly universal Cas proteins, Cas1 and Cas2, insert the prespacer into the CRISPR locus during adaptation in all systems that contain them. The mechanism of site-specific prespacer integration varies across CRISPR classes and types, and distinct differences can even be found within the same subtype. In this review, the current knowledge on the mechanisms of prespacer integration in type II-A CRISPR-Cas systems will be described. Comparisons of the currently characterized type II-A systems show that distinct mechanisms exist within different members of this subtype and are correlated to sequence-specific interactions of Cas proteins and the DNA elements present in the CRISPR array. These observations indicate that nature has fine-tuned the mechanistic details while performing the basic step of DNA integration by Cas proteins, which offers unique advantages to develop Cas1-Cas2-based biotechnology.