Abstract
CRISPR-Cas is an adaptive immune system that protects prokaryotes against foreign nucleic acids. Prokaryotes gain immunity by acquiring short pieces of the invading nucleic acid termed prespacers and inserting them into their CRISPR array. In type II-A systems, Cas1 and Cas2 proteins insert prespacers always at the leader-repeat junction of the CRISPR array. Among type II-A CRISPR systems, three distinct groups (G1, G2, and G3) exist according to the extent of DNA sequence conservation at the 3' end of the leader. However, the mechanisms by which these conserved motifs interact with their cognate Cas1 and Cas2 proteins remain unclear. Here, we performed in vitro integration assays, finding that for G1 and G2, the insertion site is recognized through defined mechanisms, at least in members examined to date, whereas G3 exhibits no sequence-specific insertion. G1 first recognized a 12-bp sequence at the leader-repeat junction and performed leader-side insertion before proceeding to spacer-side insertion. G2 recognized the full repeat sequence and could perform independent leader-side or spacer-side insertions, although the leader-side insertion was faster than spacer-side. The prespacer morphology requirements for Cas1-Cas2 varied, with G1 stringently requiring a 5-nucleotide 3' overhang and G2 being able to insert many forms of prespacers with variable efficiencies. These results highlight the intricacy of protein-DNA sequence interactions within the seemingly similar type II-A integration complexes and provide mechanistic insights into prespacer insertion. These interactions can be fine-tuned to expand the Cas1-Cas2 toolset for inserting small DNAs into diverse DNA targets.
Highlights
CRISPR and CRISPR-associated (Cas) proteins comprise a unique adaptive immune system utilized by bacteria and archaea to defend against invading nucleic acids [1]
We show that Cas1–Cas2 complexes preferentially integrate prespacers at their native leader 39 ends compared with mutated leader 39 ends or leaders from a different type II-A phylogenetic group
Divalent metal cations were tested for each Cas1– Cas2 complex, with the metal cation allowing the highest and specific activity to be selected for the rest of the study (G1, Mn21; group 2 (G2), Mg21; group 3 (G3), Mn21; Fig. S1)
Summary
CRISPR-Cas is an adaptive immune system that protects prokaryotes against foreign nucleic acids. The prespacer morphology requirements for Cas1–Cas varied, with G1 stringently requiring a 5nucleotide 39 overhang and G2 being able to insert many forms of prespacers with variable efficiencies These results highlight the intricacy of protein–DNA sequence interactions within the seemingly similar type II-A integration complexes and provide mechanistic insights into prespacer insertion. Even though previous studies have shown integration activities of different Cas1–Cas complexes into their cognate backbones, a direct comparison of Cas1–Cas complexes from all three different type II-A groups and their abilities to integrate into noncognate target DNAs is missing This information is essential to derive a complete picture of how adaptation mechanisms vary between different groups of a single CRISPR subtype. Our results unify the type II-A adaptation process into phylogenetically distinct mechanisms, in which the phylogeny was derived based on protein–DNA sequence conservations related to the CRISPR adaptation module
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