Conserved DNA motifs in the type II-A CRISPR leader region

Abstract

The Clustered Regularly Interspaced Short Palindromic Repeats associated (CRISPR-Cas) systems consist of RNA-protein complexes that provide bacteria and archaea with sequence-specific immunity against bacteriophages, plasmids, and other mobile genetic elements. Bacteria and archaea become immune to phage or plasmid infections by inserting short pieces of the intruder DNA (spacer) site-specifically into the leader-repeat junction in a process called adaptation. Previous studies have shown that parts of the leader region, especially the 3′ end of the leader, are indispensable for adaptation. However, a comprehensive analysis of leader ends remains absent. Here, we have analyzed the leader, repeat, and Cas proteins from 167 type II-A CRISPR loci. Our results indicate two distinct conserved DNA motifs at the 3′ leader end: ATTTGAG (noted previously in the CRISPR1 locus of Streptococcus thermophilus DGCC7710) and a newly defined CTRCGAG, associated with the CRISPR3 locus of S. thermophilus DGCC7710. A third group with a very short CG DNA conservation at the 3′ leader end is observed mostly in lactobacilli. Analysis of the repeats and Cas proteins revealed clustering of these CRISPR components that mirrors the leader motif clustering, in agreement with the coevolution of CRISPR-Cas components. Based on our analysis of the type II-A CRISPR loci, we implicate leader end sequences that could confer site-specificity for the adaptation-machinery in the different subsets of type II-A CRISPR loci.

CRISPR type II-A subgroups exhibit phylogenetically distinct mechanisms for prespacer insertion

“…The G2 loci show dependence on the full repeat sequence for both leader and spacer-side insertions, which are not interdependent. Thus, our study establishes that type II-A systems use different rules for prespacer insertion, based on the members characterized so far, and that these rules have coevolved with leaderrepeat and Cas1-Cas2 sequence conservations (25).…”

“…S14. A comparison of our results with in vitro results from the other studies show a mechanistic distinction of prespacer insertion, based on the type II-A groupings that we identified through the bioinformatic work (25). Specifically, the G1 results show a strong dependence on leader-repeat junction for leader-side insertion, followed by a full repeat requirement for spacer-side insertion with an order in prespacer insertion.…”

“…Prespacer DNA integration by the Cas1-Cas2 protein IC into DNA targets that mimic the CRISPR array. A, sequence logos showing conservation in the last 7 bp of the leader and first 5 bp of the repeat from all three previously identified groups of type II-A CRISPR systems (25). B, schematic showing integration of FAM-labeled prespacer into a 116-bp linear target.…”

“…Recently, we presented bioinformatic evidence showing that the 39 end of type II-A leader sequences segregates into three distinct, conserved groups (25). These three groups are defined by the last 7-base pairs (bp) of the leader sequence: group 1 (G1) with ATTTGAG, group 2 (G2) with CTRCGAG, and group 3 (G3) with NNNNNCG, where R is a purine nucleotide (nt) (A or G), and N is any nt (Fig.…”

CRISPR-Cas systems in multicellular cyanobacteria

“…Organisms possessing CRISPR-Cas systems become immune to phage or other invading DNA by the insertion of DNA sequences (spacers) into the leader-repeat junction (i.e., at the 5ʹ end of the repeat-spacer array) in a site-specific process called adaptation. The leader region, especially its 3ʹ end, is indispensable for this adaptation [41][42][43][44][45]. Therefore, it must contain sequence determinants important for adaptation.…”