Processing and integration of functionally oriented prespacers in the Escherichia coli CRISPR system depends on bacterial host exonucleases

Abstract

CRISPR-Cas systems provide bacteria with adaptive immunity against viruses. During spacer adaptation, the Cas1-Cas2 complex selects fragments of foreign DNA, called prespacers, and integrates them into CRISPR arrays in an orientation that provides functional immunity. Cas4 is involved in both the trimming of prespacers and the cleavage of protospacer adjacent motif (PAM) in several type I CRISPR-Cas systems, but how the prespacers are processed in systems lacking Cas4, such as the type I-E and I-F systems, is not understood. In Escherichia coli, which has a type I-E system, Cas1-Cas2 preferentially selects prespacers with 3′ overhangs via specific recognition of a PAM, but how these prespacers are integrated in a functional orientation in the absence of Cas4 is not known. Using a biochemical approach with purified proteins, as well as integration, prespacer protection, sequencing, and quantitative PCR assays, we show here that the bacterial 3′–5′ exonucleases DnaQ and ExoT can trim long 3′ overhangs of prespacers and promote integration in the correct orientation. We found that trimming by these exonucleases results in an asymmetric intermediate, because Cas1-Cas2 protects the PAM sequence, which helps to define spacer orientation. Our findings implicate the E. coli host 3′–5′ exonucleases DnaQ and ExoT in spacer adaptation and reveal a mechanism by which spacer orientation is defined in E. coli.

Adaptation induced by self-targeting in a type I-B CRISPR-Cas system

“…Acquisition of spacers upstream of the targeting site might be because of Cas3 flipping onto the other DNA strand and proceeding to unwind and cleave it in a 3′–5′ direction, as has been suggested for H. hispanica ( 13 ). However, it is also possible that DNases from other DNA repair or recombination pathways are involved in cleaving the DNA, because more and more reports show the involvement of host enzymes in CRISPR-Cas reactions ( 48 , 49 ); this remains to be elucidated.…”

Characterization of Primed Adaptation in the Escherichia coli type I-E CRISPR-Cas System

“…A recent study examined the nature of DNA fragments bound by Cas1-Cas2 in E. coli undergoing primed adaptation. Surprisingly, the DNA fragments did not have the ends expected based on biochemical studies of adaptation (Kim et al, 2020;Ramachandran et al, 2020), but rather had a blunt PAM-distal end and a 4 nt 3ʹ overhang at the PAM-proximal end, with the 5ʹ nucleotide at the PAM-proximal end being the last nucleotide of the PAM. These data suggest that the processing of fragments bound by Cas1-Cas2 may be different during naïve and primed adaptation.…”

“…Our data indicate that together, +1 slips and -1 slips represent the majority (60%) of primed spacer acquisition on the non-target strand for pre-spacers not associated with an AAG PAM. The mechanism of slipping is not known, but may be due to imprecise processing of Cas1-Cas2-bound pre-spacer DNA by exonucleases (Kim et al, 2020;Ramachandran et al, 2020). The remaining pre-spacers that lack an AAG PAM and are not associated with slipping events typically have suboptimal PAMs, with the third base of the PAM strongly enriched for G, and the second base moderately enriched for A or C ( Figure 7C).…”

“…Recent studies suggest that in Escherichia coli, Cas1 binding to the PAM protects the PAM-proximal end of the bound DNA from exonucleolytic digestion, whereas the PAM-distal end is not protected. The resulting difference in 3ʹ overhang length dictates the order of nucleophilic attack, and thus ensures that the PAM-proximal side is integrated closest to the leader (Drabavicius et al, 2018;Kim et al, 2020;Ramachandran et al, 2020).…”

CRISPR-Associated Primase-Polymerases are implicated in prokaryotic CRISPR-Cas adaptation

“…Some non-Cas exonucleases e.g. DnaQ, EcoT were shown to play a role in in vitro processing of prespacers to make them suitable for integration into a CRISPR array 18 , 19 . During primed adaptation, new spacer acquisition is dependent on already present spacers originating from the same invading DNA, with the target site partially mutated to escape bacterial immunity 20 , 21 .…”