Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation

Olga Musharova,Konstantin Severinov,Sofia Medvedeva,Danylo Vyhovskyi,Marko Djordjevic,Yulia Zhitnyuk,Jelena Guzina,Ekaterina Savitskaya

doi:10.1128/mbio.02169-18

Olga Musharova, Konstantin Severinov + Show 6 more

Open Access

PDF Available

https://doi.org/10.1128/mbio.02169-18

Copy DOI

Export

Save

Cite

Abstract
Highlights/Summary
Full-Text PDF
Similar Papers

Abstract

Listen

CRISPR DNA arrays of unique spacers separated by identical repeats ensure prokaryotic immunity through specific targeting of foreign nucleic acids complementary to spacers. New spacers are acquired into a CRISPR array in a process of CRISPR adaptation. Selection of foreign DNA fragments to be integrated into CRISPR arrays relies on PAM (protospacer adjacent motif) recognition, as only those spacers will be functional against invaders. However, acquisition of different PAM-associated spacers proceeds with markedly different efficiency from the same DNA. Here, we used a combination of bioinformatics and experimental approaches to understand factors affecting the efficiency of acquisition of spacers by the Escherichia coli type I-E CRISPR-Cas system, for which two modes of CRISPR adaptation have been described: naive and primed. We found that during primed adaptation, efficiency of spacer acquisition is strongly negatively affected by the presence of an AAG trinucleotide-a consensus PAM-within the sequence being selected. No such trend is observed during naive adaptation. The results are consistent with a unidirectional spacer selection process during primed adaptation and provide a specific signature for identification of spacers acquired through primed adaptation in natural populations.IMPORTANCE Adaptive immunity of prokaryotes depends on acquisition of foreign DNA fragments into CRISPR arrays as spacers followed by destruction of foreign DNA by CRISPR interference machinery. Different fragments are acquired into CRISPR arrays with widely different efficiencies, but the factors responsible are not known. We analyzed the frequency of spacers acquired during primed adaptation in an E. coli CRISPR array and found that AAG motif was depleted from highly acquired spacers. AAG is also a consensus protospacer adjacent motif (PAM) that must be present upstream from the target of the CRISPR spacer for its efficient destruction by the interference machinery. These results are important because they provide new information on the mechanism of primed spacer acquisition. They add to other previous evidence in the field that pointed out to a "directionality" in the capture of new spacers. Our data strongly suggest that the recognition of an AAG PAM by the interference machinery components prior to spacer capture occludes downstream AAG sequences, thus preventing their recognition by the adaptation machinery.

Highlights

IMPORTANCE Adaptive immunity of prokaryotes depends on acquisition of foreign DNA fragments into CRISPR arrays as spacers followed by destruction of foreign DNA by CRISPR interference machinery
As expected for primed adaptation, most spacers in each culture were acquired from plasmid (99.7%) rather than the bacterial genome, and 86.35% of plasmid-derived spacers mapped to the DNA strand that was not targeted by G8 CRISPR RNAs (crRNAs) (Fig. 1A; Table S1)
While it has been observed that regions proximal to a priming protospacer preferentially donate new spacers during primed adaptation [18, 25,26,27], there was no gradient in prespacer usage with any of the plasmids (Fig. 1A), likely due to their small size

Summary

Introduction

IMPORTANCE Adaptive immunity of prokaryotes depends on acquisition of foreign DNA fragments into CRISPR arrays as spacers followed by destruction of foreign DNA by CRISPR interference machinery. AAG is a consensus protospacer adjacent motif (PAM) that must be present upstream from the target of the CRISPR spacer for its efficient destruction by the interference machinery These results are important because they provide new information on the mechanism of primed spacer acquisition. The separation of CRISPR defense into spacer acquisition and target interference stages and the requirement for PAM means that new spacers need to arise from sequences (prespacers) associated with PAM Otherwise, they will not be able to perform their protective function. The efficiency of prespacer acquisition decreases with increasing distance from the priming site [18] No such biases are apparent during naive adaptation, and acquired spacers map to both strands of foreign DNA. It was shown that naive adaptation is affected by RecBCD activity, and acquired spacers tend to originate from regions with double-stranded breaks or replication fork stalling [19, 20]

Methods

Results

Conclusion