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

ABSTRACT 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...

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Autores principales:	Olga Musharova, Danylo Vyhovskyi, Sofia Medvedeva, Jelena Guzina, Yulia Zhitnyuk, Marko Djordjevic, Konstantin Severinov, Ekaterina Savitskaya
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2018
Materias:	CRISPR spacers CRISPR-Cas naïve adaptation primed adaptation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/7aa6c4b730ee47aeb0dc8159091991c9
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spelling	oai:doaj.org-article:7aa6c4b730ee47aeb0dc8159091991c92021-11-15T15:52:18ZAvoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation10.1128/mBio.02169-182150-7511https://doaj.org/article/7aa6c4b730ee47aeb0dc8159091991c92018-12-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02169-18https://doaj.org/toc/2150-7511ABSTRACT 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.Olga MusharovaDanylo VyhovskyiSofia MedvedevaJelena GuzinaYulia ZhitnyukMarko DjordjevicKonstantin SeverinovEkaterina SavitskayaAmerican Society for MicrobiologyarticleCRISPR spacersCRISPR-Casnaïve adaptationprimed adaptationMicrobiologyQR1-502ENmBio, Vol 9, Iss 6 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR spacers CRISPR-Cas naïve adaptation primed adaptation Microbiology QR1-502
spellingShingle	CRISPR spacers CRISPR-Cas naïve adaptation primed adaptation Microbiology QR1-502 Olga Musharova Danylo Vyhovskyi Sofia Medvedeva Jelena Guzina Yulia Zhitnyuk Marko Djordjevic Konstantin Severinov Ekaterina Savitskaya Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation
description	ABSTRACT 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.
format	article
author	Olga Musharova Danylo Vyhovskyi Sofia Medvedeva Jelena Guzina Yulia Zhitnyuk Marko Djordjevic Konstantin Severinov Ekaterina Savitskaya
author_facet	Olga Musharova Danylo Vyhovskyi Sofia Medvedeva Jelena Guzina Yulia Zhitnyuk Marko Djordjevic Konstantin Severinov Ekaterina Savitskaya
author_sort	Olga Musharova
title	Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation
title_short	Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation
title_full	Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation
title_fullStr	Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation
title_full_unstemmed	Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation
title_sort	avoidance of trinucleotide corresponding to consensus protospacer adjacent motif controls the efficiency of prespacer selection during primed adaptation
publisher	American Society for Microbiology
publishDate	2018
url	https://doaj.org/article/7aa6c4b730ee47aeb0dc8159091991c9
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Avoidance of Trinucleotide Corresponding to Consensus Protospacer Adjacent Motif Controls the Efficiency of Prespacer Selection during Primed Adaptation

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