Protospacer-Adjacent Motif Specificity during <named-content content-type="genus-species">Clostridioides difficile</named-content> Type I-B CRISPR-Cas Interference and Adaptation

ABSTRACT CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide prokaryotes with efficient protection against foreign nucleic acid invaders. We have recently demonstrated the defensive interference function of a CRISPR-Cas system from Clostridioide...

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Autores principales:	Anna Maikova, Pierre Boudry, Anna Shiriaeva, Aleksandra Vasileva, Anaïs Boutserin, Sofia Medvedeva, Ekaterina Semenova, Konstantin Severinov, Olga Soutourina
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2021
Materias:	CRISPR-Cas interference CRISPR-Cas adaptation Clostridium difficile PAM type I-B CRISPR-Cas enteropathogen Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/a1a07cf6296041e9ad107cf56f8087f0
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spelling	oai:doaj.org-article:a1a07cf6296041e9ad107cf56f8087f02021-11-10T18:37:52ZProtospacer-Adjacent Motif Specificity during <named-content content-type="genus-species">Clostridioides difficile</named-content> Type I-B CRISPR-Cas Interference and Adaptation10.1128/mBio.02136-212150-7511https://doaj.org/article/a1a07cf6296041e9ad107cf56f8087f02021-08-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.02136-21https://doaj.org/toc/2150-7511ABSTRACT CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide prokaryotes with efficient protection against foreign nucleic acid invaders. We have recently demonstrated the defensive interference function of a CRISPR-Cas system from Clostridioides (Clostridium) difficile, a major human enteropathogen, and showed that it could be harnessed for efficient genome editing in this bacterium. However, molecular details are still missing on CRISPR-Cas function for adaptation and sequence requirements for both interference and new spacer acquisition in this pathogen. Despite accumulating knowledge on the individual CRISPR-Cas systems in various prokaryotes, no data are available on the adaptation process in bacterial type I-B CRISPR-Cas systems. Here, we report the first experimental evidence that the C. difficile type I-B CRISPR-Cas system acquires new spacers upon overexpression of its adaptation module. The majority of new spacers are derived from a plasmid expressing Cas proteins required for adaptation or from regions of the C. difficile genome where generation of free DNA termini is expected. Results from protospacer-adjacent motif (PAM) library experiments and plasmid conjugation efficiency assays indicate that C. difficile CRISPR-Cas requires the YCN consensus PAM for efficient interference. We revealed a functional link between the adaptation and interference machineries, since newly adapted spacers are derived from sequences associated with a CCN PAM, which fits the interference consensus. The definition of functional PAMs and establishment of relative activity levels of each of the multiple C. difficile CRISPR arrays in present study are necessary for further CRISPR-based biotechnological and medical applications involving this organism. IMPORTANCE CRISPR-Cas systems provide prokaryotes with adaptive immunity for defense against foreign nucleic acid invaders, such as viruses or phages and plasmids. The CRISPR-Cas systems are highly diverse, and detailed studies of individual CRISPR-Cas subtypes are important for our understanding of various aspects of microbial adaptation strategies and for the potential applications. The significance of our work is in providing the first experimental evidence for type I-B CRISPR-Cas system adaptation in the emerging human enteropathogen Clostridioides difficile. This bacterium needs to survive in phage-rich gut communities, and its active CRISPR-Cas system might provide efficient antiphage defense by acquiring new spacers that constitute memory for further invader elimination. Our study also reveals a functional link between the adaptation and interference CRISPR machineries. The definition of all possible functional trinucleotide motifs upstream protospacers within foreign nucleic acid sequences is important for CRISPR-based genome editing in this pathogen and for developing new drugs against C. difficile infections.Anna MaikovaPierre BoudryAnna ShiriaevaAleksandra VasilevaAnaïs BoutserinSofia MedvedevaEkaterina SemenovaKonstantin SeverinovOlga SoutourinaAmerican Society for MicrobiologyarticleCRISPR-Cas interferenceCRISPR-Cas adaptationClostridium difficilePAMtype I-B CRISPR-CasenteropathogenMicrobiologyQR1-502ENmBio, Vol 12, Iss 4 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR-Cas interference CRISPR-Cas adaptation Clostridium difficile PAM type I-B CRISPR-Cas enteropathogen Microbiology QR1-502
spellingShingle	CRISPR-Cas interference CRISPR-Cas adaptation Clostridium difficile PAM type I-B CRISPR-Cas enteropathogen Microbiology QR1-502 Anna Maikova Pierre Boudry Anna Shiriaeva Aleksandra Vasileva Anaïs Boutserin Sofia Medvedeva Ekaterina Semenova Konstantin Severinov Olga Soutourina Protospacer-Adjacent Motif Specificity during <named-content content-type="genus-species">Clostridioides difficile</named-content> Type I-B CRISPR-Cas Interference and Adaptation
description	ABSTRACT CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide prokaryotes with efficient protection against foreign nucleic acid invaders. We have recently demonstrated the defensive interference function of a CRISPR-Cas system from Clostridioides (Clostridium) difficile, a major human enteropathogen, and showed that it could be harnessed for efficient genome editing in this bacterium. However, molecular details are still missing on CRISPR-Cas function for adaptation and sequence requirements for both interference and new spacer acquisition in this pathogen. Despite accumulating knowledge on the individual CRISPR-Cas systems in various prokaryotes, no data are available on the adaptation process in bacterial type I-B CRISPR-Cas systems. Here, we report the first experimental evidence that the C. difficile type I-B CRISPR-Cas system acquires new spacers upon overexpression of its adaptation module. The majority of new spacers are derived from a plasmid expressing Cas proteins required for adaptation or from regions of the C. difficile genome where generation of free DNA termini is expected. Results from protospacer-adjacent motif (PAM) library experiments and plasmid conjugation efficiency assays indicate that C. difficile CRISPR-Cas requires the YCN consensus PAM for efficient interference. We revealed a functional link between the adaptation and interference machineries, since newly adapted spacers are derived from sequences associated with a CCN PAM, which fits the interference consensus. The definition of functional PAMs and establishment of relative activity levels of each of the multiple C. difficile CRISPR arrays in present study are necessary for further CRISPR-based biotechnological and medical applications involving this organism. IMPORTANCE CRISPR-Cas systems provide prokaryotes with adaptive immunity for defense against foreign nucleic acid invaders, such as viruses or phages and plasmids. The CRISPR-Cas systems are highly diverse, and detailed studies of individual CRISPR-Cas subtypes are important for our understanding of various aspects of microbial adaptation strategies and for the potential applications. The significance of our work is in providing the first experimental evidence for type I-B CRISPR-Cas system adaptation in the emerging human enteropathogen Clostridioides difficile. This bacterium needs to survive in phage-rich gut communities, and its active CRISPR-Cas system might provide efficient antiphage defense by acquiring new spacers that constitute memory for further invader elimination. Our study also reveals a functional link between the adaptation and interference CRISPR machineries. The definition of all possible functional trinucleotide motifs upstream protospacers within foreign nucleic acid sequences is important for CRISPR-based genome editing in this pathogen and for developing new drugs against C. difficile infections.
format	article
author	Anna Maikova Pierre Boudry Anna Shiriaeva Aleksandra Vasileva Anaïs Boutserin Sofia Medvedeva Ekaterina Semenova Konstantin Severinov Olga Soutourina
author_facet	Anna Maikova Pierre Boudry Anna Shiriaeva Aleksandra Vasileva Anaïs Boutserin Sofia Medvedeva Ekaterina Semenova Konstantin Severinov Olga Soutourina
author_sort	Anna Maikova
title	Protospacer-Adjacent Motif Specificity during <named-content content-type="genus-species">Clostridioides difficile</named-content> Type I-B CRISPR-Cas Interference and Adaptation
title_short	Protospacer-Adjacent Motif Specificity during <named-content content-type="genus-species">Clostridioides difficile</named-content> Type I-B CRISPR-Cas Interference and Adaptation
title_full	Protospacer-Adjacent Motif Specificity during <named-content content-type="genus-species">Clostridioides difficile</named-content> Type I-B CRISPR-Cas Interference and Adaptation
title_fullStr	Protospacer-Adjacent Motif Specificity during <named-content content-type="genus-species">Clostridioides difficile</named-content> Type I-B CRISPR-Cas Interference and Adaptation
title_full_unstemmed	Protospacer-Adjacent Motif Specificity during <named-content content-type="genus-species">Clostridioides difficile</named-content> Type I-B CRISPR-Cas Interference and Adaptation
title_sort	protospacer-adjacent motif specificity during <named-content content-type="genus-species">clostridioides difficile</named-content> type i-b crispr-cas interference and adaptation
publisher	American Society for Microbiology
publishDate	2021
url	https://doaj.org/article/a1a07cf6296041e9ad107cf56f8087f0
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Protospacer-Adjacent Motif Specificity during <named-content content-type="genus-species">Clostridioides difficile</named-content> Type I-B CRISPR-Cas Interference and Adaptation

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