Protospacer-adjacent motif specificity during clostridioides difficile type i-b crispr-cas interference and adaptation

Anna Maikova, Pierre Boudry, Anna Shiriaeva, Aleksandra Vasileva, Anaïs Boutserin, Sofia Medvedeva, Ekaterina Semenova, Konstantin Severinov, Olga Soutourina

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1 Citation (Scopus)


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 entero-pathogen, 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 con-sensus. 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.

Original languageEnglish
Article numbere02136-21
Issue number4
Publication statusPublished - Aug 2021


  • Clostridium difficile
  • CRISPR-Cas adaptation
  • CRISPR-Cas interference
  • Enteropathogen
  • PAM
  • Type I-B CRISPR-Cas


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