Detection of spacer precursors formed in vivo during primed CRISPR adaptation

Anna A. Shiriaeva, Ekaterina Savitskaya, Kirill A. Datsenko, Irina O. Vvedenskaya, Iana Fedorova, Natalia Morozova, Anastasia Metlitskaya, Anton Sabantsev, Bryce E. Nickels, Konstantin Severinov, Ekaterina Semenova

    Research output: Contribution to journalArticlepeer-review

    13 Citations (Scopus)


    Type I CRISPR-Cas loci provide prokaryotes with a nucleic-acid-based adaptive immunity against foreign DNA. Immunity involves adaptation, the integration of ~30-bp DNA fragments, termed prespacers, into the CRISPR array as spacers, and interference, the targeted degradation of DNA containing a protospacer. Interference-driven DNA degradation can be coupled with primed adaptation, in which spacers are acquired from DNA surrounding the targeted protospacer. Here we develop a method for strand-specific, high-throughput sequencing of DNA fragments, FragSeq, and apply this method to identify DNA fragments accumulated in Escherichia coli cells undergoing robust primed adaptation by a type I-E or type I-F CRISPR-Cas system. The detected fragments have sequences matching spacers acquired during primed adaptation and function as spacer precursors when introduced exogenously into cells by transformation. The identified prespacers contain a characteristic asymmetrical structure that we propose is a key determinant of integration into the CRISPR array in an orientation that confers immunity.

    Original languageEnglish
    Article number4603
    JournalNature Communications
    Issue number1
    Publication statusPublished - 1 Dec 2019


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