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Detection of spacer precursors formed in vivo during primed CRISPR adaptation

Author

Listed:
  • Anna A. Shiriaeva

    (Center of Life Sciences, Skolkovo Institute of Science and Technology
    Peter the Great St. Petersburg Polytechnic University
    Rutgers University)

  • Ekaterina Savitskaya

    (Center of Life Sciences, Skolkovo Institute of Science and Technology
    Institute of Molecular Genetics, Russian Academy of Sciences)

  • Kirill A. Datsenko

    (Rutgers University)

  • Irina O. Vvedenskaya

    (Rutgers University)

  • Iana Fedorova

    (Center of Life Sciences, Skolkovo Institute of Science and Technology
    Peter the Great St. Petersburg Polytechnic University)

  • Natalia Morozova

    (Center of Life Sciences, Skolkovo Institute of Science and Technology
    Peter the Great St. Petersburg Polytechnic University)

  • Anastasia Metlitskaya

    (Institute of Molecular Genetics, Russian Academy of Sciences)

  • Anton Sabantsev

    (Peter the Great St. Petersburg Polytechnic University)

  • Bryce E. Nickels

    (Rutgers University)

  • Konstantin Severinov

    (Center of Life Sciences, Skolkovo Institute of Science and Technology
    Peter the Great St. Petersburg Polytechnic University
    Rutgers University
    Institute of Molecular Genetics, Russian Academy of Sciences)

  • Ekaterina Semenova

    (Rutgers University)

Abstract

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.

Suggested Citation

  • Anna A. Shiriaeva & Ekaterina Savitskaya & Kirill A. Datsenko & Irina O. Vvedenskaya & Iana Fedorova & Natalia Morozova & Anastasia Metlitskaya & Anton Sabantsev & Bryce E. Nickels & Konstantin Severi, 2019. "Detection of spacer precursors formed in vivo during primed CRISPR adaptation," Nature Communications, Nature, vol. 10(1), pages 1-9, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-12417-w
    DOI: 10.1038/s41467-019-12417-w
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    Cited by:

    1. Kazuto Yoshimi & Kohei Takeshita & Noriyuki Kodera & Satomi Shibumura & Yuko Yamauchi & Mine Omatsu & Kenichi Umeda & Yayoi Kunihiro & Masaki Yamamoto & Tomoji Mashimo, 2022. "Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3," Nature Communications, Nature, vol. 13(1), pages 1-14, December.

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