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Integrase-mediated spacer acquisition during CRISPR–Cas adaptive immunity

Author

Listed:
  • James K. Nuñez

    (University of California, Berkeley, Berkeley, California 94720, USA)

  • Amy S. Y. Lee

    (University of California, Berkeley, Berkeley, California 94720, USA
    Center for RNA Systems Biology, University of California, Berkeley, Berkeley, California 94720, USA)

  • Alan Engelman

    (Harvard Medical School)

  • Jennifer A. Doudna

    (University of California, Berkeley, Berkeley, California 94720, USA
    Center for RNA Systems Biology, University of California, Berkeley, Berkeley, California 94720, USA
    Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California 94720, USA
    University of California, Berkeley, Berkeley, California 94720, USA)

Abstract

Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1–Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1–Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3′-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1–Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1–Cas2-mediated adaptive immunity.

Suggested Citation

  • James K. Nuñez & Amy S. Y. Lee & Alan Engelman & Jennifer A. Doudna, 2015. "Integrase-mediated spacer acquisition during CRISPR–Cas adaptive immunity," Nature, Nature, vol. 519(7542), pages 193-198, March.
    • Handle: RePEc:nat:nature:v:519:y:2015:i:7542:d:10.1038_nature14237
    DOI: 10.1038/nature14237
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    Cited by:

    1. Mingfang Bi & Wenjing Su & Jiafu Li & Xiaobing Mo, 2024. "Insights into the inhibition of protospacer integration via direct interaction between Cas2 and AcrVA5," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    2. Bin Ma & Caiyu Lu & Yiling Wang & Jingwen Yu & Kankan Zhao & Ran Xue & Hao Ren & Xiaofei Lv & Ronghui Pan & Jiabao Zhang & Yongguan Zhu & Jianming Xu, 2023. "A genomic catalogue of soil microbiomes boosts mining of biodiversity and genetic resources," Nature Communications, Nature, vol. 14(1), pages 1-14, December.

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