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How type II CRISPR–Cas establish immunity through Cas1–Cas2-mediated spacer integration

Nature volume 550, pages 137141 (05 October 2017) | Download Citation


CRISPR (clustered regularly interspaced short palindromic repeats) and the nearby Cas (CRISPR-associated) operon establish an RNA-based adaptive immunity system in prokaryotes1,2,3,4,5. Molecular memory is created when a short foreign DNA-derived prespacer is integrated into the CRISPR array as a new spacer6,7,8,9. Whereas the RNA-guided CRISPR interference mechanism varies widely among CRISPR–Cas systems, the spacer integration mechanism is essentially identical7,8,9. The conserved Cas1 and Cas2 proteins form an integrase complex consisting of two distal Cas1 dimers bridged by a Cas2 dimer6,10. The prespacer is bound by Cas1–Cas2 as a dual-forked DNA, and the terminal 3′-OH of each 3′ overhang serves as an attacking nucleophile during integration11,12,13,14. The prespacer is preferentially integrated into the leader-proximal region of the CRISPR array1,7,10,15, guided by the leader sequence and a pair of inverted repeats inside the CRISPR repeat7,15,16,17,18,19,20. Spacer integration in the well-studied Escherichia coli type I–E CRISPR system also relies on the bacterial integration host factor21,22. In type II–A CRISPR, however, Cas1–Cas2 alone integrates spacers efficiently in vitro18; other Cas proteins (such as Cas9 and Csn2) have accessory roles in the biogenesis phase of prespacers17,23. Here we present four structural snapshots from the type II–A system24 of Enterococcus faecalis Cas1 and Cas2 during spacer integration. Enterococcus faecalis Cas1–Cas2 selectively binds to a splayed 30-base-pair prespacer bearing 4-nucleotide 3′ overhangs. Three molecular events take place upon encountering a target: first, the Cas1–Cas2–prespacer complex searches for half-sites stochastically, then it preferentially interacts with the leader-side CRISPR repeat, and finally, it catalyses a nucleophilic attack that connects one strand of the leader-proximal repeat to the prespacer 3′ overhang. Recognition of the spacer half-site requires DNA bending and leads to full integration. We derive a mechanistic framework to explain the stepwise spacer integration process and the leader-proximal preference.

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This work is supported by NIH/NIGMS awards GM118174 and GM102543 to A.K. We thank D. Neau for assistance with data collection, and K. Rajashankar and I. Kriksunov for beamtime allocation. We thank P. Nguyen, R. Battaglia and A. Dolan for technical assistance and discussions. This work is based upon research conducted at NECAT, supported by NIH/NIGMS awards P41-GM103403 and S10-RR029205; and at CHESS and MACCHESS, supported by NSF award DMR-1332208 and NIH/NIGMS award GM-103485. This research used resources of the Advanced Photon Source, a US Department of Energy facility under contract no. DE-AC02-06CH11357.

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Author notes

    • Sherwin Ng
    •  & Ki Hyun Nam

    These authors contributed equally to this work.


  1. Department of Molecular Biology and Genetics, Cornell University, 253 Biotechnology Building, Ithaca, New York 14853, USA

    • Yibei Xiao
    • , Sherwin Ng
    •  & Ailong Ke
  2. Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang, South Korea

    • Ki Hyun Nam


  1. Search for Yibei Xiao in:

  2. Search for Sherwin Ng in:

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  4. Search for Ailong Ke in:


Y.X. and A.K. designed the research. Y.X. and S.N. are the main contributors to the biochemical and crystallography experiments. Y.X., K.H.N. and A.K. collected diffraction data, determined the structure, and performed structure–function analyses. A.K. and the rest of the authors wrote the manuscript.

Competing interests

Cornell University is in the process of applying for a patent application covering the utilization of the Cas1–Cas2-mediated DNA integration mechanism, as revealed in this study, for potential genome manipulation applications that lists A.K. and Y.X. as inventors.

Corresponding author

Correspondence to Ailong Ke.

Reviewer Information Nature thanks F. Dyda, M. White and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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