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Nature. 2020 Jan;577(7789):271-274. doi: 10.1038/s41586-019-1849-0. Epub 2019 Dec 18.

Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system.

Author information

1
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.
2
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA. shsternberg@gmail.com.
3
Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA. isf2106@cumc.columbia.edu.

Abstract

Bacteria use adaptive immune systems encoded by CRISPR and Cas genes to maintain genomic integrity when challenged by pathogens and mobile genetic elements1-3. Type I CRISPR-Cas systems typically target foreign DNA for degradation via joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas34,5, but nuclease-deficient type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons6,7. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion remains unknown. Here we describe structures of a TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using cryo-electron microscopy, revealing the mechanistic basis of this functional coupling. The cryo-electron microscopy maps enabled de novo modelling and refinement of the transposition protein TniQ, which binds to the Cascade complex as a dimer in a head-to-tail configuration, at the interface formed by Cas6 and Cas7 near the 3' end of the CRISPR RNA (crRNA). The natural Cas8-Cas5 fusion protein binds the 5' crRNA handle and contacts the TniQ dimer via a flexible insertion domain. A target DNA-bound structure reveals critical interactions necessary for protospacer-adjacent motif recognition and R-loop formation. This work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposase proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome-engineering applications.

PMID:
31853065
DOI:
10.1038/s41586-019-1849-0

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