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bioRxiv · 10.1101/706143

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

Halpin-Healy, T.·Klompe, S.·Sternberg, S.·Fernandez, I.
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Abstract

Bacteria have evolved adaptive immune systems encoded by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated (Cas) genes to maintain genomic integrity in the face of relentless assault from pathogens and mobile genetic elements [1-3]. Type I CRISPR-Cas systems canonically target foreign DNA for degradation via the joint action of the ribonucleoprotein complex Cascade and the helicase-nuclease Cas3 [4,5] but nuclease-deficient Type I systems lacking Cas3 have been repurposed for RNA-guided transposition by bacterial Tn7-like transposons [6,7]. How CRISPR- and transposon-associated machineries collaborate during DNA targeting and insertion has remained elusive. Here we determined structures of a novel TniQ-Cascade complex encoded by the Vibrio cholerae Tn6677 transposon using single particle electron cryo-microscopy (cryo-EM), revealing the mechanistic basis of this functional coupling. The quality of the cryo-EM maps allowed for de novo modeling 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 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 (PAM) recognition and R-loop formation. The present work lays the foundation for a structural understanding of how DNA targeting by TniQ-Cascade leads to downstream recruitment of additional transposon-associated proteins, and will guide protein engineering efforts to leverage this system for programmable DNA insertions in genome engineering applications.

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Halpin-Healy, T., Klompe, S., Sternberg, S., Fernandez, I.. 2019-07-18. Structural basis of DNA targeting by a transposon-encoded CRISPR-Cas system. https://doi.org/10.1101/706143

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