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

Autor: Halpin-Healy, Tyler S., Klompe, Sanne E., Sternberg, Samuel H., Fernández, Israel S.
Zdroj: Nature; 1/9/2020, Vol. 577 Issue 7789, p271-274, 4p, 10 Diagrams, 3 Graphs
Abstrakt: 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. Cryo-electron microscopy structures of the TniQ–Cascade complex encoded by the Vibrio cholerae Tn6677 transposon reveal the mechanistic basis of the functional association of CRISPR- and transposon-associated machineries. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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