Abstract 1270 Visualizing Alternative Conformations of CRISPR Type I-E Cascade Upon DNA Binding using Cryo-EM

CRISPR-Cas systems are immune systems in bacteria and archaea that provide adaptive defense against pathogens. CRISPR-mediated immunity is based on the RNA-guided degradation activity of a Cas effector against a foreign nucleic acid. In type I CRISPR systems, the 450 kDa multiprotein effector complex Cascade is formed by a copy of the guiding CRISPR RNA (crRNA), along with five proteins in unequal stoichiometry: Cas51, Cas61, Cas76, Cas112 and Cas81. Cascade identifies the complementary DNA target through formation of an R-loop in which the crRNA hybridizes to the target strand of the DNA and the non-target strand is displaced. Upon R-loop formation, the helicase-nuclease Cas3 is recruited by the Cas8 subunit of Cascade, allowing processive DNA degradation of the non-target strand in the 3' to 5' direction. In vivo evidence indicates that the target strand is also degraded in the 3' to 5' direction by Cas3, suggesting that a second copy of Cas3 is recruited at the opposite end of Cascade. However, there is currently no biochemical or structural evidence of bidirectional Cas3 recruitment. To address how a second copy of Cas3 may be recruited at the opposite end of Cascade, we solved the cryo-EM structure of Cascade targeting double-stranded DNA (dsDNA). Interestingly, we observed that Cas8e adopts three different conformations within our cryo-EM dataset. While the X-ray crystal structure of Cascade bound to dsDNA contained Cas8 in a "closed and locked" conformation that is required for Cas3 recruitment, our cryo-EM structures reveal that a significant number of particles contain alternative Cas8 conformations. We observed that ∼45% of particles have Cas8 in a "closed and unlocked" conformation that is unlikely to support Cas3 recruitment at the Cas8 end of Cascade. The closed and unlocked conformation of Cas8, along with full R-loop formation, results in destabilization of the Cas6 subunit, located on the opposite end of Cascade from Cas8. Our results reveal that the overall conformation of Cascade is highly dynamic, suggesting that alternate conformations may have a role for recruitment of a second Cas3 subunit to the Cascade complex.