Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection

• Published in: Cell reports (Cambridge) 2015-03, Vol.10 (9), p.1534-1543
• Main Authors: Rutkauskas, Marius, Sinkunas, Tomas, Songailiene, Inga, Tikhomirova, Maria S., Siksnys, Virginijus, Seidel, Ralf
• Format: Article
• Language: English
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2015.01.067

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign nucleic acids. In type I CRISPR-Cas systems, invading DNA is detected by a large ribonucleoprotein surveillance complex called Cascade. The crRNA component of Cascade is used to recognize target sites in foreign DNA (protospacers) by formation of an R-loop driven by base-pairing complementarity. Using single-molecule supercoiling experiments with near base-pair resolution, we probe here the mechanism of R-loop formation and detect short-lived R-loop intermediates on off-target sites bearing single mismatches. We show that R-loops propagate directionally starting from the protospacer-adjacent motif (PAM). Upon reaching a mismatch, R-loop propagation stalls and collapses in a length-dependent manner. This unambiguously demonstrates that directional zipping of the R-loop accomplishes efficient target recognition by rapidly rejecting binding to off-target sites with PAM-proximal mutations. R-loops that reach the protospacer end become locked to license DNA degradation by the auxiliary Cas3 nuclease/helicase without further target verification. [Display omitted] •R-loop progression is monitored with close to base-pair resolution•Target recognition occurs exclusively through a directional R-loop-zipping process•Intermediate R-loops stall at mutations and collapse in a length-dependent manner•R-loop locking at protospacer end licenses DNA cleavage without further proofreading The CRISPR-Cas surveillance complex Cascade targets invading DNA for destruction with the help of a short crRNA. Rutkauskas et al. use real-time supercoiling experiments on single DNA molecules to directly reveal the impact of mismatches during the directional zipping of the crRNA on a DNA target.