RT Journal Article
SR Electronic
T1 Distinct subcellular localization of a Type I CRISPR complex and the Cas3 nuclease in bacteria
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.09.29.318501
DO 10.1101/2020.09.29.318501
A1 Sutharsan Govindarajan
A1 Adair Borges
A1 Joseph Bondy-Denomy
YR 2020
UL http://biorxiv.org/content/early/2020/10/19/2020.09.29.318501.abstract
AB CRISPR-Cas systems are prokaryotic adaptive immune systems that have been well characterized biochemically, but in vivo spatiotemporal regulation and cell biology remains largely unaddressed. Here, we used fluorescent fusion proteins to study the localization of the Type I-F CRISPR-Cas system native to Pseudomonas aeruginosa. When targeted to an integrated prophage, the crRNA-guided (Csy) complex and a majority of Cas3 molecules in the cell are recruited to a single focus. When lacking a target in the cell, however, the Csy complex is broadly nucleoid bound, while Cas3 is diffuse in the cytoplasm. Nucleoid association for the Csy proteins is crRNA-dependent, and inhibited by expression of anti-CRISPR AcrIF2, which blocks PAM binding. The Cas9 nuclease is also nucleoid localized, only when gRNA-bound, which is abolished by PAM mimic, AcrIIA4. Our findings reveal PAM-dependent nucleoid surveillance and spatiotemporal regulation in Type I CRISPR-Cas that separates the nuclease-helicase Cas3 from the crRNA-guided surveillance complex.Competing Interest StatementJ.B.-D. is a scientific advisory board member of SNIPR Biome and Excision Biotherapeutics and a scientific advisory board member and co-founder of Acrigen Biosciences.