Biochemical plasticity of the Escherichia coli CRISPR Cascade revealed by in vitro reconstitution of Cascade activities from purified Cas proteins.

ABSTRACT: The most abundant clustered regularly interspaced short palindromic repeats (CRISPR) type I systems employ a multisubunit RNA-protein effector complex (Cascade), with varying protein composition and activity. The Escherichia coli Cascade complex consists of 11 protein subunits and functions as an effector through CRISPR RNA (crRNA) binding, protospacer adjacent motif (PAM)-specific double-stranded DNA targeting, R-loop formation, and Cas3 helicase-nuclease recruitment for target DNA cleavage. Here, we present a biochemical reconstruction of the E. coli Cascade from purified Cas proteins and analyze its activities including crRNA binding, dsDNA targeting, R-loop formation, and Cas3 recruitment. Affinity purification of 6His-tagged Cas7 coexpressed with untagged Cas5 revealed the physical as

SUBMITTER: Lemak S 

PROVIDER: S-EPMC11617276 | biostudies-literature | 2024 Dec

REPOSITORIES: biostudies-literature