Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex

Abstract

Bacteria and archaea rely on CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided adaptive immune systems for sequence specific elimination of foreign nucleic acids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble with Cas (CRISPR-associated) proteins into a 405-kilodalton multi-subunit surveillance complex called Cascade (CRISPR-associated complex for antiviral defense). Cascade binds foreign DNA complementary to the crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required for target degradation. Structural models of Cascade have captured static snapshots of the complex in distinct conformational states, but conformational dynamics of the 11-subunit surveillance complex have not been measured. Here we use hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to map conformational dynamics of Cascade onto the three-dimensional structure. New insights from structural dynamics are used to make functional predictions about the mechanisms of the R-loop coordination and Cas3 recruitment. We test these predictions in vivo and in vitro. Collectively, we show how mapping conformational dynamics onto static 3D-structures adds an additional dimension to the functional understanding of this biological machine.

CRISPR RNA-guided DNA cleavage by reconstituted Type I-A immune effector complexes

“…2 and Majumdar et al 2015) and as was previously observed in Sulfolobus islandicus (Peng et al 2013) and Thermoproteus tenax (Plagens et al 2012, 2014), the Cas3′ and Cas3″ polypeptides of differing Type I-A systems are integral structural components of the effec- tor crRNPs that target destruction of invader DNA. In con- trast, the single Cas3 subunit of other systems acts in trans with the effector crRNP and only becomes recruited and activated for function upon crRNP-target DNA interaction and crRNA-induced R-loop formation of the target DNA (Elmore et al 2015; Hayes et al 2016; Jackson et al 2014; Mulepati et al 2014; van Erp et al 2015, 2018; Wang et al 2016; Xiao et al 2018; Zhao et al 2014). In this study, we reconstituted functional Pfu Type I-A crRNPs that cleave double-stranded DNA targets in a PAM (5′-GGG/CCN-3′) and crRNA-dependent manner (Figs.…”

Structure reveals mechanism of CRISPR RNA-guided nuclease recruitment and anti-CRISPR viral mimicry

“…PAM recognition by the surveillance complex destabilizes the DNA duplex and facilitates crRNA-guided strand invasion 22,35,36 . Hybridization of the crRNA-guide to the complementary DNA displaces the non-complementary strand, resulting in an Rloop structure [35][36][37][38][39][40][41][42] . The N-terminal domain of Cas8f and the opposing face of the terminal Cas7f subunit (Cas7f.6) form a positively charged "vise" that closes around dsDNA ( Figure 2B-C) 22,24 .…”

Characterization and repurposing of the endogenous Type I-F CRISPR-Cas system ofZymomonas mobilisfor genome engineering

“…In this work, we took the cas3 gene as a target to study genome engineering in Z. mobilis, and different cas3 mutant strains were successfully obtained. All Type I systems use a complex of Cas proteins to bind to a target while employ Cas3 nuclease to execute protospacer cleavage (63,64).…”