Structure and mechanism of the type I-G CRISPR effector

Abstract

Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3′ end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.

Repurposing the atypical Type I-G CRISPR system for bacterial genome engineering

“…Cas3 is universally conserved across all type I CRISPR systems, despite the differences in Cascade composition (7). In the type I-G system Cas3 is an integral subunit of Cascade rather than being recruited on target DNA binding (24,39). This association between Cas3 and Cascade has also been observed in a type I-…”

“…When submitted to a phage P1 challenge assay, cells expressing the K39A or D164A Cas3 variant showed full immunity against phage infection under induced condition, and compromised immunity at higher MOIs compared to wild-type Cas3 when expression was uninduced (Figure 2B). In previous in vitro experiments, we observed that T. sulfidiphilus Cascade in the absence of ATP (and thus helicase activity) only cleaved target DNA at the site of Cas3 loading (39). This appears to be enough in vivo to prevent target plasmid and phage replication as long as the type I-G CRISPR effector is highly expressed or MOI is low.…”

“…Two BpiI restriction sites with type I-G repeat sequence were introduced to pRAT-Duet MCS-I to get the spacer replaceable backbone of the pSPACER plasmid. 5'-phosphorylated oligos of CRISPR spacers were annealed and ligated into the BpiI digested pSPACER backbone to obtain the pSPACER vector with target spacer (39).…”

“…The type I-G CRISPR system from Thioalkalivibrio sulfidiphilus consists of four protein-coding genes and a CRISPR locus. We previously reported a biochemical and structural study of this complex (39), demonstrating that Cas3 is an integral component of this complex rather than a separate subunit that is recruited on target DNA binding. Although the Cas3 structure was not observed in the cryo-EM density, bioinformatic predictions suggest that the domain organisation is distinct from Cas3s from other type I CRISPR subtypes, with its HD nuclease domain located at the C-terminus instead of the N-terminus (7) (Figure 1).…”

Repurposing the atypical type I-G CRISPR system for bacterial genome engineering

“…Previously, we reported the structure and mechanism of the type I-G system from Thioalkalivibrio sulfidiphilus [39], demonstrating the constitutive association of Cascade with the Cas3 subunit. Here, we repurpose the T.…”