Positioning Diverse Type IV Structures and Functions Within Class 1 CRISPR-Cas Systems

Abstract

Type IV CRISPR systems encode CRISPR associated (Cas)-like proteins that combine with small RNAs to form multi-subunit ribonucleoprotein complexes. However, the lack of Cas nucleases, integrases, and other genetic features commonly observed in most CRISPR systems has made it difficult to predict type IV mechanisms of action and biological function. Here we summarize recent bioinformatic and experimental advancements that collectively provide the first glimpses into the function of specific type IV subtypes. We also provide a bioinformatic and structural analysis of type IV-specific proteins within the context of multi-subunit (class 1) CRISPR systems, informing future studies aimed at elucidating the function of these cryptic systems.

Keywords: CRISPR; Cas; Cas6; Cas7; CysH; DinG helicase; type IV.

FIGURE 1

The type IV Cas accessory proteins have evolved a Cas specific function. (A) Classification schematic of type IV CRISPR-Cas systems. A typical locus is represented for each type IV subtype. Dashed lines indicate components that are sometimes not encoded by the subtype. Shaded backgrounds highlight which gene products form the ribonucleoprotein (RNP) complex. The yellow square in the IV-C cas10-like large subunit represents an HD nuclease domain. (B) Phylogenetic tree of Cas- and non-CasDinG sequences. Posterior probabilities are shown. (C) Cartoons of Cas- and non-CasDinG sequences indicating positions of certain helicase motifs and domain architecture. Weblogos (Crooks, 2004) of the FeS cluster region in non-CasDinG (below, blue outline) and CasDinG (top, red outline) are shown. (D) Phylogenetic tree of Cas- and non-CasCysH sequences. Posterior probabilities are shown. (E) Cartoons of Cas- and non-CasCysH sequences. CasCysH is predicted to adopt the Rossman-like α - β - α fold observed in non-CasCysH structures. Positions and sequences of P- and PP-loops are indicated. Weblogos of the catalytic cysteine in non-CasCysH (bottom, teal outline) and CasCysH (top, orange outline) are shown.

FIGURE 2

Models of type IV system functions highlighting questions that remain to be answered. (A) IV-A RNP complexes likely bind DNA targets and recruit CasDinG for target unwinding and degradation. (B) IV-B RNP complexes likely interact with CasCysH to perform an unknown function. (C) The putative IV-C RNP complex likely binds a nucleic acid target and cleaves that target with the HD nuclease domain. Created with BioRender.com.