Type III CRISPR-Cas Systems: Deciphering the Most Complex Prokaryotic Immune System

Abstract

The emergence and persistence of selfish genetic elements is an intrinsic feature of all living systems. Cellular organisms have evolved a plethora of elaborate defense systems that limit the spread of such genetic parasites. CRISPR-Cas are RNA-guided defense systems used by prokaryotes to recognize and destroy foreign nucleic acids. These systems acquire and store fragments of foreign nucleic acids and utilize the stored sequences as guides to recognize and destroy genetic invaders. CRISPR-Cas systems have been extensively studied, as some of them are used in various genome editing technologies. Although Type III CRISPR-Cas systems are among the most common CRISPR-Cas systems, they are also some of the least investigated ones, mostly due to the complexity of their action compared to other CRISPR-Cas system types. Type III effector complexes specifically recognize and cleave RNA molecules. The recognition of the target RNA activates the effector large subunit - the so-called CRISPR polymerase - which cleaves DNA and produces small cyclic oligonucleotides that act as signaling molecules to activate auxiliary effectors, notably non-specific RNases. In this review, we provide a historical overview of the sometimes meandering pathway of the Type III CRISPR research. We also review the current data on the structures and activities of Type III CRISPR-Cas systems components, their biological roles, and evolutionary history. Finally, using structural modeling with AlphaFold2, we show that the archaeal HRAMP signature protein, which heretofore has had no assigned function, is a degenerate relative of Type III CRISPR-Cas signature protein Cas10, suggesting that HRAMP systems have descended from Type III CRISPR-Cas systems or their ancestors.

Keywords: CRISPR evolution; CRISPR-Cas Type III; HRAMP; RNA-guided defense; cyclic oligonucleotides; prokaryotic immunity.

Fig. 1.

Type III adaptive immunity. a) Adaptation: insertion of small fragments of invader-derived DNA into the host CRISPR array with the formation of a new spacer-repeat unit. In some systems, the spacers can be acquired from RNA through the activity of the RT domain fused to Cas1. b) Expression: maturation of crRNAs and assembly of effector complexes. c) Interference: triggering of the immune response by specific recognition of foreign RNA.

Fig. 2.

A model of the co-transcriptional cleavage by Type III effectors and activation of auxiliary nucleases triggered by the target recognition. a) The recognition of target RNA by Type III effector stimulates the activities of HD and Palm domains of the Cas10 subunit. The Palm domain catalyzes the synthesis of cyclic oligoadenylates (cOAs) while the HD domain degrades single-stranded DNA within the transcription bubble. b) cOAs activate the auxiliary nucleases that target DNA or RNA molecules. The activity of the auxiliary effectors is regulated through the degradation of cOAs by ring nucleases or, in some cases, by the auxiliary proteins. c) The avoidance of self-targeting in Type III CRISPR-Cas systems: the complementarity between the target and repeat-derived 5′-tag of crRNA prevents the activation of both the HD and the Palm domains of the Cas10 subunit.

Fig. 3.

HRAMP signature protein is a Cas10-related nuclease with the HD domain. Crystal structure of Csm1 from S. thermophilus (left panel) and the AlphaFold2 model of the HRAMP signature protein WP_013440547.1 from Halogeometricum borinquense (right panel) are shown as ribbon diagrams. Conserved structural elements are colored; dissimilar domains are shown in light grey. The positions of the His-Asp active sites are indicated by arrows (positions in Csm1 are experimentally confirmed). Below, Csm1 from S. thermophilus is shown as a part of the Type III-A CRISPR-Cas effector complex.

Fig. 4.

Comparison of structures of Type I and Type III effectors; homologous proteins are depicted by matching colors.

Fig. 5.

Proposed scenario of the origin of the Type III CRISPR-Cas systems. Adopted from Koonin et al., 2019 [70].