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.

Importance of Mobile Genetic Element Immunity in Numerically AbundantTrichodesmiumClades

“…Adaptation: recognition and incorporation of transcribed 30-50bp protospacers ( i . e ., DNA or RNA sequences of invading MGEs; typically mediated by Cas1 or Cas1-reverse transcriptase (RT) fusion proteins, respectively (66, 67)) into CRISPR arrays as spacer sequence DNA ‘memories’ of past attacks, 2 . Expression: spacer RNAs are expressed as precursor CRISPR RNA (crRNA), and 3 .…”

“…Mechanistically, Type III-B CRISPR-Cas systems operate in three steps 1. Adaptation: recognition and incorporation of transcribed 30-50bp protospacers (i.e., DNA or RNA sequences of invading MGEs; typically mediated by Cas1 or Cas1-reverse transcriptase (RT) fusion proteins, respectively (66,67)) into CRISPR arrays as spacer sequence DNA 'memories' of past attacks, 2.…”

“…The absence of a Cas1-RT fusion protein in T. thiebautii suggests that the primary adaptation targets for this system are DNA MGEs. In contrast, an HD superfamily nuclease domain in T. thiebautii Cas10 proteins indicates that the interference step is likely cleaving both RNA and transcribed DNA (49,66,68). Importantly, these DNA spacer sequences also provide 'fingerprints' of past MGE attacks that link phage/plasmid sequences with the CRISPR-Cas system containing host (e.g., (69)(70)(71)(72)(73)(74)).…”

Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications

“…The Cas10 subunit cleaves the transcribed ssDNA and activates the Csm6 nonspecific-RNase activity, while as the Csm 3 cleaves the RNA target, the RuvC domain introduces staggered double-stranded DNA (dsDNA) breaks[ 7 - 9 ]. Since the Cas10 subunit HD domain cleaves ssDNA from the transcription bubble[ 10 - 12 ], due to activation of the Palm domain, ATP is converted into four or six-member cyclic oligoadenylate (CoA) rings. These six-member CoA rings then act as a secondary messenger and activate Csm6 by binding to its CARF (CRISPR-Associated Rossman Fold) domain, causing activation of the Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domain and unleashing nonspecific RNA cleavage[ 13 - 15 ].…”

Investigating CRISPR spacer targets and their impact on genomic diversification of Streptococcus mutans

“…Type I (C and E subtypes) and Type II (A subtype) are most commonly identified in S. mutans and these systems recognize and cleave DNA. There are rare instances where Type III-A systems, that have specificity towards cleaving RNA [8], are found in S. mutans . A substantial number of S. mutans strains encode both a Type I-C and a Type II-A CRISPR-Cas system, perhaps to help with viral escape from either system.…”