Interference Requirements of Type III CRISPR-Cas Systems from Thermus thermophilus.

Among the diverse prokaryotic adaptive immunity mechanisms, the Type III CRISPR-Cas systems are the most complex. The multisubunit Type III effectors recognize RNA targets complementary to CRISPR RNAs (crRNAs). Target recognition causes synthesis of cyclic oligoadenylates that activate downstream auxiliary effectors, which affect cell physiology in complex and poorly understood ways. Here, we studied the ability of III-A and III-B CRISPR-Cas subtypes from Thermus thermophilus to interfere with plasmid transformation. We find that for both systems, requirements for crRNA-target complementarity sufficient for interference depend on the target transcript abundance, with more abundant targets requiring shorter complementarity segments. This result and thermodynamic calculations indicate that Type III effectors bind their targets in a simple bimolecular reaction with more extensive crRNA-target base pairing compensating for lower target abundance. Since the targeted RNA used in our work is non-essential for either the host or the plasmid, the results also establish that a certain number of target-bound effector complexes must be present in the cell to interfere with plasmid establishment. For the more active III-A system, we determine the minimal length of RNA-duplex sufficient for interference and show that the position of this minimal duplex can vary within the effector. Finally, we show that the III-A immunity is dependent on the HD nuclease domain of the Cas10 subunit. Since this domain is absent from the III-B system the result implies that the T. thermophilus III-B system must elicit a more efficient cyclic oligoadenylate-dependent response to provide the immunity.

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

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.

Spacer acquisition by Type III CRISPR-Cas system during bacteriophage infection of Thermus thermophilus.

Type III CRISPR-Cas systems provide immunity to foreign DNA by targeting its transcripts. Target recognition activates RNases and DNases that may either destroy foreign DNA directly or elicit collateral damage inducing death of infected cells. While some Type III systems encode a reverse transcriptase to acquire spacers from foreign transcripts, most contain conventional spacer acquisition machinery found in DNA-targeting systems. We studied Type III spacer acquisition in phage-infected Thermus thermophilus, a bacterium that lacks either a standalone reverse transcriptase or its fusion to spacer integrase Cas1. Cells with spacers targeting a subset of phage transcripts survived the infection, indicating that Type III immunity does not operate through altruistic suicide. In the absence of selection spacers were acquired from both strands of phage DNA, indicating that no mechanism ensuring acquisition of RNA-targeting spacers exists. Spacers that protect the host from the phage demonstrate a very strong strand bias due to positive selection during infection. Phages that escaped Type III interference accumulated deletions of integral number of codons in an essential gene and much longer deletions in a non-essential gene. This and the fact that Type III immunity can be provided by plasmid-borne mini-arrays open ways for genomic manipulation of Thermus phages.