The RNA- and DNA-targeting CRISPR-Cas immune systems of Pyrococcus furiosus

Abstract

Using the hyperthermophile Pyrococcus furiosus, we have delineated several key steps in CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) invader defence pathways. P. furiosus has seven transcriptionally active CRISPR loci that together encode a total of 200 crRNAs (CRISPR RNAs). The 27 Cas proteins in this organism represent three distinct pathways and are primarily encoded in two large gene clusters. The Cas6 protein dices CRISPR locus transcripts to generate individual invader-targeting crRNAs. The mature crRNAs include a signature sequence element (the 5' tag) derived from the CRISPR locus repeat sequence that is important for function. crRNAs are tailored into distinct species and integrated into three distinct crRNA-Cas protein complexes that are all candidate effector complexes. The complex formed by the Cmr [Cas module RAMP (repeat-associated mysterious proteins)] (subtype III-B) proteins cleaves complementary target RNAs and can be programmed to cleave novel target RNAs in a prokaryotic RNAi-like manner. Evidence suggests that the other two CRISPR-Cas systems in P. furiosus, Csa (Cas subtype Apern) (subtype I-A) and Cst (Cas subtype Tneap) (subtype I-B), target invaders at the DNA level. Studies of the CRISPR-Cas systems from P. furiosus are yielding fundamental knowledge of mechanisms of crRNA biogenesis and silencing for three of the diverse CRISPR-Cas pathways, and reveal that organisms such as P. furiosus possess an arsenal of multiple RNA-guided mechanisms to resist diverse invaders. Our knowledge of the fascinating CRISPR-Cas pathways is leading in turn to our ability to co-opt these systems for exciting new biomedical and biotechnological applications.

Figure 1. P. furiosus CRISPR–Cas systems

(A) Distribution of the CRISPR loci (Cr1, Cr2, Cr4–Cr8; red) and two major cas gene clusters (Cas; blue) throughout the P. furiosus genome. (B) CRISPRs comprise leader regions (L) preceding series of repeat (black) and spacer (coloured) elements. The actual numbers of spacers are indicated. The consensus crRNA repeat sequence found in the seven CRISPR loci is shown above with the Cas6-binding and cleavage sites, and the 8-nt 5′ tag crRNA sequence indicated. (C) Genome organization of predicted P. furiosus (Pf) cas genes. Subtype III-B Cmr (blue), subtype I-B Cst (yellow), subtype I-A Csa (green) and core cas genes (grey) are indicated, and recent cas gene superfamily designations [15] are indicated below [14]. The known or predicted general function of the core cas genes is indicated.

Figure 2. Cas6 and crRNA biogenesis

(A) Cas6 cleaves within the repeat regions of the CRISPR transcript to liberate the individual embedded crRNAs. The products of Cas6 cleavage (1× crRNAs) undergo 3′ end trimming. (B) The architecture of a mature crRNA. This abundant crRNA form includes the 5′ repeat-derived tag and complete guide sequence. Other mature crRNAs in P. furiosus contain some repeat sequences at their 3′ ends or slightly less than full-length guide sequences (not depicted). (C) Cas6 is an endoribonuclease critical for crRNA biogenesis. Cas6 interacts with the 5′ region of the CRISPR repeat RNA (red box) and cleaves at a distal site using an active site composed of three key amino acids (indicated). The Cas6-binding region in the crRNA interacts with the positively charged cleft of Cas6.

Figure 3. crRNAs are loaded into three distinct crRNP immune effector complexes in P. furiosus

Following release by Cas6 and further 3′ end trimming, crRNAs of specific size formed from all seven CRISPR loci become incorporated into the three distinct crRNP complexes containing Cmr, Csa or Cst Cas proteins. There is evidence that Cmr crRNPs cleave target RNAs, whereas Csa and Cst complexes cleave invader DNA.