CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes

Abstract

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated genes) is an adaptive immunity system in bacteria and archaea that functions via a distinct self-non-self recognition mechanism that is partially analogous to the mechanism of eukaryotic RNA interference (RNAi). The CRISPR-Cas system incorporates fragments of virus or plasmid DNA into the CRISPR repeat cassettes and employs the processed transcripts of these spacers as guide RNAs to cleave the cognate foreign DNA or RNA. The Cas proteins, however, are not homologous to the proteins involved in RNAi and comprise numerous, highly diverged families. The majority of the Cas proteins contain diverse variants of the RNA recognition motif (RRM), a widespread RNA-binding domain. Despite the fast evolution that is typical of the cas genes, the presence of diverse versions of the RRM in most Cas proteins provides for a simple scenario for the evolution of the three distinct types of CRISPR-cas systems. In addition to several proteins that are directly implicated in the immune response, the cas genes encode a variety of proteins that are homologous to prokaryotic toxins that typically possess nuclease activity. The predicted toxins associated with CRISPR-Cas systems include the essential Cas2 protein, proteins of COG1517 that, in addition to a ligand-binding domain and a helix-turn-helix domain, typically contain different nuclease domains and several other predicted nucleases. The tight association of the CRISPR-Cas immunity systems with predicted toxins that, upon activation, would induce dormancy or cell death suggests that adaptive immunity and dormancy/suicide response are functionally coupled. Such coupling could manifest in the persistence state being induced and potentially providing conditions for more effective action of the immune system or in cell death being triggered when immunity fails.

Keywords: CRISPR-Cas; RRM domain; adaptive immunity; dormancy; innate immunity; programmed cell death.

Figure 1. The principal building blocks of the three types of CRISPR-Cas systems. For each Cas protein, the systematic gene name and (whenever available) domain description are given. The color code for general functions is shown on the right. Homologous genes are shown by shapes of the same color, shades denote specialized activities within the same general function. Abbreviations: LS, large sunubit (including Cas10, Cas8, Cse1 and Csy1subfamilies); SS, small subunit. The # symbol indicates putative small subunits that appear to be fused to the large subunit in several Type I subtypes. The $ symbol indicates that several distinct subfamilies of the Cas7 family could be present in a Cascade complex with unknown stoichiometry. An asterisk indicates that the respective COG1517 family proteins contain a third effector (toxin) domain.

Figure 2. The RRM domain, the cornerstone of CRISPR-Cas. The figure shows RRM domain-containing proteins in CRISPR-Cas systems. The typical organization of the operons in Type I and Type III CRISPR-Cas loci is depicted by arrows with the size roughly proportional to the size of respective gene. Homologous genes are shown by arrows of same color. Color coding is the same as in the Figure 1. Gene and family names are from references and . Additional designations: LS, large subunit; SS, small subunit; R, RAMPs. RRM domains are shown by pink rectangles, with semi-transparent rectangles indicating deteriorated RRM fold. The proteins representing families with RRM domains for which structures have been solved are denoted by asterisks.

Figure 3. A parsimonious evolutionary scenario for the three types of CRISPR-Cas. Homologous genes are color-coded and identified by a family name.^, Color coding is the same as in Figure 1. Genes coding for inactivated (putative) large subunits are indicated by crosses. Major evolutionary events are shown in the corresponding branches. The figure is an updated and modified version of figure 7 in reference .

Figure 4. Hypothetical coupling of the CRISPR-mediated adaptive immunity with the persistence/cell suicide response. The genes are depicted by black arrows. Persistence/cell suicide related genes are shown in green shades. Homologous genes are shown by the arrows of same color. The PD-(D/E)xK (RecB-like) endonuclease, VapD/Cas2 endoribonuclease and predicted endoribonuclease HEPN domains^, are indicated above the corresponding gene.