(C) PLOS One [1]. This unaltered content originally appeared in journals.plosone.org.
Licensed under Creative Commons Attribution (CC BY) license.
url:https://journals.plos.org/plosone/s/licenses-and-copyright

------------



Evolutionary plasticity and functional versatility of CRISPR systems

['Eugene V. Koonin', 'National Center For Biotechnology Information', 'National Library Of Medicine', 'National Institutes Of Health', 'Bethesda', 'Maryland', 'United States Of America', 'Kira S. Makarova']

Date: 2022-01 

The principal biological function of bacterial and archaeal CRISPR systems is RNA-guided adaptive immunity against viruses and other mobile genetic elements (MGEs). These systems show remarkable evolutionary plasticity and functional versatility at multiple levels, including both the defense mechanisms that lead to direct, specific elimination of the target DNA or RNA and those that cause programmed cell death (PCD) or induction of dormancy. This flexibility is also evident in the recruitment of CRISPR systems for nondefense functions. Defective CRISPR systems or individual CRISPR components have been recruited by transposons for RNA-guided transposition, by plasmids for interplasmid competition, and by viruses for antidefense and interviral conflicts. Additionally, multiple highly derived CRISPR variants of yet unknown functions have been discovered. A major route of innovation in CRISPR evolution is the repurposing of diverged repeat variants encoded outside CRISPR arrays for various structural and regulatory functions. The evolutionary plasticity and functional versatility of CRISPR systems are striking manifestations of the ubiquitous interplay between defense and “normal” cellular functions.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Introduction

The CRISPR systems are known primarily as a new generation of genome editing tools that possess unprecedented specificity and efficiency thanks to the use of guide RNAs to recognize unique sequences in the genome [1–3]. This specificity of nucleic acid recognition also underlies the primary biological function of CRISPR in prokaryotes, namely adaptive immunity against viruses and other mobile genetic elements (MGEs) [4–7]. The CRISPR immune response involves 3 stages:

adaptation, the process of acquisition of pieces of foreign DNA (protospacers) that become spacers inserted between repeats in CRISPR arrays and are subsequently employed to produce guide RNAs that specifically target the cognate foreign nucleic acid; expression and maturation of the CRISPR (cr) RNAs, whereby the long transcript of the CRISPR array, the pre-crRNA, is processed to yield functional, mature crRNA; and interference, whereby crRNAs are exploited as guides to recognize the target DNA or RNA that is then, typically, cleaved by the nuclease moiety of the CRISPR effector complex.

Each of these stages in the CRISPR immune function is mediated by a distinct set of Cas (CRISPR associated) proteins that comprise functional modules of the CRISPR systems [8,9]. Characteristically of defense systems [10], CRISPRs are prone to fast evolution that involves not only sequence change but also numerous rearrangements and replacements of the Cas protein complexes [11,12]. In addition to the 3 core modules, most of the CRISPR systems also encompass various accessory genes, which encode proteins modulating the activity of the core CRISPR components in manners that, with a few exceptions, remain poorly characterized [13,14].

From the early days of CRISPR research, it seemed natural to surmise that CRISPR systems would be recruited for various nondefense functions, including regulation of gene expression, but probably much more [15,16]. However, more than a decade later, the information on nondefense functions of CRISPR remains scarce. Nevertheless, one remarkable theme has emerged prominently, namely the recruitment of partially degraded, defective CRISPR systems by transposons that employ them for RNA-guided transposition [17,18]. In addition, a variety of highly derived cas operons have been discovered that are often not linked to CRISPR arrays [19,20]; their functions remain enigmatic and await experimental study. Another trend that is becoming increasingly prominent is the repurposing of the CRISPR themselves, that is, crRNAs and their derivatives, for various, primarily regulatory functions rather than for target recognition followed by cleavage [21–23].

In this essay, we outline our current understanding of the evolutionary plasticity and functional versatility of CRISPR. Evolutionary plasticity refers to gain and loss of components by CRISPR systems as well as functionally consequential evolutionary changes in cas genes, for example, those that lead to enzyme inactivation. These evolutionary changes give rise to remarkable functional versatility, that is, a broad repertoire of biological functions and molecular mechanisms across the numerous variants of CRISPR systems and their derivatives. The universe of CRISPR is expanding fast with advancing genome and especially metagenome sequencing, but we believe that general trends can already be captured.

The exaptive splendor of CRISPR In this section, we discuss the well characterized as well as tentative cases of exaptation of CRISPR systems and their components, that is, their recruitment for alternative biological functions distinct from bacterial or archaeal adaptive immunity [16,26]. Diverse cases of CRISPR exaptation have been discovered (and counting), although they seem to comprise only a relatively small minority of CRISPR systems [11]. In some instances, an intact, apparently functional CRISPR systems is subverted for a new function. The dev operon of Myxobacteria that regulates the sporulation process encodes a typical subtype I-B CRISPR system including the adaptation module and the CRISPR array [58,59]. However, this CRISPR system contributes to the regulation of sporulation via a mechanism that is distinct from the typical CRISPR activity. It has been shown that the complex of Cas5, Cas7, and Cas8 proteins, a subcomplex of the Cascade, employs a distinct antisense RNA to abrogate the expression of devI, a sporulation inhibitor, and thus promote sporulation [59]. Most likely, the I-B CRIPSR system in the dev operon performs a dual function, in both adaptive immunity and expression regulation. Without targeted experimental studies, it is impossible to tell how common the dual functionality of CRISPR systems might be, opening the tantalizing possibility that such secondary recruitment occurred in many cases. Apart from the utility for the host cell, the gain of a new function that is important for the host could make it addicted to CRISPR, preventing the loss of the CRISPR locus and stabilizing the “symbiosis” between CRISPR and the host organism (more on such addiction below). Other cases of repurposing of intact, functional CRISPR systems have been identified in viruses. Some bacteriophages infecting Vibrio bacteria encode a subtype I-F CRISPR system that targets innate immunity systems of the host bacteria and thus contributes to the virus–bacterium arms race [60,61]. CRISPR systems have also been discovered in multiple megaphages that infect Bacteroidetes [62]. This is a striking illustration of the guns for hire principle. Below we discuss several additional cases of this form of exaptation, which is a persistent trend in the evolution of CRISPR derivatives (Fig 3). On many other occasions, exaptation of CRISPR involves derived forms resulting from reductive evolution. The first case in point are type IV systems, another reduced derivative of type III (Fig 2). Type IV systems possess homologs of Cas7 and Cas5 but lack Cas10 (or Cas8) and instead encompass a much smaller protein, which plays the role of the large subunit (Csf1) of the effector complex but does not contain any recognizable enzymatic domains [63–65]. Notably, in the recently discovered subtype IV-C, Csf1 protein contains a carboxyl-terminal HD nuclease domain related to the corresponding domain of Cas10. In the phylogenetic tree of Cas7, subtype IV-C is the deepest branch, suggesting that it could represent an intermediate stage of evolution of type IV from some variant of type III. Comparisons of the solved structures of the effector complexes also support the affinity of type IV and III [64,66]. The subtypes within type IV show considerable variability of gene composition. In particular, subtypes IV-A and IV-C encompass Cas11, the small subunit of the effector complex, whereas subtype IV-B systems lack this protein. Additionally, different subtypes have distinct accessory proteins, such as the DinG helicase in subtype IV-A and the inactivated homolog of APS/PAPS reductase CysH in subtype IV-B and some subtype IV-A systems. The presence of these genes that are tightly associated with type IV loci suggests distinct functionalities that remain uncharacterized, although it has been shown that DinG is required for subtype IV system interference activity against plasmids [67]. All identified type IV systems are located on plasmids, integrative conjugative elements (ICEs), prophages, and some free phages. Those spacers in CRISPR arrays of subtypes IV-A and IV-C, for which protospacers were detected, mapped primarily to genes of plasmid conjugative machinery [65]. Thus, type IV systems are most likely engaged in competition among MGE, in particular plasmid exclusion. The molecular mechanisms of their action, however, remain enigmatic given that, with the exception of the rare subtype IV-C, they all lack recognizable nuclease domains. Furthermore, there are major functional differences between subtypes IV-A and IV-C, on the one hand, and IV-B, on the other hand. The IV-A effector complexes that lack Cas11 bind crRNA similarly to other CRISPR systems, whereas subtype IV-B systems bind heterogeneous small RNAs via a filament formed by Cas11 subunits [29], suggestive of a distinct, unknown mechanism. Regardless of the mechanistic details, type IV systems are another clear-cut case of CRISPR recruitment as “guns for hire.” The next variety of derived CRISPR systems we discuss is an even more striking exhibit for the same principle. A genomic survey of CRISPR systems has shown that numerous Tn7-like transposons encode partially degraded subtypes I-B, I-F, and V-K CRISPR loci (Fig 2) [17,68,69]. These CRISPR systems have been acquired by transposons on at least 3, but probably, more independent occasions, suggesting that they confer selective advantages on the transposons. Furthermore, all transposon-encoded CRISPR systems have lost the interference capacity albeit via different routes, either by losing the cas3 genes, which encodes the helicase–nuclease protein that shreds DNA in type I systems, or by inactivation of the catalytic site of the RuvC-like nuclease in V-K. Hence, it has been proposed that this variety of CRISPR mediates RNA-guided transposition, a phenomenon that has not been described prior to these observations [68]. Indeed, this route of transposition has been experimentally demonstrated for all major varieties of transposon-encoded CRISPR systems, subtypes V-K, I-B, and I-F [70–73]. In each case, the effector complex of the CRISPR systems binds to the target via the guide crRNA and delivers the transposase which inserts the transposon within a short distance from the recognized protospacer. Accordingly, the RNA-guided transposition systems were dubbed CRISPR-associated transposase (CAST). Notably, in the case of CAST-V-K and CAST-I-F, all transposition appears to be RNA-guided (some details of the molecular mechanism are discussed below), whereas transposons encoding CAST-I-B alternate 2 modes of transposition, one of which is CRISPR independent [70]. Type IV CRISPR systems, as well as the CASTs, deviate from the adaptive immune function of CRISPR, but are typically associated with CRISPR arrays and rely on the same fundamental molecular principle, namely employing guide RNAs to ensure the specificity of target recognition. Other CRISPR derivatives seem to depart from the mainstream even further (Fig 2). In numerous species of Streptomyces, a derived subtype I-E system consisting of cas5, cas6, cas7, and cas8 genes colocalizes with a gene coding for a STAND NTPase, a putative PCD effector [14]. Given the absence of Cas3, an adaptation module or an array, this module cannot be a typical CRISPR system, but rather, can be predicted to function as a distinct defense mechanism, in conjunction with the STAND NTPase. Many Haloarchaea encode a distinct, highly diverged CRISPR derivatives dubbed haloarchaeal RAMP (HRAMP) that encompass homologs of Cas5 and Cas7 along with additional nucleases and uncharacterized proteins (Fig 2) [20]. In the case of HRAMP, the sequences of the Cas5 and Cas7 homologs have diverged to such an extent that tracing their origin to a specific subtype or even type of regular CRISPR systems is challenging, although there is somewhat higher similarity to the homologs from type III systems. The presence of apparently active nucleases implies that HRAMP is a distinct defense system that, however, can be expected to function via mechanisms distinct from those of CRISPR. CRISPR system derivatives distantly related to HRAMP have been identified in genomes of many Asgard archaea (and accordingly denoted ARAMP) [19]. In addition to the components distantly related to those of HRAMP, most of the ARAMPs encompass a distinct form of the adaptation integrase Cas1, making their mechanism even more enigmatic (Fig 2). Furthermore, Asgard genomes encode an unprecedented diversity of Cas1 homologs, some possibly associated with novel MGE [19]. All the CRISPR-like systems discussed here are limited in their spread to a particular group of prokaryotes or MGE, which supports the view that these are derived CRISPR forms. There is little doubt that, with further advance of genomics and metagenomics, many additional specialized CRISPR derivatives will be discovered.

[END]

[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001481

(C) Plos One. "Accelerating the publication of peer-reviewed science."
Licensed under Creative Commons Attribution (CC BY 4.0)
URL: https://creativecommons.org/licenses/by/4.0/


via Magical.Fish Gopher News Feeds:
gopher://magical.fish/1/feeds/news/plosone/