The ecology and evolution of microbial CRISPR-Cas adaptive immune systems.

Abstract

Over the past decade, the field of CRISPR-Cas research has received a lot of attention from the scientific community. While initially, this mostly concerned microbiologists who were fascinated by the discovery that some bacteria encode RNA-guided adaptive immune systems, this rapidly spread to other scientific disciplines following the development of groundbreaking molecular biology tools [1] and, more recently, to the public domain where the societal and ethical implications and legislation surrounding CRISPR applications are being debated. Some of the potential CRISPR applications that are currently being explored in the laboratory would involve the release of CRISPR genes into confined or open environments—for example, when CRISPR would be used to protect focal bacterial species against phage infections, when it is applied to suppress the spread of antimicrobial resistance or to control vectors of disease [2–4]. One component of the debate surrounding the societal impacts of these applications entails an assessment of the potential risks associated with these strategies (e.g. [5–7]), which requires an understanding of how CRISPR-Cas behaves in an ecological context. In this special issue, we explore this question by examining the evolutionary history of CRISPR-Cas immune systems, where they occur naturally, when they evolve and how this impacts the spread and evolution of other DNA elements. Finally, we return to the question how CRISPR-Cas may be exploited in an ecological context for the benefit of human health, and the ethical challenges that are associated with this. (a) CRISPR-Cas adaptive immune systems: a brief overview CRISPR-Cas adaptive immune systems were discovered around 15 years ago [8–11] and are estimated to exist in approximately 50% of all bacterial genomes and roughly 90% of all archaeal genomes [12]. A CRISPR immunity phenotype is genetically encoded by a so-called CRISPR locus (clustered regularly interspaced short palindromic repeats)—an array of repetitive and unique sequences (repeats and spacers, respectively), both of which are typically around 30 nt in length. Spacers are derived from (foreign) genetic elements, such as plasmids and viruses, and provide immunity to re-infection based on recognition of the cognate sequence (known as ‘protospacer’) [13–15]. Bacteria or archaea may carry a single linked array of spacers interspersed with repeats (one CRISPR locus) or multiple loci. CRISPR loci can evolve very rapidly owing to insertion of new spacers and the occasional loss of spacers or deletion of the CRISPR locus itself, which can cause very closely related strains to carry unique combinations of spacer sequences, known as a CRISPR allele. The overall length of CRISPR loci will increase and decrease with the acquisition and loss of spacers, and can vary from as little as a single spacer flanked by two repeats to hundreds of spacers and repeats [16]. As new spacers are added at the so-called leader-end of the locus, which is the sequence that contains the CRISPR promoter, CRISPR loci form an inverse chronological record of previous infections from the leader to the trailer end of the locus [17]. The extent to which different strains share the same spacer sequences in the same order (usually at the trailer end of the CRISPR locus) is commonly used to define related allele groups as a measure of their evolutionary relatedness. Apart from the genetic CRISPR memory, a functional CRISPR-Cas immune system also requires a set of CRISPR-associated genes (cas genes), which encode the protein machinery required for carrying out the immune response [18]. Cas operons vary in their cas gene composition and gene synteny, resulting in a classification of CRISPR-Cas systems into two classes, six types and 33 subtypes [19–22]. These diverse CRISPR-Cas variants differ in many of their mechanistic details, which have been discussed elsewhere [23,24], yet also have commonalities in the basic steps of the immune pathway. For example, two Cas proteins—Cas1 and Cas2—are almost invariably part of CRISPR-Cas immune systems and are responsible for inserting new spacer sequences into CRISPR arrays, sometimes assisted by other Cas proteins (reviewed in [15,25]). CRISPR transcripts are processed by either Cas proteins or housekeeping RNases [26], and the resulting processed CRISPR RNAs (crRNAs) are bound by Cas proteins to form a ribonucleoprotein complex that serves to detect and cleave complementary nucleic acid sequences [23].