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CRISPR systems, as bacterial defenses against phages, logically must display in their functioning a sequence of at least three major steps. These, in order of occurrence, are “facilitation,” adaptation and interference, where the facilitation step is the main issue considered in this commentary. Interference is the blocking of phage infections as mediated in part by CRISPR spacer sequences. Adaptation, at least as narrowly defined, is the acquisition of these spacer sequences by CRISPR loci. Facilitation, in turn and as defined here, corresponds to phage-naïve bacteria avoiding death follow first-time exposure to specific phages, where bacterial survival of course is necessary for subsequent spacer acquisition. Working from a variety of perspectives, I argue that a requirement for facilitation suggests that CRISPR systems may play secondary rather than primary roles as bacterial defenses, particularly against more virulent phages. So considered, the role of facilitation in CRISPR functioning could be viewed as analogous to the building, in vertebrate animals, of adaptive immunity upon an immunological foundation comprised of mechanisms that are both more generally acting and innate.
Clustered Regularly Interspaced Short Palindromic Repeats, or CRISPR systems, have been the subject of a substantial quantity of recent inquiry.1–6 These bacterial loci contain up to several hundred repeats of non-variable, somewhat palindromic sequences that separate stretches of variable spacer DNA. Both sequences are tens of base pairs in length and spacer sequences have been found to be homologous to “proto-spacer” sequences associated with certain phages and plasmids. This homology, in turn, is required for CRISPR-mediated interference with, for example, phage infection. CRISPR systems acquire new spacer sequences seemingly in response to phage or plasmid exposure and so, consequently, have been described as a form of bacterial adaptive immunity.
The term adaptation can be used to describe the molecular mechanism(s) of acquisition of CRISPR spacer sequences. In a broader sense, though, CRISPR adaptation entails the conversion of phagesensitive bacteria to phage-resistant ones. Such adaptation necessarily requires two perhaps distinct aspects. One consists of CRISPR adaptation in the sense of spacer acquisition while the other is the means by which bacteria avoid being killed, by phages, so that spacer acquisition can occur. That latter I will call CRISPR facilitation. I thus envisage CRISPR functioning against phages as requiring a progression of three essential steps: (I) facilitation of CRISPR adaptation, (II) actual acquisition of spacer sequences (CRISPR adaptation, as narrowly defined), and then (III) subsequent CRISPR interference with phage infection.
The CRISPR facilitation step might be accomplished by a variety of mechanisms including what I will describe as (1) “anticipatory,” (2) “deficient,” (3) “biased,” (4) “CRISPR-associated” or (5) “innate.” Keep in mind as I discuss these possibilities that they likely differ not just mechanistically but also in terms of their impact on the efficiency of CRISPR adaptation—as broadly defined—that is, the likelihood that a bacterium or bacterial population will succeed in achieving CRISPR-associated immunity against a given phage. This efficiency, in turn, may be relevant in determining what roles CRIPSR systems might play, ecologically, as just one of numerous bacterial antiphage defenses.7,8
Such speculation begs the question of what bacterial function could play this role of innate, primary phage-resistance mechanism that is capable of facilitating CRISPR adaption. One possibility is the bacterial protection provided by restriction- modification (R-M) systems. Indeed, R-M systems, which are present in many bacteria, would seem to be ideal candidates for CRISPR facilitation: (a) They allow foreign DNA into cells, thereby permitting spacer acquisition. (b) When successfully implemented, R-M systems block subsequent DNA functioning, thereby providing phage resistance. (c) And, occasionally, R-M systems fail to protect not only individual bacteria but entire bacterial populations.8,19 There would be utility, therefore, for bacteria to posses some sort of anti-phage back up to R-M systems in the course of repeated bacterial exposure to specific phage types and CRISPR systems, seemingly, could serve in just such a role.
Which of these five general mechanisms actually facilitates CRISPR adaptation will, of course, only be determined via experimental analysis. There is no reason to assume, however, that any one mechanism might preclude others, or that a mechanism that functions in one system will function for all CRISPR-carrying bacteria. Furthermore, no matter how rare CRISPR adaptation might be, so long as CRISPR systems occasionally play a key role in protecting bacterial populations from phage-mediated extinction,20 then—as a consequence of such positive selection— specific CRISPR spacer sequences should still provide an “historical record” of bacterial interactions with specific phage types.9,21,22
Thank you to Luciano Marraffini, Paul Hyman, Bob Blasdel and Cameron Thomas-Abedon for their helpful comments.