Prokaryotes encode adaptive immune systems, called CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR associated), to provide resistance against mobile invaders, such as viruses and plasmids. Host immunity is based on incorporation of invader DNA sequences in a memory locus (CRISPR), the formation of guide RNAs from this locus, and the degradation of cognate invader DNA (protospacer). Invaders can escape type I-E CRISPR-Cas immunity in
Escherichia coli K12 by making point mutations in the seed region of the protospacer or its adjacent motif (PAM), but hosts quickly restore immunity by integrating new spacers in a positive-feedback process termed “priming.” Here, by using a randomized protospacer and PAM library and high-throughput plasmid loss assays, we provide a systematic analysis of the constraints of both direct interference and subsequent priming in
E. coli. We have defined a high-resolution genetic map of direct interference by Cascade and Cas3, which includes five positions of the protospacer at 6-nt intervals that readily tolerate mutations. Importantly, we show that priming is an extremely robust process capable of using degenerate target regions, with up to 13 mutations throughout the PAM and protospacer region. Priming is influenced by the number of mismatches, their position, and is nucleotide dependent. Our findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the coevolutionary arms race with their invaders.Bacteria and Archaea are regularly exposed to bacteriophages and other mobile genetic elements, such as plasmids. To control the competing effects of horizontal gene transfer, a spectrum of resistance strategies have evolved in prokaryotes (
1). One of the most widespread and well-characterized are the CRISPR-Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated) systems, which provide bacterial “adaptive immunity” (
1–
8). Simply, CRISPR-Cas functions in three major steps. First, in a process termed “adaptation,” short sequences are derived from the invading element and incorporated into a CRISPR array (
9). CRISPR arrays are composed of short repeats that are separated by the foreign-derived sequences, termed “spacers.” Second, CRISPRs are transcribed into a pre-CRISPR RNA (pre-crRNA), which is then processed into short crRNAs, which encompass portions of the repeats and most—or all—of the spacer. Finally, as part of a Cas ribonucleoprotein complex, the crRNAs guide a sequence-specific targeting of complementary nucleic acids (for recent reviews, see refs.
1–
7).CRISPR-Cas systems are divided into three major types (I–III) and further categorized into subtypes (e.g., I-A to I-F) (
10). The mechanisms of both crRNA generation and interference differ between the types and there are even significant differences between closely related subtypes. However, Cas1 and Cas2 are the only two Cas proteins completely conserved across all CRISPR-Cas systems and they are crucial for adaptation in
Escherichia coli (
10–
12). The acquisition of new spacers is the most poorly understood stage in CRISPR-Cas immunity, mainly hindered by the paucity of robust laboratory assays to monitor this process (reviewed in ref.
9).
Streptococcus thermophilus is highly proficient at spacer acquisition and provided much of the early insight into adaptation, showing that new spacers are typically acquired at one end of the CRISPR array from either phages (
13–
15) or plasmids (
16). Recently, spacer acquisition has been detected in a variety of other systems (
11,
12,
17–
20). Adjacent to the expanding end of the array is the leader region, which harbors the promoter for pre-crRNA expression and sequences important for spacer acquisition (
12,
21). Recent studies in
E. coli in the type I-E system have shown that spacer acquisition can occur from phages and plasmids either when the Cas1 and Cas2 proteins are overexpressed or if the native
cas genes are up-regulated, because of deletion of
hns (
11,
12,
20–
22). The DNA targets (termed “protospacers”) of newly acquired spacers are consistently flanked by protospacer-adjacent motifs (PAMs), with the
E. coli type I-E consensus 5′-protospacer-CTT-3′. PAMs were originally identified computationally (
23) and were shown to play a role in interference in an early study (
14). The importance of PAMs in the recognition and selection of precursor-spacers (prespacers) during adaptation was demonstrated unequivocally using assays that were independent of interference (
12,
21). The simple overexpression of Cas1 and Cas2, in the absence of other
cas genes, demonstrated these are the only Cas proteins essential for adaptation and are likely to recognize PAMs (
12).Adaptation consists of two related stages, termed “naïve” and “primed” (
9). Naïve adaptation occurs when a bacterium harboring a CRISPR-Cas system is infected by a new foreign element that it has not previously encountered. Although the acquisition of a new spacer can result in effective protection from the element, point mutations within the protospacer or PAM allow the element to escape CRISPR-Cas targeting (
14,
24,
25). This aspect had been viewed as a weakness of CRISPR-Cas interference, but recent studies show that a positive feedback loop—called priming—occurs, which enables one or more new spacers to be acquired (
11,
20,
22). Specifically, single mutations within either the PAM or the seed region of the protospacer, although inactive for interference, promote the rapid acquisition of new spacers from the same target (
11). Priming is proposed to allow an effective response against viral or plasmid escapees through the incorporation of new spacers. Unlike naïve adaptation, priming is more complex, and in type I-E systems requires Cas1, Cas2, crRNA, the targeting complex termed Cascade [CRISPR-associated complex for antiviral defence, composed of Cse1, Cse2, Cas7, Cas5, and Cas6e (
26–
28)] and the Cas3 nuclease/helicase (
11). Interestingly, the vast majority of spacers acquired through priming are derived from the same DNA strand as the original priming spacer (
11,
20,
22). In addition, priming in
E. coli was abolished by two mutations in the protospacer and PAM regions (
11).In this study, we generated a mutagenic variant library of a protospacer and PAM region and used both individual high-throughput plasmid-loss assays and next-generation sequencing to determine the limits of both direct interference and indirect interference through priming. Our results demonstrate that direct interference tolerates mutations mostly at very specific positions in the protospacer, whereas priming tolerates extensive mutation of the PAM and protospacer regions. The results have wide evolutionary consequences for primed acquisition and could explain the retention of multiple “older” spacers in CRISPR arrays.
相似文献