Degenerate target sites mediate rapid primed CRISPR adaptation

Abstract

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 CRISPRCas 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 Our findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the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.adaptive immunity | phage resistance | crRNA | next-generation sequencing | horizontal gene transfer

Highly efficient primed spacer acquisition from targets destroyed by the Escherichia coli type I-E CRISPR-Cas interfering complex

“…In fact, when matched and mismatched protospacers are positioned on the same DNA molecule, primed adaptation initiated by recognition of a fully matching target is about 10 times more efficient than from a mismatched target. In contrast, when tested separately, mismatched rather than matched targets behave as preferred substrates for primed adaptation (18,19). We propose that the apparent lack of adaptation from target DNA molecules with protospacers fully matching crRNA spacers is a consequence of rapid destruction of such targets.…”

“…Under pressure from CRISPR-Cas, mobile genetic elements accumulate such mutations, which allows them to escape CRISPR interference (8,17). For several type I systems, spacer acquisition from DNA molecules containing "escape" protospacers is very strongly stimulated (18)(19)(20)(21)(22) compared with "naïve" adaptation, which requires just Cas1 and Cas2. In the case of the E. coli type I CRISPR-Cas system, this specific version of spacer acquisition, referred to as "primed adaptation" (18), requires not just Cas1 and Cas2 but also all other components of the effector Cascade complex (Cse1, Cse2, Cas7, Cas5, Cas6e, and crRNA) and the Cas3 nuclease.…”

Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation

“…In the Type I-E system of E. coli, Cas1 and Cas2 form a complex that binds, the protospacer (Kunne et al, 2014;Semenova et al, 2011;Wiedenheft et al, 2011;Xue et al, 2015). Priming 93 on the other hand is an extremely robust process capable of dealing with highly mutated targets with up to 13 94 mutations.…”

“…Timing of plasmid loss and spacer acquisition reveals distinct underlying processes 98 In order to find the molecular explanation for why some mutants with equal numbers of mutations show 99 priming while others do not, we performed detailed analysis of a selected set of target mutants obtained 100 previously (Fineran et al, 2014). From the available list we chose the bona fide target (WT) and 30 mutants 101 carrying an interference permissive PAM (i.e.…”

CRISPR-Cas Systems in Prokaryotes

“…It is thought that the role of primed spacer acquisition is to counter possible evasion. The hypothesis that the effector complex slides across the target DNA was proposed to explain the mechanism of primed acquisition (Datsenko et al, 2012;Fineran et al, 2014;Li et al, 2014). This hypothesis states that, after a failed interference attempt, the effector complex recruits Cas1 and Cas2, and then slides along the target strand to find suitable PAMs that enable the acquisition of new spacers.…”