Degenerate target sites mediate rapid primed CRISPR adaptation.
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 Our findings imply that even outdated spacers containing many mismatches can induce a rapid primed CRISPR response against diversified or related invaders, giving microbes an advantage in the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.
Laboratory of Microbiology and Laboratory of Systems and Synthetic Biology, Wageningen University, 6703 HB Wageningen, The Netherlands.
Peter C
PC
Fineran
Matthias J H
MJ
Gerritzen
María
M
Suárez-Diez
Tim
T
Künne
Jos
J
Boekhorst
Sacha A F T
SA
van Hijum
Raymond H J
RH
Staals
Stan J J
SJ
Brouns
D020137:Base Pair Mismatch / Q000235:genetics; D001483:Base Sequence; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D030541:Databases, Genetic; D048168:Escherichia coli K12 / Q000235:genetics* / Q000276:immunology*; D057166:High-Throughput Screening Assays; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D059372:Nucleotide Motifs / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics
D012329:RNA, Bacterial
adaptive immunity; crRNA; horizontal gene transfer; next-generation sequencing; phage resistance