The Function of Small RNA-Based viral Defense System in E. coli - Renewal 1

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CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated genes) loci are present in almost all archaea and half of eubacteria. They protect prokaryotes from foreign genetic elements. While highly diverse, all CRISPR-Cas systems function through three common steps: 1) adaptation, i.e., acquisition of short foreign DNA sequences (spacers) into CRISPR arrays; 2) production of mature protective CRISPR RNAs (crRNAs), and 3) interference, when Cas nucleases guided by crRNAs destroy nucleic acids containing complementary targets. Studies of the interference part of CRISPR response have revolutionized the field of genomic editing. The less-studied adaptation part limits global horizontal gene transfer and can be harnessed for creation of DNA-based recording devices and control of the spread of antibiotic resistance genes. Initial CRISPR immunity is built in the course of “naïve”, non-discriminate acquisition of short intracellular DNA molecules – prespacers - as spacers into CRISPR arrays. It occurs very infrequently and can lead to suicidal self-interference. A remarkable mechanism called “priming” operates in type I CRISPR-Cas systems: acquisition of spacers from DNA with sequences matching pre-existing spacers is dramatically stimulated compared to naïve acquisition from DNA devoid of such sequences. Primed adaptation is highly beneficial to the host: it rapidly leads to specific acquisition of additional interference-proficient spacers from genetic parasites and ensures that no self-targeting spacers are selected. The mechanistic relationship between interference and adaptation during priming is not fully clear. The goal of this proposal is to dissect interrelationships between interference and adaptation during priming and to identify cellular processes that feed the adaptation machinery during naïve and primed adaptation. We will use FragSeq - an innovative high-throughput approach that identifies short intracellular DNA fragments and that was developed during the previous funding period - to determine the structure of prespacers and of other in vivo adaptation intermediates generated during naïve and primed adaptation in diverse CRISPR-Cas systems classes and types, and identify non-Cas cellular proteins essential for prespacer generation and spacer acquisition. The understanding of CRISPR adaptation that will result from our work will allow us and others to optimize the efficiency of the adaptation process, facilitating construction of strains with desired spacer content/immunity profiles and, by revealing processes that limit adaptation, may help control viability of bacterial populations by inducing adaptation from cell’s own DNA and self-interference.