The Investigation Of The Spacer Acquisition Mechnism Of CRISPR-Cas System In E.Coli

CRISPR-Cas system is a unique adaptive immune system. It provides immunity against phages and plasmids in most of the bacteria and nearly all of the archaea. CRISPR-Cas system acts in three stages. Stage 1:CR1SPR spacer acquisition, specific protospacers (with an adjacent protospacer-associated motif, PAM) of double-stranded DNA from a virus or plasmid was acquired by conserved Cas1/2 comlpex, and was then incorporated into the downstream of the first repeat at the leader end of a CRISPR array on the host DNA. Stage 2:Pre-CRISPR RNA (pre-crRNA) maturation, pre-crRNA was transcribed from the leader region and further cleaved into smaller mature crRNAs that contained a single spacer and apartial repeat. Stage 3:CRISPR interference, crRNA and Cas proteins constituted an effector complex which targeted the complementary incoming foreign nucleic acid (plasmid or virus) and initiated a sequence-specific cleavage and degradation. In recent years, the research on the mechanisms and functions of the CRISPR-Cas system have made breakthrough progress, and CRISPR interference machinery has provided an efficient tool for genome engineering in both prokaryote and eukaryote.Nevertheless, there are still some doubts about CRISPR spacer acquisition. In order to find the answer, we established an efficient spacer acquisition system in E. coli BL21AI and got the expanded CRISPR locus through PCR;then we combined the high-throughput sequencing and data analysis to characterize spacer integration and the impact on the the downstream sequences of subtype I-E CRISPR-Cas system. These observations facilitated our understanding about the mechanisms of spacer integration process in E. coli greatly.