High-Throughput Sequencing Reveals Spacer Integration and Loss of Clustered Regularly Interspaced Short Palindromic Repeats-Cas Subtype I-E System in E. coli

摘要

Clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated (Cas) proteins constitute an RNA-based adaptive immune system in prokaryotes. Insertion of short sequences into the CRISPR array is termed "spacer integration." The mechanism for this process has still remained unclear to date. Here in this study, new patterns of spacer integration and loss were identified in E. coli BL21Al with overexpression of Cas1-Cas2 complex. High-throughput sequencing analysis of the amplified products from the CRISPR array identified a series of spacer integration and loss events. Results showed that the proto-spacers abundance in the donor DNA was independent of the sequence length and the abundance of the proto-spacer adjacent motif (PAM) 5'-AAG-3'. Many spacers were preferred to target specific gene hotspots and CRISPR adaptation was promoted by active replication of the proto-spacer-containing DNA. Although only a minority of spacers was mapped on the E. coli genome, 4-fold neighboring spacers were mapped to different strands of the genome more than in the same strand. Some proto-spacers were found to have bi-directional acquisition, named as complementary spacer pairs. Moreover, regular deletions of the old repeat-spacer units occurred. These results also advanced our understanding of the adaptation stage of the CRISPR-Cas subtype I-E system in the E. coli and CRISPR evolution.