Transcription, processing and function of CRISPR cassettes in <i>Escherichia coli</i>

TLDR

CRISPR/Cas systems are bacterial and archaeal interference mechanisms composed of CRISPR cassettes with variable spacers and cas genes, producing small RNAs that can confer resistance when a spacer matches a viral genome, yet in Escherichia coli laboratory strains the system appears silent with no natural phage resistance or spacer matches to well‑studied phages. The study aimed to elucidate the silent E. coli CRISPR/Cas system by systematically characterizing processed transcripts from its cassettes. An engineered E.

Abstract

CRISPR/Cas, bacterial and archaeal systems of interference with foreign genetic elements such as viruses or plasmids, consist of DNA loci called CRISPR cassettes (a set of variable spacers regularly separated by palindromic repeats) and associated cas genes. When a CRISPR spacer sequence exactly matches a sequence in a viral genome, the cell can become resistant to the virus. The CRISPR/Cas systems function through small RNAs originating from longer CRISPR cassette transcripts. While laboratory strains of Escherichia coli contain a functional CRISPR/Cas system (as judged by appearance of phage resistance at conditions of artificial co-overexpression of Cas genes and a CRISPR cassette engineered to target a λ-phage), no natural phage resistance due to CRISPR system function was observed in this best-studied organism and no E. coli CRISPR spacer matches sequences of well-studied E. coli phages. To better understand the apparently 'silent'E. coli CRISPR/Cas system, we systematically characterized processed transcripts from CRISPR cassettes. Using an engineered strain with genomically located spacer matching phage λ we show that endogenous levels of CRISPR cassette and cas genes expression allow only weak protection against infection with the phage. However, derepression of the CRISPR/Cas system by disruption of the hns gene leads to high level of protection.