engleski

In vivo evidence that R-loops are formed for CRISPR antiviral defense in Escherichia coli

CRISPR (clustered regularly interspaced short palindromic repeats) and its associated Cas proteins is a recently discovered defence mechanism against invading genetic elements in many bacteria and archea. It consists of arrays of repeats separated by spacers which are homologous to sequences of phages and plasmids. The CRISPR system in Escherichia coli is composed of eight cas genes and a CRISPR locus. Cascade is a 405 kDa complex composed of five proteins and a 61 nt CRISPR RNA (crRNA) which is involved in target recognition by using crRNA as a guide. In that process, crRNA makes base pairs with complementary DNA strand while displacing noncomplementary strand to form R-loop. It has been proposed that Cas3 binds to these structures to destroy invading DNA sequences. However, it has been showed that Cas3 alone can catalyse R-loop formation in the absence of ATP, it exhibit helicase activity in the presence of ATP, but no nuclease activity has been observed. The majority of the research reported on CRISPR-cas immunity in E. coli has been done in vitro, or in E. coli where Cas proteins were co-expressed in high quantities and spacer was provided on a plasmid. In this work we used a strain with genomically located spacer against lambda phage and tested if we can prove a role of R-loop formation and a major role of cas3 and cascade genes in phage defense mechanism by using phage plating assay. The antiviral protection was efficient only on lower temperatures, and the strongest in hns mutant were cascade genes are not under repression. We show that Cas3 can substitute for the absence of Cascade complex in R-loop formation and vice versa as long as R-loops are stable and not attacked by RnaseH. In the absence of Cas3, Cascade proteins and RnaseH, antiviral mechanism is not functional and plaques appear. These results confirm that both Cas3 and Cascade proteins are able to form R-loops in vivo.

CRISPR/Cas; R-loop; lambda phage; E. coli; Rnase H

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