Characterisation of Chromosomal-targeting by the type of I-F CRISPR/Cas system in Pectobacterium atrosepticum

Abstract

Bacteria live in environments where they are constantly under attack by bacteriophage (phage) and are subjected to horizontal gene transfer (HGT) events, such as transfer of plasmids. Bacteria have developed many systems to limit and exclude phage infection and plasmid transfer. Clustered regularly interspaced short palindromic repeats or CRISPRs and their associated (Cas) proteins, constitute a recently discovered defense system in prokaryotes. CRISPR/Cas systems are capable of acquiring short spacer sequences from foreign genetic elements and incorporating these spacers into CRISPR arrays. Acquisition of spacers generates a memory bank of past encounters with foreign invaders that can be expressed as short CRISPR RNAs (crRNAs). These crRNAs guide the Cas proteins to the nucleic acids complementary to the spacer, resulting in the cleavage of the target.

While spacers are acquired from phage and plasmids, there are examples where spacers appear to target sequences of chromosomal origin. In this thesis the effects of CRISPR/Cas targeting of chromosomal sequences, using an engineered system are examined. The system was developed from the single CRISPR/Cas system of Pectobacterium atrosepticum. Additionally, this engineered system was adapted to show that the CRISPR/Cas system could destabilise plasmid maintenance, by interference with plasmid DNA.

The results of this thesis, demonstrate that genomic DNA was targeted by the Type I-F CRISPR/Cas system of P. atrosepticum. Chromosomal-targeting was toxic and results in a restricted growth phenotype and cellular filamentation. This restricted growth is presumably due to prolonged activity of cellular stress responses to the DNA damage, caused by CRISPR/Cas, leading to cell cycle arrest. The arrest of growth by the expression of host-complementary CRISPRs was shown in liquid media, and viable counts were reduced compared to controls. The toxic phenotype was abolished, by introducing mutations into the cas operon, the CRISPR repeat structures and the protospacer (target) sequences. Spontaneous mutants that abolished the toxic phenotype were also isolated, but the genotypes of these mutants remain uncharacterised.

In conclusion, the CRISPR/Cas system of P. atrosepticum is capable of targeting the DNA of its own genome. CRISPR/Cas self-targeting is toxic to the host and leads to CRISPR/Cas degeneration; therefore, self-targeting is presumably a flaw of CRISPR/Cas systems.