Output list
Thesis
Published 2021
The CRISPR-Cpf1 induces DNA double stranded breaks (DSBs) that need to be repaired by either homology-directed repair (HDR) or non-homologous end joining (NHEJ) repair mechanisms. Most prokaryotes lack the NHEJ repair system and repair DSBs by HDR when template DNA is provided. The human bacterial commensal Escherichia coli can repair DNA DSB damage by HDR or by the alternative method of microhomology mediated end joining (MMEJ). MMEJ can enable end resection after RecBCD exonuclease activity deletes DNA between microhomologous regions. This can result in the formation of small to undesirable large deletions between these microhomologies. Up until this point, a specific and precise gene knockout tool that does not induce extensive deletions has not been developed for use in E. coli or other NHEJ deficient bacteria. Hence, a novel strategy was developed in this study to induce DSBs by CRISPR-Cpf1, inhibit RecBCD exonuclease activity and enable repair by the expression of compatible plasmid-borne genes supplied in trans. This strategy required the development of a compatible plasmid system that could encode an arabinose inducible FnCpf1 effector from a chloramphenicol resistant p15A oriV replicon and a T7 RNA polymerase expressed crRNA (to guide FnCpf1 to its DNA target site) from a Cpf1 guide module residing on an ampicillin resistant promoterless ColE1 plasmid vector to induce DSB formation within E. coli BL21 DE3 lacZω. Surprisingly, the results revealed that the Cpf1 induction did not compromise cell viability in the induced compared to the uninduced state. In addition, a LacI de-repressible plasmid-borne multi-component NHEJ repair system containing the gam gene from phage λ (to repress the RecBCD complex), the Ku DNA end-binding protein from Mycobacterium smegmatis (to protect DSB ends from exonuclease attack) and the bacteriophage T4 DNA ligase gene (to promote ligation of DSB termini) was developed to enhance DSB end resection. The repair system was incorporated into the broad host range (BHR) plasmid pBBR1 which could be conjugally transferred into a wide variety of bacterial backgrounds. Further work needs to be conducted now to investigate if the constructed system can be used to effectively edit the genomes of not only E. coli but also other Gram-negative bacteria.