DNA-Guided Genome Manipulation in Escherichia coli

Citation

Huang, Shan (2023) DNA-Guided Genome Manipulation in Escherichia coli. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/3yab-jg50. https://resolver.caltech.edu/CaltechTHESIS:08072022-205043055

Abstract

Argonaute proteins (Agos) were initially discovered in eukaryotes as key players in RNA interference (RNAi) pathways and later found in prokaryotes. Some prokaryotic argonautes (pAgos) have been shown to mediate nucleic acid-guided cleavage of DNA targets, reminiscent of the nucleic acid-guided DNase activity of the CRISPR/Cas9 system. It has been postulated that pAgo variants might be used as a novel genome-editing tool. However, genome manipulation induced by pAgo-mediated DNA cleavage has never been established. To demonstrate that pAgo-mediated DNA cleavage can introduce genomic mutations in Escherichia coli, we first created a recombination system and showed that CbAgo, a pAgo from Clostridium butyricum, can be directed by plasmid-encoded guide sequences to cleave the genome target site and induce chromosome recombination between downstream direct repeat sequences. Results from testing different pAgo variants suggest that the recombination rate correlates well with pAgo DNA cleavage activity, and the mechanistic study suggests the recombination involves DSB generation and RecBCD processing. In RecA-deficient E. coli strain, guide-directed CbAgo cleavage on chromosomes severely impairs cell growth, which can be utilized as counter-selection to assist Lambda-Red recombineering. These findings demonstrate the guide-directed cleavage of pAgo on the host genome is mutagenic and can lead to different outcomes according to the function of the host DNA repair machinery. Furthermore, we created a dCbAgo-based deaminase and showed that it can not only act as a random mutagen in vivo but also has the potential to be directed by plasmid-encoded guide sequences. We anticipate the novel DNA-guided interference by pAgo only or by its fusion protein to be useful in broader genetic manipulation. My work of engineering fluorescent protein-based nicotine biosensors via computational design and experimental evolution is also described in the thesis.

Thesis Files