Developing and characterizing phage-encoded homologous recombination machinery as a genome editing tool

Abstract

The ability to efficiently and precisely modify the genome of living organisms is one of the most important technological advances of this generation, and propels the accelerating progress of basic research, biotechnology, and medicine. Among the many tools developed to create genomic modifications, those that utilize homologous recombination are among the simplest and most versatile. However, homologous recombination using the endogenous machinery is inefficient in most cells. These methods become useful in practice through the addition of modified or exogenous components that dramatically increase homologous recombination efficiency. Functional sets of these additional components have not been found for many types of cells, leaving homologous recombination methods inefficient and genome editing laborious. A major recent branch within the field of homologous recombination uses DNA nicks or double-stranded DNA breaks to improve the efficiency of homologous recombination. However, this strategy alone is not sufficient to enable reliable or multiplexed editing in cells that inefficiently repair DNA nicks or breaks. Most prokaryotic species fall into this category, thus strain engineering technologies in many bacteria remain similar to those developed two decades ago. A different and promising conceptual approach for improving homologous recombination efficiency relies on the expression of exogenous DNA repair and recombination proteins, which can make up for the natural deficiencies of these hosts. The best example of this technology uses the Lambda-Red proteins from Lambda phage, which when exogenously expressed in E. coli, improves genome editing efficiencies to such a great extent that these proteins are often used alone for the majority of editing procedures (without DNA nicks or dsDNA breaks). However, this technology does not function in bacterial species beyond E. coli, preventing its widespread utility across prokaryotes. The major objective of the research in this thesis was to investigate the limited portability of the family of proteins called “single-stranded DNA annealing proteins” (SSAPs), which includes Lambda-Red Beta, the major component of Lambda-Red, in order to develop a more broadly useful genome editing method. In this thesis we identify the host single-stranded DNA binding protein, SSB, as a previously unknown host factor contributing to the limited portability of SSAPs, develop new homologous recombination technologies using SSAPs and SSBs with increased efficiency and portability, and develop efficient and multiplexed editing strategies to study complex phenotypes in bacteria including antibiotic resistance and strain fitness. Chapter One briefly describes the conceptual framework that shaped these research projects. Chapter Two reviews the limitations of alternative genome editing tools, and describes previous research related to expressing exogenous proteins to improve homologous recombination efficiency. Chapter Three describes the major work of the thesis, an investigation into the species-specific functionality of SSAPs, and the development of genome editing tools with increased portability. Chapter Four describes an application of SSAPs for fitness optimization and identification of key causal alleles contributing to a complex phenotype. Chapter Five discusses future opportunities for research within this field, and outstanding challenges that - if addressed - have the potential to result in a new generation of genome editing tools.