Significance of the Nuclear Gene RAD54 in Mitochondrial Genome Stability of Saccharomyces cerevisiae

Mitochondria are essential organelles in eukaryotic organisms that synthesize the energy-providing molecule, ATP, through the process of oxidative phosphorylation. As explained by the endosymbiotic theory, mitochondria contain mitochondrial DNA (mtDNA), distinct from nuclear DNA (nDNA). When mitochondrial function is impaired, and mtDNA stability is compromised, detrimental neuromuscular and neurodegenerative disorders such as Mitochondrial Encephalomyopathy, Lactic acidosis and Stroke-like episodes (MELAS) and Leber’s Hereditary Optic Neuropathy (LHON) have the potential to occur. The purpose of this study was to determine the role of the nuclear gene RAD54 in maintaining mtDNA stability in the budding yeast, Saccharomyces cerevisiae. Although the role of Rad54p in maintaining nDNA stability is understood, its impact on mtDNA stability is relatively unknown. RAD54 is a member of the RAD52 epistasis group, coding for a protein vital to the initial steps of homologous recombination and double-stranded break (DSB) repair. Given that members of the RAD52 epistasis group have been shown to contribute to homologous recombination and DSB repair in mtDNA of S. cerevisiae, we hypothesized that loss-of-function RAD54 would decrease the rate at which homologous recombination in mtDNA occurred (Stein, Kalifa & Sia, 2015). A phenotypic respiration loss assay was performed in a rad54Δ strain to determine the frequency of spontaneous mutations in mtDNA that blocked the oxidative phosphorylation process. The mutant strain demonstrated a 1.56-fold decrease in spontaneous respiration loss when compared to wild type (p-value = 0.0574). Interestingly, previous research has demonstrated that the nature of these spontaneous mutations is due to large deletions in the mtDNA. To investigate the role of Rad54p in preventing these deletions from occurring, a direct repeat-mediated deletion (DRMD) assay was performed. The DRMD assay demonstrated a significant 3.23-fold increase in nDNA homologous recombination events (p-value = 0.0158) and a statistically insignificant 1.08-fold increase in mtDNA homologous recombination events (p-value = 0.8741) between rad54Δ and wild type strains. Given the present findings of this study, it appears the nuclear gene RAD54 does not play a significant role in maintaining mtDNA stability in respiration loss or DRMD assays

Lack of RNA-DNA oligonucleotide (chimeraplast) mutagenic activity in mouse embryos

There are numerous reports of the use of RNA-DNA oligonucleoticles (chimeraplasts) to correct point mutations in vitro and in vivo, including the human apolipoprotein E gene (ApoE). Despite the absence of selection for targeting, high efficiency conversion has been reported. Although mainly used to revert deleterious mutations for gene therapy applications, successful use of this approach would have the potential to greatly facilitate the production of defined mutations in mice and other species. We have attempted to create a point mutation in the mouse ApoE gene by microinjection of chimeraplast into the pronuclei of 1-cell mouse eggs. Following transfer of microinjected eggs we analysed 139 E12.5 embryos, but obtained no evidence for successful conversion. (c) 2005 Wiley-Liss, Inc

Engineering yeast genomes and populations

The field of synthetic biology seeks to use design principles of life to create new genes, organisms and populations to both better understand biology as well as generate species with useful properties. Budding yeast has been a workhorse for synthetic biology, as well as an important model organism in the broader fields of molecular biology and genetics. This thesis aimed to create genome engineering tools for the manipulation of genomes, with direct applications in yeast. I focused developing high-throughput and highly efficient methods for making genomic modifications in yeast to allow for the generation of large libraries of precisely modified yeast genomes. By manipulation of endogenous DNA recombinases and mismatch repair enzymes in yeast, we were able to develop an oligonucleotide only method for genome engineering to generate libraries as large as 10^5 individuals with a frequency of modification as high as 1%. Additionally, we validated the use of RNA-guided CRISPR/Cas9 endonucleases to make changes in yeast genomes, resulting in frequencies of genome modification >90% in transformed populations. We further optimized this method to generate larger libraries as high as 10^5 individuals and explored a proof of concept epistasis experiment involving thermotolerance. Lastly, the propagation of changes to successive generations is useful when engineering organisms on the population level. To this end we explored the use of RNA-guided gene drives to bias inheritance in S. cerevisiae. We show that inheritance of these selfish elements can be biased to over 99% and is reversible

Optimizing the Design of Oligonucleotides for Homology Directed Gene Targeting

BACKGROUND: Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit. METHODOLOGY/PRINCIPAL FINDINGS: In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA) polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex. CONCLUSION AND SIGNIFICANCE: A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases) in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA

Toward Multiplex Genome Engineering in Mammalian Cells

Given the explosion in human genetic data, new high-throughput genetic methods are necessary for studying variants and elucidating their role in human disease. In Chapter I, I will expand on this concept and describe current methods for genetically modifying human cells. In E. coli, Multiplex Automatable Genome Engineering (MAGE) is a powerful tool that enables the targeting of multiple genomic loci simultaneously with synthetic oligos that are recombined at high frequencies in an optimized strain. MAGE as a method has two components: organism-specific optimization of oligo recombination parameters and a protein capable of increasing recombination frequencies

Transgenic approaches for the investigation of putative airway stem cells as potential targets for gene correction therapy

Since the discovery of the CFTR gene over a decade ago, Cystic Fibrosis (CF) has been regarded as amenable to intervention by gene therapy. The ultimate aim of gene therapy must be the correction, within cells capable of repopulating the tissue, of the genetic defect in its chromosomal context. Towards that end, a mouse model designed to evaluate the efficiency of gene correction was created, and a transgenic approach was taken to the investigation of a putative progenitor cell population in the adult murine respiratory tract. Before gene correction systems can be considered as valid therapeutic agents, their utility in the cells and tissues of living animals must be demonstrated. Thus, an in vivo system permitting the simple quantification of correction frequency in a wide range of tissues would be a valuable resource for the gene correction community. The generation and analysis of a transgenic mouse carrying an inactivated, but potentially correctable, reporter transgene is described. The full potential of a gene correction strategy to provide a single-dose, permanent solution to a genetically-diseased tissue will only be realised once the therapy is able to target resident stem cells. For CF lung disease, this will require the prior identification of stem cells in the respiratory epithelium. Previous work has indicated that potential stem cells are spatially coincident with small groups of cells expressing high levels of keratin 5 (K5) protein in the proximal murine trachea. In order to investigate lineage arising from this putative stem cell niche, transgenic mice have been generated which express an inducible form of Cre recombinase from the K5 promoter. Preliminary experiments demonstrate recombination of a conditional reporter gene after induction of Cre activity in K5-expressing tissue. Comparison of the inducible system with a constitutive K5 promoter-driven Cre line validated the choice of the former, as the clarity of data obtained from the conventional system was undermined as a result of K5 promoter activity causing reporter gene activation prior to the onset ofthe experiment. In the course of these studies it became evident that the conventional, constitutive Cre line gave rise to segregating patterns of reporter gene activation. While some mice displayed the expected K5-derived expression profile, other animals demonstrated ubiquitous expression. Universal activation of the conditional reporter was detected only in animals derived from females carrying the Cre transgene, and was found to be the result of unanticipated production of Cre protein in the maternal germline. This transgenic line is unusual and valuable in offering a choice of tissuespecific and generalised recombination of floxed alleles