Rapid technological breakthroughs originating from fundamental microbiology research have yielded novel genome engineering tools that greatly facilitate our ability to modify specific genomic... Show moreRapid technological breakthroughs originating from fundamental microbiology research have yielded novel genome engineering tools that greatly facilitate our ability to modify specific genomic sequences in living cells and organisms. As a corollary, these technologies are also starting to permeate the realm of medicine when applied as a form of “genomic surgery”. These genetic therapies aim at tacking the root cause of human pathologies, inherited or acquired, by correcting or modulating the genetic content or expression, respectively, present in target cells, tissues and organs. To this end, delivery vehicles capable of introducing, in an efficient and safe manner, the increasingly sophisticated (epi)genome editing reagents are in demand, especially when considering in vivo genetic therapies. The research presented in this thesis reveals the feasibility and utility of using regular and high-specificity nicking RGNs for achieving efficient and accurate genetic modification of human cells involving targeted gene knockouts and knock-ins. Moreover, it establishes the suitability of the HC-AdV platform for the versatile investigation of prime editing systems. Finally, this thesis establishes causal relationships between specific chromatin states and the activities and fidelities attained by base editing and prime editing complexes in human cells, which has consequences for their further development and optimal deployment. Show less
Complexity is not necessarily expected from monogenic diseases but for many cardiovascular diseases (CVD), simple genotype-phenotype relationship may be far from reality. As millions of people... Show moreComplexity is not necessarily expected from monogenic diseases but for many cardiovascular diseases (CVD), simple genotype-phenotype relationship may be far from reality. As millions of people globally die of CVD, it is important to find models to study CVD that recapitulate the conditions as manifest in humans, most importantly for these cases of unexpected complexity. For these, simple gene mutation or deletion in mice has often failed. Combining human induced pluripotent stem cell (hiPSC) with genetic editing technologies is providing new opportunities to bridge the gap, with many hPSC-CM models now showing promising results for testing drugs, discovering molecular pathways associated with disease and other types of (gene) therapies. The work in this thesis contributes to this area of research. Show less
The past two decades have seen the growing development and consequent vast application of next-generation genome editing tools in fundamental and applied research. Nowadays GE based on RNA-guided... Show moreThe past two decades have seen the growing development and consequent vast application of next-generation genome editing tools in fundamental and applied research. Nowadays GE based on RNA-guided nucleases (e.g., engineered CRISPR-Cas9 nucleases) are the most common tools for targeted genetic modification. Nevertheless, these technologies are in need of increased efficiency and accuracy, especially looking forward to translation into diverse clinical applications. The work presented in this thesis focuses on improving the efficiency and accuracy of genome editing, particularly in cells with high therapeutic potential, such as induced pluripotent stem cells (iPSCs), by investigating the feasibility of using adenoviral vectors to test novel genome editing approaches and by exploring the possible applications of a scarless strategy. Show less
This research delves into the utilization of CRISPR-Cas tools for advanced genome editing. It unveils a novel method known as Ligation-Assisted Homologous Recombination (LAHR), which achieves... Show moreThis research delves into the utilization of CRISPR-Cas tools for advanced genome editing. It unveils a novel method known as Ligation-Assisted Homologous Recombination (LAHR), which achieves efficient editing and serves as a robust complement to the established Cas9-induced HDR techniques. The Prime Editor (PE) technology is scrutinized, with a focus on the issue of low editing efficiency. To counter this challenge, an improved variant, PE plus (PE+), is introduced, demonstrating a marked increase in editing efficiency. The study further presents a unique cell selection tool using LbuCas13a, which exhibits widespread RNA cleavage in human cells, hinting at its potential use in cancer therapy. The concluding section foresees a transition in genome editing tools from being DNA repair-dependent to -independent, while also addressing pivotal challenges such as off-target editing and the formulation of effective delivery strategies. Show less
Increasing the efficiency of gene targeting (GT) as a genome editing tool in plants has been an important goal in plant biotechnology. Improvements have been made using sequence-specific nucleases... Show moreIncreasing the efficiency of gene targeting (GT) as a genome editing tool in plants has been an important goal in plant biotechnology. Improvements have been made using sequence-specific nucleases such as CRISPR/Cas9 to induce DNA double strand breaks at target loci and activate repair via homologous recombination (HR). GT can then be achieved by HR-mediated integration of an artificial repair template, sharing homology with the target locus. Further improvements have been made with the in planta GT method, in which the repair template is pre-inserted in the genome and can be excised by nucleases. Although these improvements led to substantial increases in GT efficiency, GT is still not efficient enough to be feasible for crop biotechnology. This thesis describes strategies to further improve GT efficiency in the model plant Arabidopsis thaliana. One of these strategies was to perform in planta GT in meiocytes, cells that already have a higher rate of HR. Another strategy was to find new Arabidopsis mutants with increased GT frequencies and to identify genes involved in this phenotype. In the end, this may lead to a better understanding of the mechanisms underlying GT and these may be used to realize higher GT frequencies in plants. Show less
By investigating the interaction between different types of nucleases (i.e. nicking versus cleaving), donor DNA structures and target chromatin environments, this thesis provides important insights... Show moreBy investigating the interaction between different types of nucleases (i.e. nicking versus cleaving), donor DNA structures and target chromatin environments, this thesis provides important insights into how to improve the three crucial parameters of genome editing: efficiency, specificity and fidelity. The work presented in this thesis expand the range of possibilities for high-fidelity genetic manipulation of human cells. Show less
Accurate and efficient genome editing is primarily dependent on the generation of a sequence-specific, genomic double-stranded DNA break (DSB) combined with the introduction of an exogenous DNA... Show moreAccurate and efficient genome editing is primarily dependent on the generation of a sequence-specific, genomic double-stranded DNA break (DSB) combined with the introduction of an exogenous DNA template into target cells. The exogenous template, called donor DNA, normally contains the foreign sequences flanked by DNA regions sharing sequence identity ("homologous") to those bracketing the target site. The strategies for mediating the formation of DSBs at the predefined genomic loci, have been undergoing intense investigation since the introduction of sequence-customizable zinc-finger nuclease (ZFN) technology. More recently, prokaryotic protein-based transcription activator-like effector nucleases (TALENs) and RNA-guided nucleases (RGNs) derived from CRISPR-associated protein (Cas9) complexes have substantially broadened the availability and applicability of designer nuclease-mediated genome editing. A potential alternative research line to the use of designer nucleases, is to investigate whether specific DNA structures can, by themselves, serve as triggers of the DNA damage response and, in doing so, elicit targeted gene repair. Such an approach would simplify genome editing protocols, such as, by reducing the number of reagents needed to be introduced into target cells. In this thesis, the roles of these secondary structures as well as designer nucleases and donor-DNA templates, delivered via adenoviral vectors, is described. Show less
