Browsing by Subject "CRISPR-Cas9"

Now showing 1 - 4 of 4
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    Computational methods to evaluate and interpret genome-wide perturbation screens
    (2021-08) Rahman, AHM Mahfuzur
    Genome-wide perturbation screens are a powerful tool to learn about biological systems. They allow us to systematically delete or mutate individual genes or combinations of genes, measure the impact of these perturbations, and learn how biological systems are functionally organized. This has been powerfully demonstrated in the model organism yeast, where all individual genes were knocked out, and the effect of gene deletion on yeast was methodically studied. In the last decade, our lab has been involved in multiple major efforts to knock out combinations of genes in yeast and showed its efficacy at uncovering functional relationships between genes. In the past few years, many different groups (including ours) have been undertaking similar efforts in human cells using pooled CRISPR/Cas9 screening approaches. These endeavors have produced a wealth of genome-wide perturbation datasets. In this dissertation, we focus on the development of computational methods to benchmark and interpret genome-scale perturbation datasets from yeast to humans. We begin with a discussion of methods for interpreting higher-order genetic interactions in yeast. We first extend this by exploring the impact of environmental perturbation on genetic interactions using 14 different chemical conditions. This study highlights the robustness of the global reference genetic interaction network in yeast, as the functional rewiring in the presence of changing environments is rare and less functionally informative. Next, we describe methods for scoring high-throughput trigenic interaction experiments in yeast. This method and associated software tool enables quantification of higher-order interactions involving triple mutant combinations and was used to map the first large-scale network of higher-order interactions in any species. The latter half of the thesis focuses on computational approaches for generating, scoring, and interpreting the results of genome-wide perturbation screens in human cells. One important aspect of this is to be able to systematically evaluate and compare different datasets and associated methods. To this end, we develop a method named FLEX to interpret functional information in a CRISPR screen dataset and systematically compare different competing datasets or methods. We employ FLEX on a genome-wide single knockout dataset, the DepMap data, and demonstrate that it reveals a major functional bias for mitochondrial genes, which we hypothesize is related to protein stability. Another focus of our work on methods for interpreting CRISPR screens is to develop approaches that can help to quantify the reproducibility of these perturbation screens. Specifically, we developed a method called JEDER that estimates error rates for CRISPR screens and establishes a way to evaluate replicated CRISPR screens without knowledge of any external gold standard. We highlight the importance of reporting relevant reproducibility metrics by demonstrating the increased difficulty in reproducing differential effects (e.g. genetic interactions) as compared to primary effects (e.g. single mutant fitness). Given accurate methods for scoring and quality control of CRISPR screens, these technologies can be applied to map large-scale genetic interaction maps for human cells. In the final chapter, this thesis describes the results of our computational analysis of the first genome-wide interaction network for human cells. We establish a set of genomic features that relate to gene essentiality and evaluate how different functional standards and genomic or proteomic data relate to different types of interactions. Finally, we summarize different functional neighborhoods and how well they are captured by the current genetic interaction map and suggest approaches to drive future interaction screening efforts.
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    Engineering Gene Expression in Plants with Programmable Transcriptional Activators
    (2022-05) Zinselmeier, Matthew
    Agriculture is a centuries old practice that has selected upon natural variation over time. Highly productive cultivars today are the result of this selection. DNA sequencing has revealed the genetic blueprint for many of these crop species, allowing for precise selection of variants for breeding. Crops must survive and reproduce efficiently to be utilized by humans for agriculture. To accomplish this, crops put the DNA blueprint into action through gene expression, allowing for development and survival in the face of stress. Thus, understanding and controlling gene expression will be important for engineering highly productive crops around the world. In nature, transcription factors (TFs) are responsible for regulating gene expression. TFs are comprised of a DNA binding domain and transcription regulatory domain. The DNA binding domain will bind to a region in the genome, while the regulatory domains interact with other proteins capable of either initiating or blocking transcription. As programmable nuclease technology like CRISPR-Cas9 was elucidated, Programmable Transcription Activators (PTAs) were developed to function as engineered transcription factors controlling gene expression. PTAs can be targeted anywhere in the genome to activate expression of a target gene promoter. PTAs can also activate the expression of multiple genes at once. In Chapter One the status of PTA technology is reviewed, with systems showing promise in plant backgrounds given consideration. To ultimately use PTAs for basic plant research, we first set out to optimize PTAs for efficient performance across plant species. The VP64 activation domain is the most frequently used activation domains regardless of PTA system. This domain is derived from a human herpes virus yet is still used in many plant systems. To address this we designed, built, and tested a library ofplant-derived activation domains for activity in plant cells, as described in Chapter Two. The AvrXa10, Dof1, and DREB2 ADs proved to be efficient across a variety of plant species. We also demonstrate the use of the DREB2 AD to activate distal enhancers in A. thaliana protoplasts, showcasing the versatility of plant-optimized PTAs. Finally, PTAs were used to engineer a circuit for genetic biocontainment as described in Chapter 3. In certain crop species, there is a desire to prevent gene flow between closely related individuals. A system was devised called Engineered Genetic Incompatibility (EGI) to prevent crop transgenes from escaping into closely related weedy populations. To engineer this system a gene is identified that is capable of producing lethality when overexpressed using PTAs. Gene editing is then performed to remove the PTA binding site to comprise the EGI organism; a PTA is expressed alongside the modified promoter within an individual to prevent self-targeting. Upon crossing with a wild-type individual, hybrid progeny will contain one copy of the PTA and one copy of the unedited promoter resulting in lethal overexpression. Our efforts to implement EGI in the model organism A. thaliana are described in detail including selection of target genes, testing sgRNA activity, generating transgenic lines, and promoter mutagenesis. The work described in this thesis illustrates the development and application of PTA technology in agriculture to engineer more productive crops through controlling gene expression.
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    Genetic Engineering of Primary Human Natural Killer (NK) Cells for Enhanced Cancer Immunotherapy
    (2021-06) Pomeroy, Emily
    Natural killer (NK) cells are a critical component of the innate immune system due to their ability to kill a variety of target cells, including cancer cells. This innate anti-tumor phenotype has driven intense interest in the use of NK cells for cancer immunotherapy, but this has seen limited success in the clinic. Enhancing NK cell cytotoxicity by augmenting activating signals or eliminating inhibitory signals could significantly improve NK-based cancer immunotherapy. We have developed highly efficient methods for editing the genome of human NK cells. Specifically, to target inhibitory signals for elimination, we have developed methods for CRISPR-Cas9-based gene knockout. We have also created platforms for delivery of activating signals using either CRISPR-Cas9 in combination with recombinant adeno-associated virus (rAAV) and a non-viral approach for engineering using DNA transposons. We targeted relevant genes (ADAM17 and PDCD1) for knockout and delivering activating receptors CD16a and a CD19-specific chimeric antigen receptor (CAR). Importantly, we show direct functional consequences of engineering steps, using preclinical in vitro and in vivo models. Furthermore, we demonstrate the clinical scalability of all methods. The focus of this work was to develop methods for engineering primary human NK cells, with the goal of creating clinical products to treat human disease. Future work will focus on combining approaches to generate NK cells expertly equipped to kill cancer.
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    Models and Gene Therapy for GM1-Gangliosidosis and Morquio Syndrome Type B
    (2018-12) Przybilla, Michael
    GM1-gangliosidosis and Morquio syndrome type B are lysosomal diseases caused by deficiencies in the lysosomal enzyme β-galactosidase (β-gal). β-gal is responsible for catabolizing the terminal β-linked galactose residues in GM1 and GA1 ganglioside, keratan sulfate, and oligosaccharides. If β-gal enzyme activity is deficient, these macromolecules accumulate within the lysosomes, resulting in either severe neurodegeneration in GM1-gangliosidosis or severe skeletal dysplasia in Morquio syndrome type B. Sadly, no therapies for these debilitating diseases exist, so the development of novel treatments is of the utmost importance; however, to be able to test these new treatments, animal models are necessary. Previous murine models of GM1-gangliosidosis were generated using an inefficient method to disrupt the Glb1 gene by introducing foreign DNA into the coding sequence. While useful, these mutations do not recapitulate those that could be found in patients with GM1-gangliosidosis. Utilizing CRISPR-Cas9 genome editing, the mouse β-gal encoding gene was targeted to generate mutations that resulted in two novel mouse models of β-gal deficiencies (Chapter II). In one line, a 20 bp deletion was generated to remove the catalytic nucleophile of the β-gal enzyme, resulting in a mouse devoid of β-gal enzyme activity (β-gal-/-). This resulted in ganglioside accumulation and severe cellular vacuolation throughout the central nervous system (CNS). β-gal-/- mice also displayed severe neuromotor and neurocognitive dysfunction, and as the disease progressed, the mice became emaciated and succumbed to the disease by 10 months of age (Chapter III). Overall, this model phenotypically resembles a patient with infantile GM1-gangliosidosis. In the second model, a missense mutation commonly found in patients with Morquio syndrome type B, GLB1W273L, was introduced into the mouse Glb1 gene (Glb1W274L). Mice harboring this mutation showed a significant reduction in β-gal enzyme activity (8.4-13.3% of wildtype) but displayed no marked phenotype after one year of observation (Chapter IV). This is the first description of using CRISPR-Cas9 genome editing to generate mouse models of a lysosomal disease. With these models in hand, preliminary experiments were conducted to test the functionality of a novel gene therapy to treat these diseases (Chapter V). Previous studies in lysosomal diseases have shown that tissue-specific expression of lysosomal enzyme ameliorates the disease pathology, including improvement of neurocognitive function. Here, a gene therapy system was designed to integrate the human GLB1 cDNA into the albumin locus by creating a double-strand break in the DNA by an AAV8-encoded nuclease. Theoretically, this integration of GLB1 cDNA would be achieved by co-injecting a second AAV8 vector encoding the transgene that is flanked by homologous sequence to the albumin locus, allowing for homology directed repair to incorporate the sequence. 30 days post treatment, plasma enzyme activity was 4.8-fold higher than heterozygous levels. However, by four months post-treatment, β-gal enzyme activity in plasma from treated β-gal-/- mice decreased to heterozygous levels. Four months following injection, β-gal enzyme activity in a subset of treated β-gal-/- mice was observed in the liver and spleen. Motor function testing on the rotarod showed that the amount of enzyme being produced does not prevent the neurological symptoms of the disease. This preliminary data shows that this gene therapy system can produce functional β-gal enzyme that is secreted into the plasma and is capable of being taken up into peripheral tissue. Future studies focused on optimizing the dose of AAV to provide a higher enzyme level will be important for the success of this therapy for β-galactosidase deficiencies.