Browsing by Subject "CRISPR"

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Cationic Polymers and Polymeric Micelles as Plasmid DNA and CRISPR-Cas9 Ribonucleoprotein Delivery Vehicles
(2019-11) Tan, Zhe
Millions of people are currently suffering from genetic diseases and disorders worldwide and the traditional protein-based treatments are both expensive and require repetitive injections to maintain long-term effects. Gene therapy, as an alternative, holds great potential by direct delivery of genetic materials such as nucleic acids and genome editing machineries into human body to achieve long-term therapeutic protein expression, malfunctioning gene silencing, and native gene alternation. As a crucial step towards gene therapy, the delivery of genetic materials remains a major challenge, and affordable, efficient, and well-defined delivery vehicles are urgently needed. Synthetic polymers have been explored as plasmid DNA delivery vehicles for decades owing to their low production cost, chemical flexibility, low immunotoxicity, and the ability to encapsulate biomacromolecules. However, the precise control of polymeric vehicle properties by structure-tuning is a general challenge. Hence, the fundamental understanding of polymeric vehicle’s structure-property relationships is of great importance. In addition, polymers as well-documented nucleic acid delivery vehicles, are largely underexplored for their ability to encapsulate, stabilize and deliver RNA-protein conjugates, such as CRISPR/Cas9 ribonucleoprotein, a recently emerged versatile genome editing tool, presumably due to the inherent structural differences between long, semiflexible DNA and globular RNA-protein payload. Herein, several classes of polymeric delivery vehicles were synthesized and investigated, namely, cationic linear polymers, block copolymers, and polymeric micelles. Initially, a systematic comparison of linear polymers and micelles were performed, and vehicle architecture were shown to largely affect DNA complexation ability, complex physical properties, and biological performance. Later on, polymeric micelles were explored as well-defined CRISPR/Cas9 ribonucleoprotein delivery vehicles, and the solvent condition was found to be a key factor that affect particle complexation and gene-editing efficiency. Finally, polymers with liver-targeting ability and cellular membrane-penetration property have been developed and studied for their gene delivery efficiency and cytotoxicity.
Development of RNA Viral Vectors for Plant Genome Engineering
(2022-02) Ellison, Evan
Genetic variation is a key principle in the improvement of agricultural crops. For thousands of years, crop productivity, resilience and adaptability has been slowly improved by selection of favorable alleles. An increasing understanding of molecular genetics underlying key traits has contributed to the continuing progress in crop development. Dissecting and exploiting plant molecular genetics is greatly enhanced by the ability to precisely create genetic variation at pre-selected sites. Creating novel genetic variation through gene editing is reliant on technology that creates the desired modification at the target site and delivery of the reagents to plant cells. RNA guided endonucleases, such as CRISPR/Cas9, have enabled an unprecedented ability for site-specific genetic modification. Delivery of reagents, however, is still largely reliant on tissue culture regeneration to fix targeted genetic modifications in the genome. Tissue culture regeneration is a technically difficult process that can easily take several months or years to complete. The work described here outlines approaches to deliver genome editing reagents using RNA viruses. Chapter 1 discusses background information related to plant viruses and viral vectors. Chapter 2 describes a collaborative effort to develop a novel gene editing reagent delivery vector, Foxtail Mosaic Virus (FoMV), and its application in gene editing of monocot and dicot plant species. Genetic modifications obtained in chapter 2 only occurred in somatic cells, and still require laborious tissue-culture to fix in the germline. Chapter 3 describes my approaches to improve Tobacco Rattle Virus (TRV) for highly efficient heritable genome editing in the model species Nicotiana benthamiana. This work was improved further in chapter 4 by using viral vectors and an improved method for heritable genome editing in Solanum lycopersicum (tomato). Together, these improvements to RNA viral vectors provide an efficient and rapid means for delivery of genome engineering reagents. The time to generate targeted modifications that are fixed in the genome, for both model and crop species, is reduced from years to only a few months which enables genome editing at scale. The ease at which targeted genetic modifications can now be generated will enable important progress in crop improvement.
An E. coli cell-free transcription- translation system: modeling gene expression and characterizing CRISPR elements and gene circuits
(2019-09) Marshall, Ryan
Cell-free transcription-translation systems are versatile tools for rapid prototyping and characterization of biological systems and processes. Proteins can be expressed and measured in a matter of hours, whereas in vivo experiments often take days to weeks because they require protein purification or live cell transformations and cultures. TXTL systems, however, are still lacking in simple models that quantitatively describe the behavior of reactions. Here, we present an model of the all E. coli TXTL system using ordinary differential equations, encompassing the limited concentrations of transcription and translation machineries, capturing the linear and saturated regime of gene expression. Many biochemical constants are determined through experimental assays. We then show how this TXTL system was used to characterize CRISPR technologies. CRISPR-Cas systems have huge potential to be used as tools for genome engineering, as well as gene silencing and regulation. We characterize a set of sgRNAs, CRISPR nucleases, anti- CRISPR proteins, and determine protospacer-adjacent motifs. Finally, we use the TXTL system to execute gene circuits, including an IFFL and an integral controller.
Eeffective pDNA and CRISPR RNP delivery promoted by design of cationic bottlebrush and combinatorial polymers synthesis
(2022-07) Dalal, Rishad
The field of gene therapy has grown in response of the millions of people who suffer from genetic diseases worldwide. As genetic payloads need a delivery carrier, cationic polymeric vectors have grown in promise as delivery vehicles that are more cost-effective, scalable, and stable in comparison to viral vectors. The field of polymeric gene delivery has focused on improving delivery efficiency through chemical and structural modifications. Herein, we have made steps towards understanding how architectural modifications and how structure property relationships can improve the field of gene delivery. Initially it was found that when comparing cationic homopolymers, a bottlebrush architecture outperformed a linear analog in over pDNA delivery efficacy. Follow-up studies explored how bottlebrush end-group hydrophilicity can play a role in balancing colloidal stability, gene expression, and cellular viability. In addition to architectural understanding, studies to understand how structure-property relationships within linear polymers were explored in which a combinatorial library of 36 polymers was synthesized and used to deliver pDNA and CRISPR-Cas9 RNP. Machine learning aided in optimizing and analyzing structural relationships relative to expression outputs. Overall, we were able to create guides in improving gene expression through the optimization of polymer macromolecular structure and unique chemical understanding per biological payload.
Engineered immune cells to treat enzymopathies and cancers
(2022-08) Laoharawee, Kanut
The ability to edit the genome of human cells has potentially countless applications in advanced medicine. There are currently two major genome engineering methods: non-targeted genome engineering and targeted genome engineering. Non-targeted genome engineering comprises of viral and non-viral tools. Targeted genome engineering comprises of Zinc-Finger Nucleases, TALENs, and CRISPR-based tools. Inarguably, these tools provide new avenues to create therapies to treat diseases such as enzymopathies and cancers.We have developed methods for editing the genome of human B cells and T cells. Specifically, we precisely inserted a therapeutic transgene into these cells by developing methods for CRISPR-Cas9 along with recombinant adeno-associated virus (rAAV) to mediate insertion of α-L-iduronidase (IDUA). Furthermore, we tested the efficacy of the engineered B and T cells to express IDUA enzyme to treat Hurler syndrome, a form of enzymopathy. We demonstrate that successful engraftment of the engineered cells in a mouse model of Hurler syndrome shows direct positive outcomes and is superior to current standard treatments. Importantly, this platform has potential use not only for Hurler syndrome or enzymopathies, but also other diseases. We also developed a method for editing the genome of human monocytes. Specifically, we developed a highly efficient method that allowed CRISPR-Cas-based in the form of mRNA to edit the genome of monocytes by deploying a pan-RNase inhibitor along with CRISPR-based mRNA with gRNA. We have also created a platform for delivery of CRISPR-Cas9 in combination with rAAV to insert a transgene into monocytes. Importantly, we targeted SIRPA gene for knockout and showed enhanced functional properties in the engineered monocytes against CD47-expressing cancer cells in vitro. The focus of this work was to develop methods for engineering primary human immune cells with the goal of creating clinical products to treat enzymopathies and cancers. Future work will focus on further optimizations to improve efficacy of the engineered methods and treatments.
Improved Base Editing Technologies With Novel Editors and Assays
(2018-10) St. Martin, Amber
Genome engineering is a rapidly evolving area of study. One driver of the breakneck speed with which the field is moving forward is the application of CRISPR/Cas9. Since its introduction in 2013, CRISPR/Cas9 has completely changed the ease and utility of genome engineering and has revolutionized the field. The use of CRISPR/Cas9 to directly edit genes, increase or decrease gene expression, or even image genomic loci is widely accepted and extensively used in models ranging from bacteria to mammals. The recent development of second-generation CRISPR editing tools has opened even more doors into how the human genome can be manipulated. Past methods to introduce a single-base substitution into genomes involved creating a double-strand break and taking advantage of the cellular repair pathway homologous recombination to incorporate a donor plasmid into the genomic sequence. These methods are inefficient and can result in introduction of the donor template into numerous unrelated loci throughout the genome, unwanted insertions or deletions, or chromosomal translocations. Base editing unlocks a method to introduce single-base substitutions without the need for a donor template or the creation of a double-strand break. By fusing rat APOBEC1, a natural cytosine deaminase, to Cas9 nickase and uracil DNA glycosylase inhibitor, the Liu lab was the first to create an RNA-guided base editor that can change target cytosines to thymines. In just a year and a half, significant improvements have been made on this front, including making the original base editor more efficient and specific and even introducing editors with the power to mutate adenosines to guanosines. Despite the advancements constantly being made to this technology, there is still room for improvement. The current base editors such as BE3 can edit unintended cytosines in the target sequence at high rates. In addition, the combination of the nickase activity of Cas9 and the abasic site created after deamination of the target cytosine can create a pseudo double-strand break resulting in creation of unwanted insertions or deletions. Finally, targeting at endogenous loci continues to hover between 30% and 80%, depending on the method used and model genome being targeted. Targeting rates could benefit from growth, especially as this technology is being considered for therapeutic applications. A bottleneck in the process of developing new and improved base editing technology is the time and effort that is required to quantify editing efficiencies. Most studies use next-generation sequencing to quantify editing rates. The preparation of samples and quality control required can take up to six weeks. If using a core at a larger university or research institution there is additional time spent waiting in a queue to use a sequencing instrument. The field is in need of a rapid method to quantify base editing in real time that is transferable to multiple cellular systems. Here I report two bicistronic, fluorescence-based systems for the quantification of base editing activity. By changing a 5’-TT-3’ dinucleotide motif to a 5’-TC-3’ dinucleotide motif in eGFP or mCherry, I simultaneously ablate fluorescence and create an APOBEC-preferred mutational hotspot. When the cytosine is reverted back to a thymine, fluorescence is restored. This tight off-to-on system allows for real-time quantification of base editing activity through fluorescence microscopy or flow-cytometry. After creating a novel base editing reporter system, I hypothesized that I could use the newly designed assay to create more efficient and specific base editors. Using members of the human APOBEC3 family of enzymes, I created a suite of novel base editors. These base editors have advantages over rat APOBEC1-based editors in that the structure of many APOBEC3s are known, allowing for easier structure-guided evolution to improve their editing activity. As a proof of concept, I used this knowledge to evolve APOBEC3H haplotype II into a more efficient base editor by making only a few amino acid substitution mutations. In addition, we were able to create base editors using APOBEC3A and the catalytic domain of APOBEC3B that surpass BE3 in editing efficiency. Taken together, these data contribute positively to the genome engineering field and open new doors for continuing development of this technology.
Insulin signals through IGF-IR in insulin receptor knockout breast cancer cell line
(2020-07) Monteiro, Marvis
Breast cancer is a common malignancy observed more in females than in males. In breast cancer there is an upregulation of the IGF system. Upregulation of insulin and InsR are associated with poor patient prognosis. In order to understand the role of InsR in breast cancer biology, an InsR knockout cell line was created from MCF-7L breast cancer cells. Clone 35 showed loss of InsR expression, despite this loss, clone 35 showed activation of p-Akt and p-MAPK on stimulation with insulin. The hypothesis was developed that in the InsR knockout cell line insulin bound to IGF-IR and activated signaling. This hypothesis was proven by developing a knockout model, then using InsR and IGF-IR specific inhibitors on clone 35 to suggest the involvement of IGF-IR in activation through insulin. The following research substantiate the claims and provide a new understanding in the role of InsR and IGF-IR in breast cancer biology.
Methods of Enhancing Triterpenoid Production in Yeast
(2023-07) Scott, Samuel
Microbial cell factories, particularly those using eukaryotic yeasts, are ideal platforms for producing plant secondary metabolites, including flavonoids, alkaloids, and terpenoids. Accordingly, this study aimed to increase triterpenoid production in the Saccharomyces cerevisiae strain BY4743 through CRISPR/Cas9 and cultivation engineering. The ROX1, DGK1, and PAH1 genes were targeted for knockout experiments. Sanger sequencing showed all three targets were successfully mutated; however, only the DGK1 knockout strain had a significant change in triterpenoid production at 130% compared to the wild-type. Various cultivation strategies were also explored, but none increased triterpenoid production significantly. Additionally, to illustrate the potential applications of engineered yeast, five uncharacterized oxidosqualene cyclases (OSCs) from Erysimum cheiranthioides were tested in the ROX1 knockout strain, revealing one responsible for producing the steroid core of medicinal cardenolides. In summary, this thesis provides engineered yeast strains with improved MVA pathway derivative production potential and comprehensive CRISPR/Cas9 methodologies for S. cerevisiae.
Novel precise genome editing technologies
(2020-09) Aird, Eric
Genome engineering, the ability to manipulate and modify genomes, has become a standard tool in life sciences and beyond. Programmable nucleases such as CRISPR-Cas9 have afforded the ability to target particular regions in genomes to make targeted changes. While genome editing technologies continue to flourish, the ability for CRISPR-Cas9 to deliver precise genomic modifications is in part hindered by the lower efficiencies of homology-directed repair (HDR). Additionally, delivery of these genome modifying reagents is hampered by current technological constraints. This dissertation describes our unique approaches to developing tools to improve both of these aspects of genome engineering. HUH endonucleases are a family of ssDNA binding proteins that bind sequence specifically to its target and have been co-opted for biotechnological purposes. We developed a Cas9-HUH fusion that increases precise editing outcomes on the order of 2- to 3-fold through covalent tethering of the HDR template. We also demonstrate application of this platform in concert with the development of a molecular tension sensor based on bioluminescent energy transfer (BRET). Next, we pivot to improving delivery of genome editors utilizing adeno-associated virus (AAV). We describe efforts to specifically target AAV to explicit cell types of interest via HUH-antibody selectivity. Building upon this method, we present a novel iteration of a next generation CRISPR-Cas9 based technology termed prime editor. We developed a split prime editor capable of being delivered by dual AAVs in vivo. Overall, the technologies and methodologies developed in this dissertation can readily be incorporated in various CRISPR-Cas workflows to enhance precise genome editing or specific targeting of various genome modifying reagents such as prime editor.
Optimal Stoichiometry of the PS Gene-editing System
(2023-11) Saveraid, Hanna G; Przybilla, Michael J; Ou, Li; Whitley, Chester B
Introduction: The PS Gene-editing System has been shown to increase IDUA expression and improve therapeutic outcomes in murine models of Hurler Syndrome. However, the optimal ratio of bipartite AAV vectors in the system is unknown. Results: Mice (+/- or -/- for IDUA) treated with a 1:6 ratio of AAV1:AAV2 in the PSG System had the highest IDUA activity in plasma and liver, followed by 1:13 and then 1:27 ratio. All treated mice had higher enzyme levels than control mice. Conclusion: This work demonstrates that a 1:6 ratio is optimal; however, all ratios resulted in supraphysiological levels of IDUA activity. More research is needed to determine if other ratios (e.g., 1:1), are more effective.
Quinine Copolymer Reporters For Enhanced Gene Editing And Raman Imaging
(2022-01) Van Bruggen, Craig
After decades of development, gene therapy has finally reached the forefront of medicine and has led to new cures for genetic disorders and the development of life-saving vaccines. The field has been buoyed by the development of more precise and user-friendly targeted nucleases, such as those used for clustered regularly interspersed palindromic repeats (CRISPR)-based editing. These useful gene-editing technologies, however, are still stymied by the challenge of delivering exogenous nucleic acids and proteins into the cells of interest. The emerging gene therapy industry is investing heavily in developing more efficient and safe non-viral vehicles as alternatives to costly and immunogenic viral vectors. Cationic polymers are promising non-viral vectors due to their manufacturing scalability, their chemical stability, and their synthetic tunability. Improvements in delivery efficiency are necessary, however, for widespread adoption of polymeric vehicles for gene therapy. One challenge in improving performance, however, is the difficulty and limited methodology for elucidating the intracellular mechanics of polymeric vehicles. In this thesis, I describe my research focused on the development of a novel quinine-containing polymer, called a Quinine Copolymer Reporter (QCR), that enhanced transient transfections of cultured cells with plasmids and improved gene editing of cultured cells through the simultaneous delivery of the CRISPR-associated protein Cas9 and DNA donor template. In addition, I describe collaborative research performed with colleagues in the research group of Prof. Renee Frontiera that characterized a band in quinine’s Raman spectrum that is diagnostic of its chemical environment. Using this chemical sensitivity in conjunction with Raman microscopic imaging, we help elucidated the intracellular unpackaging mechanisms of the QCR-nucleic acid complexes.
Recombinant Engineering Strategies for the Production of Therapeutic Coagulation Factor Proteins
(2023-09) Feser, Colby
Dysregulated coagulation is a common clinical condition secondary to one of numerous blood dyscrasias and can be restored by therapeutic intervention with agents rich in coagulation factor proteins such as whole blood, fresh frozen plasma, prothrombin complex concentrates, and single coagulation factor concentrates. These agents are predominantly sourced from volunteer donors and consequently face supply, product uniformity, and pathogen contamination hurdles. These hurdles are a motivating factor behind the exploration of recombinant manufacturing alternatives that leverage the innate capabilities of a host organism combined with modifications to its genomic, transcriptomic, or proteomic profile to produce therapeutic proteins. Strategies for producing complex human proteins, especially coagulation factors, are highly homologous and consistently employ non-human mammalian cell lines and plasmid-based gene transfer techniques to engineer transgenic cell lines. This resource-intensive strategy must be carried out for each new protein of interest, a critical limitation for complex multi-protein cocktails, and can yield proteins with non-human post-translational modifications; a complication that can lead to reduced protein activity and immunogenic reactions. We sought to address these limitations by exploring a strategy in which a flexible transcriptional activation system could be applied to the expression of diverse single or multi-protein cocktails from human cells that are inherently capable of carrying out human post-translational modifications. Towards this goal, our pilot study investigated the Synergistic Activation Mediators (SAMs), a transcriptional activation system designed around the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nuclease. Utilizing a plasmid-based strategy to express the CRISPR/Cas9-SAM system components our study validated the compatibility of the system with human cells, confirmed its programmability for mixed coagulation factor gene and protein expression, and optimized the SAM architecture for peak transcriptional activity. Our follow up study then took steps to engineer transgenic human cell lines stably expressing these optimized elements. Resulting transgenic cell lines were then programmed for expression of mixed coagulation factor gene and protein targets validating the flexibility of both the cells and the expression strategy. To expand the utility of our engineering approach we then replicated our engineering approach in a murine cell line, programmed it for expression of a murine specific coagulation factor protein, and validated subsequent gene overexpression. Our collective efforts establish proof-of-principle for a novel engineering strategy for the streamlined production of recombinant coagulation factor proteins with promise to address therapeutic gaps in the field of coagulation management. Further, the overall versatility of the SAM system including its trans-species compatibility extend its use beyond coagulation factor proteins to broad applications including therapeutic protein production, cellular network modulation and a multitude of basic, translational, and clinical areas of investigation.
Reducing Environmental Risk in Genetically Modified Perennial Ryegrass
(2022-11) Cors, Jonathan; Watkins, Eric; Smanski, Michael; Casas Mollano, Juan; Zinselmeier, Matthew
Engineered genetic incompatibility can allow for release of genetically modified turfgrass.
Understanding and engineering the molecular regulation of nectar production in field pennycress (Thlaspi arvense).
(2020-09) Thomas, Jason
Anthropogenic climate change and the growing world population are putting pressure on our agroecosystems. Sustainable farming efforts are needed when intensive agriculture systems are fallow and prone to erosion and nutrient leaching. We can mitigate these issues while increasing farmer income by planting field pennycress (Thlaspi arvense). Pennycress forms penny shaped pods containing oil-rich seeds with diverse uses from jet fuel to cooking oil. As an overwintering cover crop, pennycress grows from fall to late spring, avoiding land-use competition with summer annual cash crops. Pennycress provides an ecosystem service to pollinator populations, which are needed for fruit and vegetable production. Pollinators are suffering due partially to losses of habitat and floral resources. Fortunately, pennycress flowers provide nectar as a floral resource when most agricultural landscapes are barren. Additionally, pollinator visitation increases pennycress seed yield. Therefore, the purpose of the study was to understand the genetics behind nectar production and develop pennycress plants with altered floral traits. Microscopy was performed on pennycress flowers to characterize the structure of the nectar-producing glands called nectaries. Additionally, pennycress nectary transcriptomes were determined using transcriptomic sequencing which led to the identification of genes and metabolic pathways. In both cases, the strong similarity in nectar production was confirmed between pennycress and Arabidopsis thaliana, a model plant and close pennycress relative. Because of the close relationship, it was possible to characterize Arabidopsis genes that can later be used to find orthologs in pennycress. The Arabidopsis gene At5g60760 annotated as ‘a P-LOOP containing nucleoside triphosphate hydrolases superfamily protein’ (hereafter AT5G60760) is a putative inositol kinase highly expressed in nectaries. Through assaying mutant AT5G60760 gene expression and nectar production, it was determined that AT5G60760 negatively regulates nectar production. By using findings from nectary genetics, such as the function of AT5G60760 and the pennycress nectary transcriptome, 13 genes were mutated using CRISPR/Cas9 to alter traits relating to pollinator attraction in field pennycress. We have identified two homozygous mutant lines and conducted phenotyping. The auxin response factor 8 (arf8) mutant flowers and petals are larger and produce more nectar than wild type. The cell wall invertase 4 (cwinv4) mutants do not produce nectar and have greatly reduced invertase activity in nectaries. In the future, these plants can be grown in field settings to test pollinator attraction, assay pollinator health, and measure pennycress seed yield.
Using CRISPR to Model MCT1 Deficiency in Pluripotent Stem Cells.
(2024) Reutzel, Bryan
Monocarboxylate transporter 1 (MCT1) plays a key role in transporting monocarboxylates such as lactate, pyruvate, and ketone bodies across the neurovascular unit/blood brain barrier (NVU/BBB). Human MCT1 is a highly conserved 500 amino acid protein embedded in the plasma membrane which contains twelve transmembrane segments with its amino and carboxyl termini in the cytoplasm (Figure). It acts as a facilitative carrier that transports a monocarboxylate and H + in equimolar amounts down a concentration gradient. Some carboxylate drugs may enter the brain via MCT1 and because of its role in metabolism, it has become the target for transport inhibitors (α-cyano-4-hydroxycinnamate, MD-1, AZD3965). Despite the important function of MCT1 in moving metabolically relevant substrates, there have been reports of mutations in the SLC16A1 gene, which codes for MCT1, and render the protein product dysfunctional. Patients with these mutations present with metabolic ketoacidosis that sometimes include seizures, vomiting, and mild to moderate developmental delay. There are currently no in vitro models of MCT1 deficiency in human cells. We hypothesized that creating a cell line of human stem cells in which the SLC16A1 gene is altered would be useful for characterizing the role of MCT1 in the metabolism of cells of the NVU. We therefore used CRISPR/Cas9 editing of human induced pluripotent stem cells (iPSCs). We were able to isolate a heterozygous KO cell line, and were further able to use the isolated line to attempt to create a full/homozygous knockout. The characterization of the resulting cell lines of this study will provide insight into how this pathology may affect the NVU, as well as provide a model system to further investigate this genetic disease.

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