<p>The ends of long bones that articulate with respect to one another are lined with a crucial connective tissue called articular cartilage. This tissue plays an essential biomechanical function in synovial joints, as it serves to both dissipate load and lubricate articulating surfaces. Osteoarthritis is a painful and debilitating disease that drives the deterioration of articular cartilage. Like many chronic diseases, pro-inflammatory cytokines feature prominently in the onset and progression of osteoarthritis. Because cartilage lacks physiologic features critical for regeneration and self-repair, the development of effective strategies to create functional cartilage tissue substitutes remains a priority for the fields of tissue engineering and regenerative medicine. The overall objectives of this dissertation are to (1) develop a bioactive scaffold capable of mediating cell differentiation and formation of extracellular matrix that recapitulates native cartilage tissue and (2) to produce stem cells specifically tailored at the scale of the genome with the ability to resist inflammatory cues that normally lead to degeneration and pain. </p><p>Engineered replacements for musculoskeletal tissues generally require extensive ex vivo manipulation of stem cells to achieve controlled differentiation and phenotypic stability. By immobilizing lentivirus driving the expression of transforming growth factor-β3 to a highly structured, three dimensionally woven tissue engineering scaffold, we developed a technique for producing cell-instructive scaffolds that control human mesenchymal stem cell differentiation and possess biomechanical properties approximating those of native tissues. This work represents an important advance, as it establishes a method for generating constructs capable of restoring biological and mechanical function that may circumvent the need for ex vivo conditioning of engineered tissue substitutes.</p><p>Any functional cartilage tissue substitute must tolerate the inflammation intrinsic to an arthritic joint. Recently emerging tools from synthetic biology and genome engineering facilitate an unprecedented ability to modify how cells respond to their microenvironments. We exploited these developments to engineer cells that can evade signaling of the pro-inflammatory cytokine interleukin-1 (IL-1). Our study provides proof-of-principle evidence that cartilage derived from such engineered stem cells are resistant to IL-1-mediated degradation. </p><p>Extending on this work, we developed a synthetic biology strategy to further customize stem cells to combat inflammatory cues. We commandeered the highly responsive endogenous locus of the chemokine (C-C motif) ligand 2 gene in pluripotent stem cells to impart self-regulated, feedback-controlled production of biologic therapy. We demonstrated that repurposing of degradative signaling pathways induced by IL-1 and tumor necrosis factor toward transient production of cytokine antagonists enabled engineered cartilage tissue to withstand the action of inflammatory cytokines and to serve as a cell-based, auto-regulated drug delivery system.</p><p>In this work, we combine principles from synthetic biology, gene therapy, and functional tissue engineering to develop methods for generating constructs with biomimetic molecular and mechanical features of articular cartilage while precisely defining how cells respond to dysfunction in the body’s finely-tuned inflammatory systems. Moreover, our strategy for customizing intrinsic cellular signaling pathways in therapeutic stem cell populations opens innovative possibilities for controlled drug delivery to native tissues, which may provide safer and more effective treatments applicable to a wide variety of chronic diseases and may transform the landscape of regenerative medicine.</p> / Dissertation

abstract: The portability of genetic tools from one organism to another is a cornerstone of synthetic biology. The shared biological language of DNA-to-RNA-to-protein allows for expression of polypeptide chains in phylogenetically distant organisms with little modification. The tools and contexts are diverse, ranging from catalytic RNAs in cell-free systems to bacterial proteins expressed in human cell lines, yet they exhibit an organizing principle: that genes and proteins may be treated as modular units that can be moved from their native organism to a novel one. However, protein behavior is always unpredictable; drop-in functionality is not guaranteed. My work characterizes how two different classes of tools behave in new contexts and explores methods to improve their functionality: 1. CRISPR/Cas9 in human cells and 2. quorum sensing networks in Escherichia coli. 1. The genome-editing tool CRISPR/Cas9 has facilitated easily targeted, effective, high throughput genome editing. However, Cas9 is a bacterially derived protein and its behavior in the complex microenvironment of the eukaryotic nucleus is not well understood. Using transgenic human cell lines, I found that gene-silencing heterochromatin impacts Cas9’s ability to bind and cut DNA in a site-specific manner and I investigated ways to improve CRISPR/Cas9 function in heterochromatin. 2. Bacteria use quorum sensing to monitor population density and regulate group behaviors such as virulence, motility, and biofilm formation. Homoserine lactone (HSL) quorum sensing networks are of particular interest to synthetic biologists because they can function as “wires” to connect multiple genetic circuits. However, only four of these networks have been widely implemented in engineered systems. I selected ten quorum sensing networks based on their HSL production profiles and confirmed their functionality in E. coli, significantly expanding the quorum sensing toolset available to synthetic biologists. / Dissertation/Thesis / Doctoral Dissertation Bioengineering 2017

At the post-transcriptional level, the splicing of the pre-mRNA plays a vital role in cell fate determination and respond to biotic and abiotic stresses. Through alternative splicing, mRNAs variants can be produced from a single gene. SF3B is a heptameric protein complex that is essential for pre-mRNA splicing. It contains seven subunits: SF3b155, SF3b130, SF3b145, SF3b49, SF3b14b, P14/SF3b14a and SF3b10 and they play an important role in BS (branch point sequence) recognition. P14/SF3b14a interacts with the branch point Adenosine (BPA), directing the binding of U2 complex. Several studies performed on the mutations of SF3b complex as it is associated with many diseases. Further studies are needed to deeply analyze the molecular function of P14/SF3b14a in plant growth and development. CRISPR/Cas9 system employed in gene editing among eukaryotes. The capability of the system is not only limited to the scope of bioengineering but also for functional studies of genes. CRISPR/Cas9 system assists in revealing the function of genes and the genetic networks through establishing a functional knockout and can help in understanding the molecular basis behind these processes. Here, we report the successful targeted mutagenesis of SF3b14a/p14 gene in Oryza sativa and the recovery of homozygous and heterozygous mutants. Phenotypic analyses have shown that SF3b14a/p14 is hypersensitive to abiotic stresses compared to the wild type plants. Further physiological and molecular studies are needed to reveal the role of p14 during plant growth and development, and responses to abiotic stresses.

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.

Plants employ sophisticated molecular machineries to fine-tune their responses to growth, developmental, and stress cues. Plants cellular response influences gene expression through regulating processes like transcription and splicing. To increase the genome coding potential and further regulate the expression, pre-mRNA is alternatively spliced. Serine/Arginine-rich (SR) proteins, a family of pre-mRNA splicing factors, recognize splicing cis-elements and regulate both constitutive and alternative splicing. Recent studies reported only 22 SR proteins encoded in the genome of rice (Oryza sativa), which are classified into 6 subfamilies. Oryza s. SC subfamily 106 kDa (Os-Sc106) locus is homologous to the human SR protein SFSR11 (SRp54). Os-Sc106 contains SR proteins characteristics, and was not included among the rice SR proteins. The clustered regularly interspaced short palindromic repeats (CRISPR) and its associated protein 9 (Cas9) system, an RNA-guided endonuclease complex that introduces a double-strand break (DSB) into the DNA. Innovative scientific advances in genome engineering have made CRISPR/Cas9 an excellent system to conduct functional knockout studies of genes in most biological systems including plants. In this study, I targeted the rice Os-Sc106 locus at exon1, and 3 via CRISPR/Cas9 system. Genotyping analyses revealed the recovery of Os-Sc106 mutants including complete functional knockouts such as sf11h-2, sf11h-8, and sf11h-55. Phenotypic analyses show that Os-Sc106 mutants (sf11h-2, 8, 55, and 57) are oversensitive under abiotic stress in comparison to WT plants, suggesting that Os-Sc106 locus encodes a protein that is important for regulating plant stress responses.

Several tools were developed to help researchers facilitate clinical translation of the use of engineered nucleases towards their disease gene of interest. Two major issues addressed were the inability to accurately predict nuclease off-target sites by user-friendly \textit{in silico} methods and the lack of a high-throughput, sensitive measurement of gene editing activity at endogenous loci. These objectives were accomplished by the completion of the following specific aims. An online search interface to allow exhaustive searching of a genome for potential nuclease off-target sites was implemented. Previously discovered off-target sites were collated and ranking algorithms developed that preferentially score validated off-target sites higher than other predictions. HEK-293T cells transfected with newly developed TALENs and ZFNs targeting the beta-globin gene were analyzed at the off-target sites predicted by the tool. Many samples of genomic DNA from cells treated with different ZFNs and TALENs were analyzed for off-target effects to generate a greatly expanded training set of bona fide off-target sites. Modifications to the off-target prediction algorithm parameters were evaluated for improvement through Precision-Recall analysis and several other metrics. An analysis pipeline was developed to process SMRT reads to simultaneously measure the rates of different DNA repair mechanisms by directly examining the DNA sequences. K562 cells were transfected with different types of nucleases and donor repair templates in order to optimize conditions for repairing the beta-globin gene. This work will have significant impact on future studies as the methods developed herein allow other laboratories to optimize nuclease-based therapies for single gene disorders.

The complexity of life exhibited in humans and other living creatures has drawn many to investigate the principles associated with organismal growth and development. A few broad questions: How do tissues develop into specified organs? How are these tissues maintained? Do they become different tissues? Scientific research has incessantly been seeking answers to these as well as a plethora of other questions. While on a quest to better understand developmental biology, investigators discovered unique populations of stem cells within a variety of tissues, which retain both varying degrees of developmental plasticity and their potential for self-regeneration. This thesis provides a brief review discussing the development and history of stem cells in medicine and associated research on these cells and their potential clinical applications. Substantial attention has been paid to pluripotent embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) which are able to be recapitulate ESC properties through the in vitro reprogramming of somatic cells. While, the ethical and legal issues have greatly hindered the use of ESCs this has made the benefit of iPSCs so great. An interconnected network of pluripotency-associated genes, integrates external signals and exerts control to maintain the state of pluripotency. Recent research has proven the pluripotency regulatory network to be flexible such that the underlying principles promise unprecedented opportunities for scientific study and regenerative medicine. Additional topics reviewed here include vast clinical applications of stem cells as well as their notable limitations.

<p>Electrowetting-on-dielectric (EWD) digital microfluidics is a droplet-based fluid handling technology capable of radically accelerating the pace of genome engineering research. EWD-based laboratory-on-chip (LoC) platforms demonstrate excellent performance in automating labor-intensive laboratory protocols at ever smaller scales. Until now, there has not been an effective means of gene transfer demonstrated in EWD microfluidic platforms. This thesis describes the theoretical and experimental approaches developed in the demonstration of an EWD-enabled electrotransfer device. Standard microfabrication methods were employed in the integration of electroporation (EP) and EWD device architectures. These devices enabled the droplet-based bulk transformation of E. coli with plasmid and oligo DNA. Peak on-chip transformation efficiencies for the EP/EWD device rivaled that of comparable benchtop protocols. Additionally, ultrasound induced in-droplet microstreaming was developed as a means of improving on-chip electroporation. The advent of electroporation in an EWD platform offers synthetic biologists a reconfigurable, programmable, and scalable fluid handling platform capable of automating next-generation genome engineering methods. This capability will drive the discovery and production of exotic biomaterials by providing the instrumentation necessary for rapidly generating ultra-rich genomic diversity at arbitrary volumetric scales.</p> / Dissertation

Zinc finger nucleases (ZFNs) induce double-strand DNA breaks at specific recognition sites. ZFNs can dramatically increase the efficiency of incorporating desired insertions, deletions, or substitutions in living cells. These tools have revolutionized the field of genome engineering in several model organisms and cell types including zebrafish, rats, and human pluripotent stem cells. There have been numerous advances in ZFN engineering and characterization strategies, some of which are detailed in this work. The central theme of this dissertation is improving the activity and specificity of engineered zinc finger nucleases with the ultimate goal of increasing the safety and efficacy of these tools for human therapy. As a first step, I undertook a large-scale effort to demonstrate that the modular assembly method of ZFN synthesis has a significantly higher failure rate than previously reported in the literature. This strongly suggested that engineering of ZFNs should better account for context-dependent effects among zinc fingers. The second advance reported in this dissertation is a method for biasing repair of zinc finger protein-induced DNA breaks toward homology-driven rather than error-prone repair in the presence of a donor template. Catalytically inactivating one monomer of a ZFN dimer results in a zinc finger nickase (ZFNickase) whose cleavage preference is directed at only one DNA strand. In human cell reporter assays, these ZFNickases exhibit a higher likelihood of repair by homology-driven processes, albeit with reduced absolute rates of correction. With further optimization, zinc finger nickases could provide a safer alternative to ZFNs in the context of gene correction therapies. Third, realizing there was no robust method for determining off-target cleavage sites of ZFNs in a genome-wide manner, I validated a collaborator’s novel in vitro selection system in human cells by identifying eight new potential off-target cleavage sites for a ZFN pair currently being used in clinical trials. Although it is unlikely these low-frequency mutations would be deleterious to patients, these results demonstrated that ZFNs induced more off-target effects than had been appreciated by previous work in the field. Collectively, the findings of this dissertation have contributed to more robust strategies for designing and evaluating ZFNs.

Engineered DNA-binding proteins enable targeted manipulation of the genome. Zinc fingers are the most well characterized DNA-binding domain and for many years research has focused on understanding and manipulating the sequence-specificities of these proteins. Recently, major advances in the ability to engineer zinc finger proteins, as well as the discovery of a new class of DNA-binding domains - transcription activator-like effectors (TALEs), have made it possible to rapidly and reliably engineer proteins targeted to any sequence of interest. With this capability, focus has shifted to exploring the applications of this powerful technology. In this dissertation I explore three important applications of engineered DNA-binding proteins.