Theses and Dissertations from UMD
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Item DEVELOPING AND IMPROVING CRISPR-BASED GENOME EDITING TECHNOLOGIES IN PLANTS(2022) Sretenovic, Simon; Qi, Yiping; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Earlier genome editing technologies were developed based on programmable nucleases including zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), both requiring tedious protein engineering. By contrast, clustered regularly interspaced short palindromic repeats (CRISPR) systems, such as CRISPR-Cas9, has revolutionized the genome editing field in the past decade due to ease of guiding Cas9 endonuclease to the target site by programmable guide RNAs. However, not every target site can be edited due to Cas9 endonucleases’ recognition of so-called protospacer adjacent motif (PAM) when binding to the target site. For example, the PAM for the widely used SpCas9 is NGG (N=A, C, T or G). This drastically limits targeting scope in the genomes. Thus, researchers have developed engineered Cas9 variants recognizing more relaxed PAMs and tested them in mammalian cell lines. Repair of Cas9 endonuclease-induced double strand breaks through non-homologous end joining (NHEJ) DNA repair pathway typically generates unspecified insertions and deletions, which is a missed opportunity for introducing precise edits. To confer precise genome editing, CRISPR-Cas9 derived base editors and prime editors have been developed. In this work, expanding the plant genome editing scope with engineered Cas9 variants, improving precise cytosine and adenine base editing in plants as well as demonstrating prime editing in plant cells were pursued. The technologies were tested in the model crop, rice, in transiently transformed protoplasts and stably transformed T0 lines. Findings suggest that engineered Cas9 variants can drastically expand the targeting scope for targeted mutagenesis and base editing in plants. Additionally, newer genome editing technologies such as base editors and prime editors can be applied in plants to achieve precise genome editing with varying efficiencies. These validated and useful CRISPR-Cas9 genome editing toolkits have been deposited to the public vector depository, Addgene. Adoption of these genome editing technologies by plant scientists and breeders will enable basic research discoveries and accelerate breeding of next generation crops, ensuring global food security amidst climate change and increasing global population.Item DESIGN OF ENZYME CASCADE AND CRISPR-CAS9 NETWORKS FOR ENABLING BIOELECTRONIC COMMUNICATION(2017) Bhokisham, Narendranath; Bentley, William E; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)There is an increasing emphasis on building closed loop systems in human health where real time monitoring and analysis is connected to feedback and treatment. Building such systems requires bridging the information loop across the different signal modalities of biology and electronics. In this work, I have created two different networks at biology-electronic interface to enable the communication from biology to electronics and vice versa. The first network is a multi-step enzyme cascade assembled on a microchip to enable conversion of biologic information into electro-chemical information. I first devised a modular construction approach using microbial transglutaminase (mTG) based conjugation chemistry where multiple enzyme components are assembled on an abiotic surface in a ‘plug and play’ fashion. Integration of bio-components with electronics requires a scaffold material for functionalization of the bio-electronic interface. To address this challenge, I engineered a self-assembling Tobacco Mosaic Virus-Virus Like Particle into a 3D scaffold displaying desired functional groups at the interface. Using the 3D scaffolds and mTG mediated conjugation chemistry, I assembled a synthetic 3-enzyme cascade on a microchip for conversion of methyl cycle intermediates into homocysteine, an electrochemically readable molecule. The modular construction approach and the scaffold materials that I developed can enable facile assembly of multi-subunit bio-components and diversify the range of metabolites that can be detected on a microchip for use in biosensing applications. Next, I focused on mediating communication from electronics to specific genes in the genome of biological systems. An electrogenetic promoter that is responsive to the electrical stimuli was reported in E. coli. In this work, I integrated the precise gene targeting capabilities of the CRISPR-Cas9 system with the electrogenetic promoter to target specific host transcriptional processes. I displayed temporal silencing of several host defense mechanisms against the electrical signals resulting in an overall positive feedback for electrogenicity in E. coli. A more sophisticated control of host transcriptional processes by the CRISPR-Cas9 system is a valuable addition to the existing electrogenetic toolbox, one that could enhance the interoperability of electrogenetic systems and mediate bio-electronic communication between strains and species.