Engineering high-throughput approaches for CRISPR gene therapy and synthetic biology
- UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
- Tinku Supakar (Creator)
- Institution
- The University of North Carolina at Greensboro (UNCG )
- Web Site: http://library.uncg.edu/
- Advisor
- Eric Josephs
Abstract: The field of synthetic biology has witnessed rapid advancements in recent years, driven by the integration of high-throughput engineering approaches. This dissertation delves into the design, development, and implementation of high-throughput tools tailored for synthetic biology for various applications while addressing various challenges and complexities associated with each application. Firstly, we have focused on applying high throughput engineering approach in gene editing tools such as CRISPR. CRISPR based technologies primarily are used for precision gene editing, mutating a DNA sequence based on the CRISPR effector’s guide RNA or gRNA. Beyond gene editing, a burgeoning yet less-explored application of CRISPR effectors is in CRISPR-based antiviral biotechnologies. However, the rapid proliferation and mutation rates of viruses introduce unique complexities such as need of expanding recognition across clinical strain variants, enhancing viral detection sensitivity, and limiting mutagenic escape which are not addressed by current gene editing oriented CRISPR guide RNA (gRNA) design tools. To address this challenge, here, we have developed a computational algorithm for the design of efficient gRNAs, termed polyvalent guide RNAs (pgRNAs), which are optimized for simultaneous activity at multiple viral targets by utilizing the inherent tolerance of certain CRISPR effectors to mismatches between their guide RNA (gRNA) spacer sequences and its target sites. Next, we present a highly parallelized method, compartmentalized CRISPR reactions (CCR), for screening large numbers of gRNA/target/off-target combinations simultaneously in vitro for both CRISPR effector activity and specificity, by confining the complete CRISPR reaction of gRNA transcription and CRISPR target cleavage within individual water-in-oil microemulsions. This approach overcomes the limitations of traditional CRISPR gRNAs screening, which has low throughput. Additionally, we demonstrate that CCR can be used to screen hundreds of thousands of extended gRNA (x-gRNAs) for highly active and highly specific variants of the standard gRNA sequences that can completely block cleavage at off-target sequences while maintaining high levels of on-target activity. Lastly, we have focused on scaling microfluidic systems which are used in many advanced applications in medical diagnostics, lab-on-chips, and laboratory automation. Microfluidic valves play a key role within microfluidic systems by regulating fluid flow through distinct microchannels. While microfluidic systems are often limited to planar structures, 3D printing enables new capabilities to generate complex designs for fluidic circuits with higher densities and integrated components. However, the control of fluids within 3D structures presents several difficulties, making it challenging to scale effectively and many fluidic devices are still often restricted to quasi-planar structures. Here, we have performed systematic computational and experimental characterization of a modified re-entrant honeycomb structure to generate a modular metamaterial for an active device that allows us to directly regulate flow through integrated, multiplexed fluidic channels “one-at-a-time,” in a manner that is highly scalable. In conclusion, these high throughput techniques developed in this research, including multiplexed CRISPR-based antivirals, parallelized and compartmentalized in vitro CRISPR screening, and scaling microfluidic systems with novel metamaterial designs will enhance the efficiency and scalability of molecular biology methodologies across biotechnology, medicine, and diagnostics.
Engineering high-throughput approaches for CRISPR gene therapy and synthetic biology
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Created on 8/1/2024
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Additional Information
- Publication
- Dissertation
- Language: English
- Date: 2024
- Keywords
- 3D Printed Fluidic Valve, CRISPR gRNA Screen, CRISPR-Based Antivirals, High-Throughput Engineering Approach, Mechanical Metamaterial
- Subjects
- Synthetic biology
- CRISPR (Genetics)
- Metamaterials