Minimization of Cas9 and Perspectives on Genetically Engineered Microorganisms and Their Regulation

CRISPR technology has revolutionized the way biology is conducted. In a few years, scientists have broken through barriers that have hamstrung the field for decades, thanks to the conceptually simple ability to target specific sequences in an organism’s genome. That ability alone has led to enormous leaps in our understanding of complex traits, biological pathways, disease, and evolution. Additionally, genome editing with CRISPR has ushered in a new age of therapeutics and genetic engineering. As new applications of CRISPR technology emerge, we are beginning to push the boundaries of what it is capable of, and new modalities of CRISPR are required to overcome new challenges. For example, despite its reputation of being the “flagship” CRISPR molecule, the S. pyogenes Cas9 (a.k.a. SpCas9), is still too large to be genetically encoded and delivered by adeno-associated viruses (AAVs). As AAVs are one of the predominant delivery mechanisms for gene therapies, precluding SpCas9 from its repertoire is a significant deficiency./nSpCas9 has been studied extensively to date. However, certain aspects of the molecule and its mechanisms are still unknown and evade our understanding even as we expand our gene-editing toolkit to include new Cas9s, new CRISPR systems, and new platforms. One of the questions associated with SpCas9 is its multi-domain architecture, which is atypically complex and large for bacterial proteins. While the general molecular mechanism of Cas9 is understood thanks to structural and kinetics studies, the evolution of the overall protein and its domains are less understood. This is especially interesting considering how little sequence identity Cas9s share among orthologs while still possessing a similar three-dimensional architecture./nAdditionally, SpCas9’s combination of stability, precision, and versatility makes it a prime candidate for protein engineering. Researchers have tried adding new functions to SpCas9, improving its performance, and even making it more suitable for certain use cases using rational design principles. However, making the protein smaller has been an under-utilized concept with limited success. Minimizing SpCas9 while still retaining its DNA targeting function has two main functions: a) understand the essentiality of its domains for DNA targeting, and b) develop a novel protein scaffold that is smaller and more feasible for AAV and other forms of cellular delivery. In Chapter 2 of this thesis, I discuss a project to probe the amino acid deletion landscape of SpCas9, in an effort to biochemically characterize its domains, and also to arrive at a minimal DNA-binding protein module./nCRISPR-Cas9 has undoubtedly changed the biotechnology world by advancing genetic engineering and breaking through technical barriers that have plagued the field for decades. Simultaneously, the maturation of several other synthetic biology tools has also converged into a new era of biotechnology. Genome editing, sequencing, bioinformatics, gene synthesis, etc. have brought upon a revolution of new bioengineered products to the market. Leading the charge are genetically engineered microorganisms, or GEMs. GEMs are being developed for the purpose of making biofuels, commodity chemicals, materials, food and food additives, pesticides, and fertilizers. Many of these products have promise as more efficient, eco-friendly, and overall more beneficial alternatives to conventional industries./nHowever, adoption and deployment of these technologies are not as simple as building them. Understandably, bioengineered products require regulatory oversight to ensure safety and efficacy. In the U.S., biotechnology products are regulated by a tri-agency framework consisting of the Food and Drug Administration (FDA), U.S. Department of Agriculture (USDA), and the Environmental Protection Agency (EPA). These agencies have distinct roles and evaluation criteria when assessing new biotech products for market approval. However, one of the key issues with the current regulatory landscape in the U.S. is its complicated and circuitous nature that often delay and/or drive up costs of product development. To maximize the impact of all the innovation and benefit of scientific advances happening in the laboratory, regulation needs to be more streamlined without compromising safety./nIn Chapter 3 of this thesis, I describe the current state of GEMs in the U.S., from the development and regulation perspectives. I discuss the product areas in which GEMs are emerging as feasible and scalable alternatives to conventional industrial processes, such as fuels, food, and agriculture. I also attempt to explain the current tri-agency regulatory framework as set by the FDA, USDA, and EPA. Finally, I discuss ways in which both GEM regulators and product developers must work hand in hand to enact large-scale solutions to major modern problems like climate change and food insecurity.

Advisor: Savage, David