Directed evolution of CRISPR-associated transposases for programmable gene insertion in human cells

Abstract

Advances in genome editing have enabled the generation of virtually any small sequence change in the genome, transforming our ability to treat genetic disorders. Installing large DNA sequences at specified genomic sites, however, has remained a longstanding challenge. Programmable gene-sized DNA insertion offers many applications in therapeutics and life sciences research, including the ability to treat genetically diverse loss-of-function diseases with mutation-agnostic strategies. In this thesis, I describe progress toward achieving efficient, programmable gene insertion in human cells, focusing specifically on enhancing the DNA insertion activity of CRISPR-associated transposases (CASTs), a class of RNA-programmable mobile genetic elements found in bacteria. In Chapter 1, I provide an overview of genome editing technologies, with an emphasis on established gene integration methods and their limitations. I also discuss how directed evolution and protein engineering techniques have been applied to broaden the utility of genome editing agents. In Chapter 2, I describe my work using directed evolution to substantially improve the DNA integration activity of a type I-F CAST in human cells. CASTs use nuclease-deficient CRISPR machinery to guide DNA integration at genomic locations specified by guide RNAs. Despite offering many attractive qualities as genome editing tools, wild-type CASTs reported to date support minimal integration in human cells. Based on past studies of mobile genetic elements, we reasoned that this low efficiency may stem from naturally evolved suboptimal transposition catalysis. To enable efficient CAST integration in human cells, we developed a PACE selection to rapidly evolve CAST variants with enhanced transposition activity. We applied PACE to a prototypical type I-F CAST system from Pseudoalteromonas, generating evolved CAST variants with >200-fold average improved integration activity in human cells. The evolved CAST system (evoCAST) achieved therapeutically relevant efficiencies (10-30%) for kilobase-scale DNA cargoes across diverse genomic targets while maintaining high product purity. Collectively, our findings establish a platform for the laboratory evolution of CASTs and advance a versatile approach for programmable gene integration in living systems. In Chapter 3, I describe my work developing a type V-K CAST with enhanced activity in human cells. evoCAST is derived from a type I-F CAST, which is a large molecular complex that can be challenging to effectively deliver to therapeutically relevant cell types. Type V-K CASTs, which are evolutionarily distinct from type I-F CASTs, contain fewer molecular components and a smaller coding size. Using the CAST PACE platform, I generated an evolved type V-K CAST with >50-fold improved activity in human cells. These findings demonstrate the generalizability of CAST PACE to diverse CAST systems and establish a simpler CAST for efficient human-cell genome editing. In Chapter 4, I briefly summarize additional efforts to expand the CAST toolkit for genome editing in human cells. The efficiency improvements observed in evoCAST largely derived from PACE of the transposase module, which catalyzes DNA insertion. Here I discuss two efforts to optimize the DNA-targeting module of evoCAST. First, we employed protein engineering and PACE to develop human-cell active “chimeric” CASTs that use a highly active DNA-targeting module from a divergent type I-F CAST to guide the evoCAST transposase module. Second, I explored using nuclease-deficient Cas9 to guide the evoCAST transposase module, enabling DNA insertion to be directed by a well-established, single-component CRISPR-associated effector. I conclude by offering perspectives on the future of gene integration in biological research and therapeutics, discussing areas of potential improvement along with opportunities for clinical implementation.