2024 Theses Doctoral

Engineering CRISPR-associated transposons for RNA-guided gene insertion in human cells

Lampe, George

Genome editing technologies have advanced from methods that rely on nucleases to catalyze programmed double-strand breaks (DSBs), which are known to cause deleterious side effects, to next-generation reagents that perform more controlled chemistry using DSB-independent approaches. Base editing and prime editing are ideally suited for small-scale modifications, but methods to achieve large-payload gene insertion have been lacking.

Recent technology development efforts have advanced strategies that employ eukaryotic transposases, bacterial recombinases/transposases, and retroelements, yet these enzymes broadly suffer from either inflexibility of target site requirements or non-specific, genome-wide insertion profiles. The ability to precisely and safely insert kilobase-scale DNA cargos at user-defined loci remained challenging due to a dearth of programmable transposase tools. CRISPR-associated transposon (CAST) systems represent a unique opportunity to solve this longstanding challenge. CASTs are diverse (types I-B, I-D, I-F, and V-K) heteromeric, macromolecular machineries that require multivalent protein-protein interactions, each of which represents a potential kinetic bottleneck, inefficiency, or failure point when porting from bacteria to mammalian cells.

Through meticulous, step-by-step engineering involving functional assays, bioinformatic mining, homolog screening, structure-guided engineering, and directed evolution, we have iteratively improved overall editing efficiencies of type I-F CASTs, identified an essential bacterial co-factor, and reached editing efficiencies that approach therapeutic relevance. Together, this work represents a critical advancement towards a broad platform for targeted genomic integration of large DNA payloads.