Base Editing and Prime Editing: Engineered Proteins That Precisely Correct Pathogenic Mutations in Cells, Animals, and Patients

In this lecture, I describe the development and therapeutic application of two precision gene editing technologies that install or correct targeted mutations without requiring double-strand DNA breaks, thereby minimizing undesired consequences of chromosomal cleavage. We developed base editors, proteins that directly perform chemistry on individual DNA bases in living cells to install or correct mutations at targeted positions in genomic DNA. We recently engineered CRISPR-free, all-protein base editors that enabled the first purposeful changes in the sequence of mitochondrial DNA in living cells. By integrating base editors with ex vivo and in vivo delivery strategies that deliver therapeutic proteins, we rescued animal models of human genetic diseases including sickle-cell disease, progeria, and spinal muscular atrophy (SMA). Single-AAV base editing systems enhance the safety and practicality of in vivo base editing. Our development of engineered virus-like particles (eVLPs) provide additional in vivo delivery methods for gene editing proteins that minimize off-target editing and the risk of oncogenic DNA integration. Base editors are in at least six clinical trials to treat diseases including familial hypercholesterolemia, sickle-cell disease, beta-thalassemia, and T-cell leukemia. The first clinical outcomes from ex vivo base editing and from in vivo base editing have also been reported, demonstrating benefit to T-cell leukemia patients and to hypercholesterolemia patients, respectively. I will also describe prime editors, engineered proteins that directly write new genetic information into a specified DNA site, replacing the original sequence, without requiring double-strand DNA breaks or donor DNA templates. Prime editing can mediate any base substitutions, deletions, and/or insertions of up to ~200 base pairs in living cells in vitro and in vivo, and has been applied to directly install or correct pathogenic alleles that previously could not be corrected in therapeutically relevant cells. We illuminated the cellular determinants of prime editing outcomes, and used the resulting insights to develop new prime editing systems with substantially higher editing efficiencies and product purities. Most recently, we used phage-assisted continuous evolution (PACE) to evolve a suite of sixth-generation prime editors (PE6a-6g), which each evolved to specialize in different types of prime edits. The combination of prime editing and site-specific recombinases enable programmable gene-sized (>5 kb) integration and inversion at loci of our choosing in human cells. Prime editing has recently been used to rescue animal models of genetic diseases including sickle-cell disease, metabolic liver diseases, and genetic blindness, and is anticipated to be cleared for clinical trials in 2024. Base editing and prime editing enable precise target gene correction, in addition to target gene disruption, in a wide range of organisms with broad implications for the life sciences and therapeutics.