David Liu

Base Editing and Prime Editing: Chemistry on the Genome Without Double-Strand Breaks

Most genetic variants that contribute to disease are challenging to correct efficiently and without excess byproducts using standard nuclease-based genome editing methods. In this lecture I describe the development of two approaches to precision genome editing that do not require double-strand DNA breaks, donor DNA templates, or HDR.  Through a combination of protein engineering and protein evolution, we developed two classes of base editors (CBE and ABE), proteins that enable all four types of transition mutations (C to T, T to C, A to G, and G to A) to be efficiently and cleanly installed or corrected at target positions in genomic DNA without making double-strand DNA breaks. Base editing has been used by many laboratories around the world in a wide range of organisms. By integrating base editors with in vivo delivery strategies, we have addressed animal models of human genetic disease, in some cases with phenotypic rescue and lifespan extension.

I will also describe prime editing, a versatile and precise genome editing method that directly writes new genetic information into a specified DNA site using a catalytically impaired Cas9 fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit. We performed >175 edits in human cells including targeted insertions, deletions, and all 12 types of point mutations without requiring double-strand breaks or donor DNA templates. We applied prime editing in human cells to correct efficiently and with few byproducts the primary genetic causes of sickle cell disease (requiring a transversion in HBB) and Tay-Sachs disease (requiring a deletion in HEXA), to install a protective transversion in PRNP, and to precisely insert various tags and epitopes into target loci. Four human cell lines and primary post-mitotic mouse cortical neurons support prime editing with varying efficiencies. Prime editing offers efficiency and product purity advantages over HDR, complementary strengths and weaknesses compared to base editing, and much lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing substantially expands the scope and capabilities of genome editing, and in principle can correct most known pathogenic human genetic variants.