Most genetic variants that contribute to disease are challenging to correct efficiently and without excess byproducts in various cell types using programmable nucleases. 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 (Komor et al. Nature 2016; Gaudelli et al. Nature 2017). We also engineered a novel double-strand DNA deaminase discovered by Joseph Mougous’s lab into a mitochondrial base editor, enabling the first precision edits in the mitochondrial DNA of living cells (Mok et al. Nature 2020). Base editing has been used by laboratories around the world in a wide range of organisms and cell types. By integrating base editors with in vivo delivery strategies, we have addressed animal models of human genetic diseases such as progeria, with a high degree of 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 (Anzalone et al. Nature 2019). 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 lower off-target editing than Cas9 nuclease at known Cas9 off-target sites. Prime editing further expands the scope and capabilities of genome editing.
David R. Liu is the Richard Merkin Professor, Director of the Merkin Institute of Transformative Technologies in Healthcare, and Vice-Chair of the Faculty at the Broad Institute of Harvard and MIT; Thomas Dudley Cabot Professor of the Natural Sciences and Professor of Chemistry and Chemical Biology at Harvard University. His major research interests include the engineering, evolution, and in vivo delivery of genome editing proteins such as base editors and prime editors to study and treat genetic diseases; the evolution of proteins with novel therapeutic potential using phage-assisted continuous evolution (PACE); and the discovery of bioactive synthetic small molecules and synthetic polymers using DNA-templated organic synthesis and DNA-encoded libraries. Base editing (named one of four 2017 Breakthrough of the Year finalists by Science), prime editing, PACE, and DNA-templated synthesis are four examples of technologies pioneered in his laboratory.
For details of his research and recent publication, please visit HERE
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