Base Editing and Prime Editing: Genome Editing Without Double-Strand Breaks

Date: Wednesday, March 17, 2021
Speaker: David Liu, Ph.D.
Richard Merkin Professor and Director of the Merkin Institute of Transformative Technologies in Healthcare; Core Institute Member and Vice-Chair of the Faculty, Broad Institute; Thomas Dudley Cabot Professor of the Natural Sciences, and Professor of Chemistry & Chemical Biology, Harvard University; Investigator, Howard Hughes Medical Institute

Talk title: Base Editing and Prime Editing: Genome Editing Without Double-Strand Breaks

Abstract: 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 (Koblan et al. Nature 2021). 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.

Links to relevant publications:

  • Programmable Editing of a Target Base in Genomic DNA Without Double-Stranded DNA Cleavage. Komor, A. C.; Kim, Y. B.; Packer, M. S.; Zuris, J. A.; Liu, D. R. Nature533, 420-424 (2016). PDF, SIExpanded Extended Data Figure 6
  • Programmable Base Editing of A*T to G*C in Genomic DNA Without DNA Cleavage. Gaudelli, N. M.; Komor, A. C.; Rees, H. A.; Packer, M. S.; Badran, A. H.; Bryson, D. I.; Liu, D. R. Nature551, 464-471 (2017). PDFSI
  • A Bacterial Cytidine Deaminase Toxin Enables CRISPR-Free Mitochondrial Base Editing. Mok, B. Y; de Moraes, M. H; Zeng, J; Bosch, D. E; Kotrys, A. V; Raguram, A.; Hsu, F; Radley, M.C; Peterson, S. B; Mootha, V.K; Mougous, J.D; Liu, D. R. Nature. 583,631-637(2020). PDF, SI
  • In Vivo Adenine Base Editing Corrects Hutchinson-Gilford Progeria Syndrome.Koblan L. W.; Erdos, M. R.; Wilson, C.; Cabral, W. A.; Levy, J. M.; Xiong, Z.-M.; Taarez, U. L.; Davison, L.; Gete, Y. G.; Mao, X.; Newby, G. A.; Doherty, S. P.; Lin, C. Y.; Gordon, L. B.; Cao, K.; Collins, F. S.; Brown, J. D.; Liu, D. R. Nature in press, available online (2021). PDF
  • Search-and-replace genome editing without double-strand breaks or donor DNA. Anzalone, A.V.; Randolph, P.B.; Davis, J.R.; Sousa, A.A.; Koblan, L.W.; Levy, J.M.; Chen, P.J.; Wilson, C.; Newby, G.A.; Raguram, A.; Liu, D.R. Nature,  576, 149–157(2019). PDF SI