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Phage-assisted evolution and protein engineering yield compact, efficient prime editors.
Doman, Jordan L; Pandey, Smriti; Neugebauer, Monica E; An, Meirui; Davis, Jessie R; Randolph, Peyton B; McElroy, Amber; Gao, Xin D; Raguram, Aditya; Richter, Michelle F; Everette, Kelcee A; Banskota, Samagya; Tian, Kathryn; Tao, Y Allen; Tolar, Jakub; Osborn, Mark J; Liu, David R.
Afiliación
  • Doman JL; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
  • Pandey S; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
  • Neugebauer ME; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
  • An M; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
  • Davis JR; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
  • Randolph PB; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
  • McElroy A; Department of Pediatrics, University of Minnesota Medical School, Minneapolis, MN, USA.
  • Gao XD; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
  • Raguram A; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
  • Richter MF; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
  • Everette KA; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
  • Banskota S; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
  • Tian K; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
  • Tao YA; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
  • Tolar J; Department of Pediatrics, University of Minnesota Medical School, Minneapolis, MN, USA.
  • Osborn MJ; Department of Pediatrics, University of Minnesota Medical School, Minneapolis, MN, USA.
  • Liu DR; Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA; Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. Electronic address: dr
Cell ; 186(18): 3983-4002.e26, 2023 08 31.
Article en En | MEDLINE | ID: mdl-37657419
Prime editing enables a wide variety of precise genome edits in living cells. Here we use protein evolution and engineering to generate prime editors with reduced size and improved efficiency. Using phage-assisted evolution, we improved editing efficiencies of compact reverse transcriptases by up to 22-fold and generated prime editors that are 516-810 base pairs smaller than the current-generation editor PEmax. We discovered that different reverse transcriptases specialize in different types of edits and used this insight to generate reverse transcriptases that outperform PEmax and PEmaxΔRNaseH, the truncated editor used in dual-AAV delivery systems. Finally, we generated Cas9 domains that improve prime editing. These resulting editors (PE6a-g) enhance therapeutically relevant editing in patient-derived fibroblasts and primary human T-cells. PE6 variants also enable longer insertions to be installed in vivo following dual-AAV delivery, achieving 40% loxP insertion in the cortex of the murine brain, a 24-fold improvement compared to previous state-of-the-art prime editors.
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Texto completo: 1 Bases de datos: MEDLINE Asunto principal: Bacteriófagos / Ingeniería de Proteínas Límite: Animals / Humans Idioma: En Revista: Cell Año: 2023 Tipo del documento: Article País de afiliación: Estados Unidos
Texto completo
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Texto completo: 1 Bases de datos: MEDLINE Asunto principal: Bacteriófagos / Ingeniería de Proteínas Límite: Animals / Humans Idioma: En Revista: Cell Año: 2023 Tipo del documento: Article País de afiliación: Estados Unidos