CRISPR base editing enables the creation of targeted single-base conversions without generating double-stranded breaks. However, the efficiency of current base editors is very low in many cell types. We reengineered the sequences of BE3, BE4Gam, and xBE3 by codon optimization and incorporation of additional nuclear-localization sequences. Our collection of optimized constitutive and inducible base-editing vector systems dramatically improves the efficiency by which single-nucleotide variants can be created. The reengineered base editors enable target modification in a wide range of mouse and human cell lines, and intestinal organoids. We also show that the optimized base editors mediate efficient in vivo somatic editing in the liver in adult mice.
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Sequence Read Archive
This work was supported by a project grant from the NIH/NCI (CA195787-01), a U54 grant from the NIH/NCI (U54OD020355), a project grant from the Starr Cancer Consortium (I10-0095), a Research Scholar Award from the American Cancer Society (RSG-17-202-01), and a Stand Up to Cancer Colorectal Cancer Dream Team Translational Research Grant (SU2C-AACR-DT22-17). Stand Up to Cancer is a program of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, a scientific partner of SU2C. M.P.Z. is supported in part by National Cancer Institute (NCI) grant NIH T32 CA203702. E.M.S. was supported by a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the NIH under award number T32GM07739 to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD–PhD Program and an F31 Award from the NCI/NIH under grant number 1 F31 CA224800-01. E.R.K. is supported by an F31 NRSA predoctoral fellowship from the NCI/NIH under award number F31CA192835. F.J.S.-R. was supported by the MSKCC TROT program (5T32CA160001) and is supported as an HHMI Hanna Gray Fellow. S.W.L. is supported as the Geoffrey Beene Chair of Cancer Biology and as an Investigator of the Howard Hughes Medical Institute. D.F.T. is supported by the Helmholtz Association (VH-NG-1114) and by the German Research Foundation (DFG) project B05, SFB/TR 209 'Liver Cancer'. L.E.D. was supported by a K22 Career Development Award from the NCI/NIH (CA 181280-01). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We thank H. Varmus (Weill Cornell Medicine) for providing cells.