Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction1,2. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks1,2. Here we report the development of ‘base editing’, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting ‘base editors’ convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15–75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
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Sequence Read Archive
High-throughput sequencing data have been deposited in the NCBI Sequence Read Archive database under accession code SRP072434. Plasmids encoding BE1, BE2, and BE3 are available from Addgene (plasmids 73018, 73019, 73020, 73021).
This work was supported by US National Institutes of Health (NIH) R01 EB022376 (formerly R01 GM065400), F-Prime Biomedical Research Initiative (A28161), and the Howard Hughes Medical Institute. A.C.K. is a Ruth L. Kirchstein National Research Service Awards Postdoctoral Fellow (F32 GM 112366-2). Y.B.K. holds a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship (NSERC PGS-D). M.S.P. is an NSF Graduate Research Fellow and was supported by the Harvard Biophysics NIH training grant T32 GM008313. J.A.Z. was a Ruth L. Kirschstein National Research Service Award Postdoctoral Fellow (F32 GM 106601-2). We thank B. Hyman and E. Hudry for providing immortalized mouse astrocytes containing APOE4.
Extended data figures
This file contains a Supplementary Discussion, Supplementary Notes, Supplementary Sequences, Supplementary Tables 1-9, Supplementary References and Supplementary Figure 1.