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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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.
A.C.K. and D.R.L. have filed a provisional patent application on this work. D.R.L. is a consultant and co-founder of Editas Medicine, a company that seeks to develop genome-editing therapeutics.
Extended data figures and tables
a, Gel-based deaminase assay showing activity of rat APOBEC1 (rAPOBEC1), lamprey CDA1 (pmCDA1), human AID (hAID), human APOBEC3G (hAPOBEC3G), rAPOBEC1-GGS-dCas9, rAPOBEC1-(GGS)3-dCas9, and dCas9-(GGS)3-rAPOBEC1 on ssDNA. Enzymes were expressed in a mammalian cell lysate-derived in vitro transcription-translation system and incubated with 1.8 μM dye-conjugated ssDNA and USER enzyme (uracil DNA glycosylase and endonuclease VIII) at 37 °C for 2 h. The resulting DNA was resolved on a denaturing polyacrylamide gel and imaged. The positive control is a sequence with a U synthetically incorporated at the same position as the target C. b, Coomassie-stained denaturing PAGE of the expressed and purified proteins used in c–f. c–f, Gel-based deaminase assay showing the deamination window of base editors with deaminase–Cas9 linkers of GGS (c), (GGS)3 (d), XTEN (e), or (GGS)7 (f). Following incubation of 1.85 μM deaminase–dCas9 fusions complexed with sgRNA with 125 nM dsDNA substrates at 37 °C for 2 h, the dye-conjugated DNA was isolated and incubated with USER enzyme at 37 °C for 1 h to cleave the DNA backbone at the site of any Us. The resulting DNA was resolved on a denaturing polyacrylamide gel, and the dye-conjugated strand was imaged. Each lane is numbered according to the position of the target C within the protospacer, or labelled with ‘–’ if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8. For uncropped gel data, see Supplementary Fig. 1.
a, Protospacer and PAM sequences (blue) of seven disease-relevant mutations. The disease-associated target C in each case is indicated with a subscripted number reflecting its position within the protospacer. For all mutations except both APOE4 SNPs, the target C resides in the template (non-coding) strand. b, Deaminase assay showing each dsDNA 80-mer oligonucleotide before (–) and after (+) incubation with BE1, DNA isolation, and incubation with USER enzymes to cleave DNA at positions containing U. Positive control lanes from incubation of synthetic oligonucleotides containing U at various positions within the protospacer with USER enzymes are shown with the corresponding number indicating the position of the U. Editing efficiencies were quantitated by dividing the intensity of the cleaved product band by that of the entire lane for each sample. For uncropped gel data, see Supplementary Fig. 1.
The protospacer and PAM (blue) of a 60-mer DNA oligonucleotide containing eight consecutive Cs is shown at the top. The oligonucleotide (125 nM) was incubated with BE1 (2 μM) for 2 h at 37 °C. The DNA was isolated and analysed by high-throughput sequencing. Shown are the percent of total reads for the most frequent nine sequences observed. The vast majority of edited strands (>93%) have more than one C converted to T.
a, Protospacer (black and red) and PAM (blue) sequences of the six mammalian cell genomic loci targeted by base editors. Target Cs are indicated in red with subscripted numbers corresponding to their positions within the protospacer. b, Synthetic 80-mers with sequences matching six different genomic sites were incubated with BE1 then analysed for base editing by high-throughput sequencing. For each site, the sequence of the protospacer is indicated to the right of the name of the site, with the PAM highlighted in blue. Underneath each sequence are the percentages of total DNA sequencing reads with the corresponding base. We considered a target C as ‘editable’ if the in vitro conversion efficiency is >10%. Note that maximum yields are 50% of total DNA sequencing reads since the non-targeted strand is unaffected by BE1. Values are shown from a single experiment. c, HEK293T cells were transfected with plasmids expressing BE1 and an appropriate sgRNA. Three days after transfection, genomic DNA was extracted and analysed by high-throughput sequencing at the six loci. Cellular C to T conversion percentages, defined as the percentage of total DNA sequencing reads with Ts at the target positions indicated, are shown for BE1 at all six genomic loci. Values and error bars of all data from HEK293T cells reflect the mean and standard deviation of three independent biological replicates performed on different days.
Cellular C to T conversion percentages by BE2 are shown for each of the six targeted genomic loci in HEK293T cells and U2OS cells. HEK293T cells were transfected using Lipofectamine 2000, and U2OS cells were nucleofected. Three days after plasmid delivery, genomic DNA was extracted and analysed for base editing at the six genomic loci by HTS. Values and error bars reflect the mean and standard deviation of two (U2OS) or three (HEK293T) biological experiments performed on different days.
Cellular C to T conversion percentages by BE2 and BE3 are shown for HEK293 sites 3 and 4 in HEK293T cells before and after passaging the cells. HEK293T cells were nucleofected with plasmids expressing BE2 or BE3 and an sgRNA targeting HEK293 site 3 or 4. Three days after nucleofection, the cells were harvested and split in half. One half was subjected to high-throughput sequencing analysis, and the other half was allowed to propagate for approximately five cell divisions, then harvested and subjected to high-throughput sequencing analysis. Values and error bars reflect the mean and standard deviation of two biological experiments performed on different days.
Shown here are the C to T and G to A mutation rates at 3,200 distinct cytosines and guanines surrounding the six on-target and 34 off-target loci tested, representing a total of 14,700,000 sequence reads derived from approximately 1.8 × 106 cells. a, Cellular non-target C to T and G to A conversion percentages by BE1, BE2, and BE3 are plotted individually against their positions relative to a protospacer for all 3,200 cytosines/guanines. The side of the protospacer distal to the PAM is designated with positive numbers, while the side that includes the PAM is designated with negative numbers. b, Average non-target cellular C to T and G to A conversion percentages by BE1, BE2, and BE3 are shown, as well as the highest and lowest individual conversion percentages.
Extended Data Figure 8 Additional data sets of BE3-mediated correction of two disease-relevant mutations in mammalian cells.
For each site, the sequence of the protospacer is indicated to the right of the name of the mutation, with the PAM highlighted in blue and the base responsible for the mutation indicated in red bold with a subscripted number corresponding to its position within the protospacer. The amino acid sequence above each disease-associated allele is shown, together with the corrected amino acid sequence following base editing in green. Underneath each sequence are the percentages of total sequencing reads with the corresponding base. Cells were nucleofected with plasmids encoding BE3 and an appropriate sgRNA. Two days after nucleofection, genomic DNA was extracted from the nucleofected cells and analysed by high-throughput sequencing to assess pathogenic mutation correction. a, The Alzheimer’s disease-associated APOE4 allele is converted to APOE3r in mouse astrocytes by BE3 in 58.3% of total reads only when treated with the correct sgRNA. Two nearby Cs are also converted to Ts, but with no change to the predicted sequence of the resulting protein. Identical treatment of these cells with wild-type Cas9 and a 200-nt ssDNA donor results in 0.2% correction, with 26.7% indel formation. b, The cancer-associated p53 Y163C mutation is corrected by BE3 in 3.3% of nucleofected human breast cancer cells only when treated with the correct sgRNA. Identical treatment of these cells with wild-type Cas9 and donor ssDNA results in no detectable mutation correction with 8.0% indel formation.
Extended Data Figure 9 Genetic variants from ClinVar that, in principle, can be corrected by base editing.
The NCBI ClinVar database of human genetic variations and their corresponding phenotypes (see main text ref. 4) was searched for genetic diseases that can be corrected by current base editing technologies. The results were filtered by imposing the successive restrictions listed on the left. The x axis shows the number of occurrences satisfying that restriction and all above restrictions on a logarithmic scale.
This file contains a Supplementary Discussion, Supplementary Notes, Supplementary Sequences, Supplementary Tables 1-9, Supplementary References and Supplementary Figure 1. (PDF 2410 kb)
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Komor, A., Kim, Y., Packer, M. et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016). https://doi.org/10.1038/nature17946
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