Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage

Abstract

Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. 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 breaks. 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.

Extended Data Figure 1. Effects of deaminase, linker length, and linker composition on base editing

a, Gel-based deaminase assay showing activity of rAPOBEC1, pmCDA1, hAID, 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), (d), (e), and (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 with – if no target C is present. 8U is a positive control sequence with a U synthetically incorporated at position 8. For gel source data, see Supplementary Figure 1.

Extended Data Figure 2. BE1 is capable of correcting disease-relevant mutations in vitro

a, Protospacer and PAM sequences (red) 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 gel source data, see Supplementary Figure 1.

Extended Data Figure 3. Processivity of BE1

The protospacer and PAM (red) 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 analyzed 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.

Extended Data Figure 4. BE1 base editing efficiencies are dramatically decreased in mammalian cells

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 analyzed for base editing by HTS. 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 analyzed by HTS 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.

Extended Data Figure 5. Base editing efficiencies of BE2 in U2OS and HEK293T cells

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 analyzed for base editing at the six genomic loci by HTS. Values and error bars reflect the mean and standard deviation of at least two biological experiments done on different days.

Extended Data Figure 6. Base editing persists over multiple cell divisions

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 HTS analysis, and the other half was allowed to propagate for approximately five cell divisions, then harvested and subjected to HTS analysis. Values and error bars reflect the mean and standard deviation of at least two biological experiments.

Extended Data Figure 7. Non-target C/G mutation rates

Shown here are the C to T and G to A mutation rates at 2,500 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 2,500 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 analyzed by HTS 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 wt Cas9 and donor ssDNA 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 wt 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 editingg

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.

Figure 1. BE1 mediates specific, guide RNA-programmed C→U conversion in vitro

a, Base editing strategy. DNA with a target C (red) at a locus specified by a guide RNA (green) is bound by dCas9 (blue), which mediates local DNA strand separation. Cytidine deamination by a tethered APOBEC1 enzyme (red) converts the single-stranded target C→U. The resulting G:U heteroduplex can be permanently converted to an A:T bp following DNA replication or DNA repair. b, Deamination assay showing a BE1 activity window of approximately five nt. Samples were prepared as described in the Methods. Each lane is labeled according to the position of the target C within the protospacer, or with “–” if no target C is present, counting the base distal from the PAM as position 1. c, Deamination assay showing the sequence specificity and sgRNA-dependence of BE1. The DNA substrate in b was incubated with BE1 and the correct sgRNA, a mismatched sgRNA, or no sgRNA. The positive control sample used a synthetic DNA substrate with a U at position 7. For gel source data, see Supplementary Figure 1.

Figure 2. Effects of sequence context and target C position on base editing efficiency in vitro

a, Effect of changing the sequence surrounding the target C on editing efficiency in vitro. The deamination yield of 80% of targeted strands (40% of total sequencing reads from both strands) for C₇ in the protospacer sequence 5′-TTATTTCGTGGATTTATTTA-3′ was defined as 1.0, and the relative deamination efficiencies of substrates containing all possible single-base mutations at positions 1-6 and 8-13 are shown. b, Positional effect of each NC motif on editing efficiency in vitro. Each NC target motif was varied from positions 1 to 8 within the protospacer as indicated in the sequences shown on the right. The PAM is shown in blue. The graph shows the percentage of total DNA sequencing reads containing T at each of the numbered target C positions following incubation with BE1. Note that the maximum possible deamination yield in vitro is 50% of total sequencing reads (100% of targeted strands). Values and error bars reflect the mean and standard deviation of three (for a) or two (for b) independent biological replicates performed on different days.

Figure 3. Base editing in human cells

a, Possible base editing outcomes in mammalian cells. Initial editing results in a U:G mismatch. Recognition and excision of the U by uracil DNA glycosylase (UDG) initiates base excision repair (BER), which leads to reversion to the C:G starting state. BER is impeded by BE2 and BE3, which inhibit UDG. The U:G mismatch is also processed by mismatch repair (MMR), which preferentially repairs the nicked strand of a mismatch. BE3 nicks the non-edited strand containing the G, favoring resolution of the U:G mismatch to the desired U:A or T:A outcome. b, HEK293T cells were treated as described in the Methods. The percentage of total DNA sequencing reads with Ts at the target positions indicated are shown for treatment with BE1, BE2, or BE3, or for treatment with wt Cas9 with a donor HDR template. c, Frequency of indel formation (see Methods) is shown following the treatment in b. Values are listed in Supplementary Table 6. For b and c, values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days.