Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage

Abstract

The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

Extended Data Figure E1. Genotypes of 57 ABEs described in this work

Mutations are colored based on the round of evolution in which they were identified.

Extended Data Figure E2. Base editing efficiencies of additional early-stage ABE variants

a, Table of 19 human genomic DNA test sites (left) with corresponding locations on human chromosomes (right). The sequence context (target motif) of the edited A in red is shown for each site. PAM sequences are shown in blue. b, A•T to G•C base editing efficiencies in HEK293T cells of various wild-type RNA adenine deaminases fused to Cas9 nickase at six human genomic target DNA sites. Values reflect the mean and standard deviation of three biological replicates performed on different days. c, A•T to G•C base editing efficiencies in HEK293T cells of ABE2 editors with altered fusion orientations and linker lengths at six human genomic target DNA sites. d, A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of ABE2 editors fused to catalytically inactivated alkyl-adenosine glycosylase (AAG) or endonuclease V (EndoV), two proteins that bind inosine in DNA. e, A•T to G•C base editing efficiencies of ABE2.1 in HAP1 cells at site 1 with or without AAG. Values and error bars in (b) and (c) reflect the mean and s.d. of three independent biological replicates performed on different days.

Extended Data Figure E3. High-throughput DNA sequencing analysis of HEK293T cells treated with ABE2.1 and sgRNAs targeting each of six human genomic sites

One representative replicate is shown. Data from untreated HEK293T cells are shown for comparison.

Extended Data Figure E4. Base editing efficiencies of additional ABE2 and ABE3 variants, and the effect of adding A142N to TadA*–dCas9 on antibiotic selection survival in E. coli

a, A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of ABE2 variants with different engineered dimeric states. A control ABE variant containing two wild-type TadA domains and no evolved TadA* domains (ABE0.2) did not result in A•T to G•C editing at the six genomic sites tested, confirming that dimerization alone is insufficient to mediate ABE activity. b, A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of ABE3.1 variants differing in their dimeric state (homodimer of TadA*–TadA*–Cas9 nickase, or heterodimer of wild-type TadA–TadA*–Cas9 nickase), in the length of the TadA–TadA linker, and in the length of the TadA–Cas9 nickase linker. See Extended Data Figure E1 for ABE genotypes and architectures. c, Colony-forming units on 2xYT agar with 256 μg/mL of spectinomycin of E. coli cells expressing an sgRNA targeting the I89T defect in the spectinomycin resistance gene and a TadA*-dCas9 editor lacking or containing the A142N mutation identified in evolution round 4. Successful A•T to G•C base editing at the target site restores spectinomycin resistance. Values and error bars in (a) and (b) reflect the mean and s.d. of three independent biological replicates performed on different days.

Extended Data Figure E5. Base editing efficiencies of additional ABE5 variants

a, A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of two ABE3.1 variants with two pairs of mutations isolated from spectinomycin selection of the round 5 library. b, A•T to G•C base editing efficiencies in HEK293T cells at six human genomic target DNA sites of ABE5 variants with different linker lengths. See Extended Data Figure E1 for ABE genotypes and architectures. Values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days.

Extended Data Figure E6. Base editing efficiencies of ABE7 variants at 17 genomic sites

A•T to G•C base editing efficiencies in HEK293T cells at 17 human genomic target DNA sites of ABE7.1-7.5 (a), and ABE7.6-7.10 (b). See Extended Data Figure E1 for ABE genotypes and architectures. c, A•T to G•C base editing efficiencies in U2OS cells at six human genomic target DNA sites of ABE7.8-7.10. The lower editing efficiencies observed in U2OS cells compared with HEK293T cells are consistent with transfection efficiency differences between the two cell lines; we observed transfection efficiencies of ~40–55% in U2OS cells under the conditions used in this study, compared to ~65–80% in HEK293T cells. Values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days.

Extended Data Figure E7. Activity window of late-stage ABEs

a, Relative A•T to G•C base editing efficiencies in HEK293T cells of late-stage ABEs at protospacer positions 1–9 in two human genomic DNA sites that together place an A at each of these positions. Values are normalized to the maximum observed efficiency at each of the two sites for each ABE = 1. b, Relative A•T to G•C base editing efficiencies in HEK293T cells of late-stage ABEs at protospacer positions 1–18 and 20 across all 19 human genomic DNA sites tested. Values are normalized to the maximum observed efficiency at each of the 19 sites for each ABE = 1. For (a) and (b), values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days.

Extended Data Figure E8. Rounds of evolution and engineering increased ABE processivity

The calculated mean normalized linkage disequilibrium (LD) between nearby target As at 6 to 17 human genomic target DNA sites for the most active ABEs emerging from each round of evolution and engineering. Higher LD values indicate that an ABE is more likely to edit an A if a nearby A in the same DNA strand (the same sequencing read) is also edited. LD values are normalized from 0 to 1 in order to be independent of editing efficiency. Values and error bars reflect the mean and s.d. of normalized LD values from three independent biological replicates performed on different days.

Extended Data Figure E9. Analysis of cellular RNAs in ABE7.10-treated cells compared with untreated cells

RNA from HEK293T cells treated with ABE7.10 and the sgRNA targeting site 1, or from untreated HEK293T cells, was isolated and reverse transcribed into cDNA. The cDNAs corresponding to four abundant cellular RNAs (encoding beta-actin, beta-catenin, GAPDH, and RB1), and two cellular RNAs with sequence homology to the tRNA anticodon loop that is the native substrate of E. coli TadA (encoding MN1 and RSLD1), were amplified and analyzed by HTS. Within each amplicon, the mutation frequency at each adenine position for which the mutation rate was ≥ 0.2% is shown for two ABE7.10-treated biological replicates (ABE-treated 1 and ABE-treated 2) and for two untreated biological replicates (untreated 1 and untreated 2). The start of each amplicon is shown.

Extended Data Figure E10. High-throughput DNA sequencing analysis of HEK293T cells treated with five late-stage ABE variants and an sgRNA targeting -198T in the promoter of HBG1 and HBG2

One representative replicate is shown of DNA sequences at the HBG1 (a) and HBG2 (b) promoter targets. ABE-mediated base editing installs a -198T→C mutation on the strand complementary to the one shown in the sequencing data tables. Data from untreated HEK293T cells are shown for comparison.

Figure 1. Scope and overview of base editing by an A•T to G•C base editor (ABE)

a, Base pair changes required to correct pathogenic human SNPs in the ClinVar database. b, The deamination of adenosine (A) forms inosine (I), which is read as guanosine (G) by polymerase enzymes. c, ABE-mediated A•T to G•C base editing strategy. ABEs contain a hypothetical deoxyadenosine deaminase, which is not known to exist in nature, and a catalytically impaired Cas9. They bind target DNA in a guide RNA-programmed manner, exposing a small bubble of single-stranded DNA. The hypothetical deoxyadenosine deaminase domain catalyzes A to I formation within this bubble. Following DNA repair or replication, the original A•T base pair is replaced with a G•C base pair at the target site.

Figure 2. Protein evolution and engineering of ABEs

a, Strategy to evolve a DNA deoxyadenosine deaminase starting from TadA. A library of E. coli harbors a plasmid library of mutant ecTadA (TadA*) genes fused to dCas9 and a selection plasmid requiring targeted A•T to G•C mutations to repair antibiotic resistance genes. Mutations from surviving TadA* variants were imported into an ABE architecture for base editing in human cells. b, Genotypes of a subset of evolved ABEs. For a list of 57 evolved TadA* genotypes, see Extended Data Figure E1. The dimerization state (monomer, TadA*–TadA* homodimer, or wild-type TadA–evolved TadA* heterodimer) and linker length (in amino acids) are also listed. c, Three views of the E. coli TadA deaminase (PDB 1Z3A) aligned with S. aureus TadA (not shown) complexed with tRNA^Arg2 (PDB 2B3J). The UAC anticodon loop of the tRNA is the native substrate of wild-type TadA.

Figure 3. Evolved ABEs mediate A•T to G•C base editing at human genomic DNA sites

A•T to G•C base editing efficiencies in HEK293T cells of round 1 and round 2 ABEs (a), round 3, round 4, and round 5 ABEs (b), and round 6 and round 7 ABEs (c) at six human genomic DNA sites. d, Editing efficiencies in HEK293T cells of round 6 and round 7 ABEs at an expanded set of human genomic sites. Values and error bars reflect the mean and s.d. of three independent biological replicates performed on different days. Homodimer indicates fused TadA*–TadA*–Cas9 nickase architecture; heterodimer indicates fused wtTadA–TadA*–Cas9 nickase architecture. ABEs in (b) are homodimers except ABE5.3; ABEs in (c) and (d) are all heterodimers.

Figure 4. Product purity of late-stage ABEs

Product distributions and indel frequencies at two representative human genomic DNA sites in HEK293T cells treated with ABE7.10 or ABE7.9 and the corresponding sgRNA, or in untreated HEK293T cells. 22,746-111,215 sequencing reads were used at every position.

Figure 5. Comparison of ABE7.10-mediated base editing and Cas9-mediated HDR, and application of ABE7.10 to two disease-relevant SNPs

a, A•T to G•C base editing efficiencies in HEK293T cells treated either with ABE7.10, or with Cas9 nuclease and an ssDNA donor template (following the CORRECT HDR method) targeted to five human genomic DNA sites. b, Indel formation in HEK293T cells treated as described in (a). c, Application of ABE to install a disease-suppressing SNP, or to correct a disease-inducing SNP. Top: ABE7.10-mediated -198T→C mutation (on the strand complementary to the one shown) in the promoter region of HBG1 and HBG2 genes in HEK293T cells. The target A is at protospacer positon 7. Bottom: ABE7.10-mediated reversion of the C282Y mutation in the HFE gene in LCL cells. The target A is at protospacer position 5.