Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity

Abstract

Cytosine base editors (CBEs) are larger and can suffer from higher off-target activity or lower on-target editing efficiency than current adenine base editors (ABEs). To develop a CBE that retains the small size, low off-target activity and high on-target activity of current ABEs, we evolved the highly active deoxyadenosine deaminase TadA-8e to perform cytidine deamination using phage-assisted continuous evolution. Evolved TadA cytidine deaminases contain mutations at DNA-binding residues that alter enzyme selectivity to strongly favor deoxycytidine over deoxyadenosine deamination. Compared to commonly used CBEs, TadA-derived cytosine base editors (TadCBEs) offer similar or higher on-target activity, smaller size and substantially lower Cas-independent DNA and RNA off-target editing activity. We also identified a TadA dual base editor (TadDE) that performs equally efficient cytosine and adenine base editing. TadCBEs support single or multiplexed base editing at therapeutically relevant genomic loci in primary human T cells and primary human hematopoietic stem and progenitor cells. TadCBEs expand the utility of CBEs for precision gene editing.

Fig. 1. Phage-assisted evolution of a cytidine deaminase from TadA-8e.

a, Evolutionary trajectory of a TadA-based cytidine deaminase from the tRNA deaminase, TadA. b, PACE overview. The selection phage (purple) encodes the evolving protein. E. coli hosts (gray) contain (1) a mutagenesis plasmid to diversify the phage (red) and (2) a plasmid system that regulates the expression of pIII (blue, encoded by gIII). Only variants with the desired activity trigger production of pIII and propagate. Phage without the desired activity cannot propagate and are diluted out of the lagoon. c, Selection circuit for cytidine deamination. TadA-8e variants are encoded on the SP (purple). The E. coli harbor three plasmids that establish the selection circuit, in addition to the mutagenesis plasmid: P1 contains the Cas9-UGI components of the base editor. Upon phage infection, the full base editor is reconstituted though the split Npu intein system (yellow). P2 encodes the guide RNA and gIII, which is under transcriptional control of the T7 promoter. P3 contains T7 RNA polymerase that is inactivated by fusion to a degron tag. C•G-to-T•A editing activity inserts a stop codon between T7 RNAP and the degron to yield active T7 RNAP, which leads to transcription of gIII and phage propagation. d, Two versions of the CBE circuit used in this work. In both cases, C•G-to-T•A editing inserts a stop codon before the degron tag, leading to active T7 RNAP. The less stringent circuit requires a C•G-to-T•A edit on the coding strand (top) and can tolerate one undesired A-to-G edit. The more stringent circuit requires a C•G-to-T•A edit on the non-coding, transcription template strand and cannot tolerate any undesired A•T-to-G•C edit. e, PANCE of a deoxycytidine deaminase from TadA-8e. The ProD (stronger, less stringent) or ProA (weaker, more stringent) promoter used in each PANCE passage is shown. At each passage, phage are diluted 1:50 unless indicated otherwise. After several rounds of evolution, phage titers stabilize despite increasing dilution rates between passages, suggesting the evolution of deoxycytidine deamination activity. ssDNA, single-stranded DNA. Source data

Fig. 2. Evolved TadA* variants catalyze deoxycytidine deamination.

a, Summary of TadA-8e variants evolved and characterized in this work. The variants are representative of conserved mutations after nine passages of PANCE or after 159 hours of PACE. For a full list of mutations, see Supplementary Figs. 2 and 3. b, Method for assessing base editing of target plasmids in E. coli. Cells are co-transformed with a target plasmid (blue) and a base editor plasmid (purple). Base editor expression is induced with arabinose. After 16 hours, cells are harvested, and the target plasmid is analyzed by high-throughput sequencing. c, Base editing in E. coli of a protospacer matching the selection circuit target site. C•G-to-T•A edits are shown in blue. A•T-to-G•C edits are shown in magenta. Dots represent individual biological replicates, and bars represent mean ± s.d. from four independent biological replicates. d, Locations of evolved mutations in the cryo-EM structure of ABE8e (PDB: 6VPC). Source data

Fig. 3. Characterization of evolved TadCBEs with SpCas9 domains in mammalian cells.

The specified base editors using SpCas9 nickase domains in the BE4max architecture or ABE8e with 2×UGI were transfected along with each of nine guide RNAs targeting the protospacers shown in each graph. Target cytosines are blue, target adenines are magenta, and PAM sequences are underlined. C•G-to-T•A base editing is shown in shades of blue. A•T-to-G•C base editing is shown in shades of magenta. Dots represent individual values, and bars represent mean ± s.d. of three independent biological replicates. HEK293T site 3 is abbreviated HEK3, and HEK293T site 4 is abbreviated HEK4. Source data

Fig. 4. Characterization of base editing window and Cas-independent off-target DNA and RNA editing by TadCBEs.

a, Base editing activity window for ABE8e with 2×UGI, TadCBEa and TadCBEa V106W across nine different target genomic sites in HEK293T. Dots represent average editing across all sites containing the specified base at the indicated position within the protospacer. Individual data points used for this analysis are in Fig. 3 and Supplementary Figs. 9 and 11. b, Method for measuring Cas-independent off-target DNA editing with the orthogonal R-loop assay. c, Average Cas-independent off-target editing across all cytosines within six orthogonal R-loops (SaR1–SaR6) generated by dead S. aureus Cas9. d, Off-target RNA editing. RNA was harvested from HEK293T cells 48 hours after transfection with the indicated base editor. After cDNA synthesis, CTNNB1, IP90 and RSL1D1 were amplified and analyzed by high-throughput sequencing. For c and d, dots represent individual biological replicates, and bars represent mean ± s.d. of three (c) or four (d) independent biological replicates. Source data

Fig. 5. Characterization of TadCBEs using a genomically integrated mESC target sequence library.

a, Overall efficiency and selectivity of base editors analyzed through editing of the library. Data show the average fraction of edited sequencing reads across all library members between protospacer positions −9 to 20, where positions 21–23 are the PAM. b, BE4max, TadCBEa–e, TadCBEd V106W and TadDE editing profiles across 10,638 genomically integrated target sites. The editing window is defined as the protospacer positions for which average editing efficiency is ≥30% of the average peak editing efficiency. Window plots for all variants tested in the library experiment can be found in Supplementary Fig. 28. c, Sequence motifs of TadCBEd and TadCBEd V106W for cytosine and adenine base editing outcomes determined by performing regression on editing efficiencies. Opacity of sequence motifs is proportional to the test R on a held-out set of sequences. Complete sequence motif plots for all variants are in Supplementary Fig. 30. Source data

Fig. 6. Base editing at therapeutically relevant loci by TadCBEs in primary human T cells and HSPCs.

mRNA encoding the indicated base editor or GFP as a negative control was electroporated into human T cells (n = 4 donors) along with two synthetic guide RNAs targeting CXCR4 (a) or CCR5 (b) at the specified protospacers. Target cytosines are blue, target adenines are magenta, and PAM sequences are underlined. After 3 days, genomic DNA was harvested from T cell lysates and analyzed by high-throughput sequencing. The gray boxes indicate the desired location of stop codon installation in CXCR4 and CCR5. The targeted cytidine to yield TAG (CXCR4) and TAA (CCR5) stop codons upon cytosine base editing is underlined. c, mRNA encoding the indicated base editor or GFP as a negative control was electroporated into HSPCs along with a synthetic guide RNA targeting the BCL11A enhancer. After 3 days, genomic DNA was harvested from cell lysates and analyzed by high-throughput sequencing. C•G-to-T•A base editing is shown in shades of blue, and A•T-to G•C-base editing is shown in shades of magenta. Dots represent individual biological replicates, and bars represent mean ± s.d. from n = 4 donors (a and b) or n = 3 donors (c). Source data