DNA deaminase enzymes play key roles in immunity and have recently been harnessed for their biotechnological applications. In base editors (BEs), the combination of DNA deaminase mutator activity with CRISPR–Cas localization confers the powerful ability to directly convert one target DNA base into another. While efforts have been made to improve targeting efficiency and precision, all BEs so far use a constitutively active DNA deaminase. The absence of regulatory control over promiscuous deaminase activity remains a major limitation to accessing the widespread potential of BEs. Here, we reveal sites that permit splitting of DNA cytosine deaminases into two inactive fragments, whose reapproximation reconstitutes activity. These findings allow for the development of split-engineered BEs (seBEs), which newly enable small-molecule control over targeted mutator activity. We show that the seBE strategy facilitates robust regulated editing with BE scaffolds containing diverse deaminases, offering a generalizable solution for temporally controlling precision genome editing.
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Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity
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High-throughput RNA-seq. data are deposited at the Gene Expression Omnibus database (accession number GSE181109). Individual amplicon sequencing data are available as Supplementary Information and raw reads will be available upon request. Novel plasmids used in this study are available from Addgene: AID’-BE4max (Addgene no. 174696), CMV-AID’-seBE4max-IRES (Addgene no. 174697), CMV-A3A-seBE4max-IRES (Addgene no. 174698), CMV-evoA1-seBE4max-IRES (Addgene no. 174699), Lenti-evoA1-BE4max (Addgene no. 174700), Lenti-evoA1-seBEn_empty (Addgene no. 174701) and Lenti-evoA1-seBEc (Addgene no. 174702). Nucleic acid sequences of all constructs used in this study are provided in a note at the end of the Supplementary Information. Source data are provided with this paper.
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We are grateful to M. Weitzman and K. Musunuru for helpful discussions. This work was in part supported by the Penn Center for Genomic Integrity and the US National Institutes of Health (NIH) through grant nos. R01-GM138908 and R01-HG010646 (to R.M.K.). K.N.B is an NSF Graduate Research Fellow. N.H.E. was supported by NIH T32-GM007170 and F30-HG011578. A.M.G. was supported by grant no. NIH K08-CA212299.
K.N.B., J.S. and R.M.K. through the University of Pennsylvania have filed a patent application on aspects of this work. R.M.K. is on the Scientific Advisory Board for Life Edit, Inc.
Peer review information Nature Chemical Biology thanks Reuben Harris, Moritoshi Sato and other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
(a) Construct schematics for AID* and AID*-INS variants (analogous to those in Fig. 1b) that were used to determine the impact of optGFP insertion in E. coli. Numbers above the constructs represent the amino acid sequence of the deaminase itself. (b) Construct schematics for co-expression or individual expression of AID*-SPL2N and AID*-SPL2C fragments in E. coli. (c) Left—an in vitro assay to measure deaminase activity on a 3’-fluorescein (FAM) labeled oligonucleotide substrate. UDG, uracil DNA glycosylase. Right—a representative denaturing gel (100 nM DNA, variable enzyme concentration) is shown, along with substrate and product controls (C and U, respectively). Analysis of product formation with three independent replicates was used to define the dose-response curve provided in Fig. 1d.
(a) Construct schematics for A3A and A3A-INS2 variants used to determine the impact of optGFP insertion in E. coli. Numbers above the constructs represent the amino acids of the deaminase itself. (b) Left—an in vitro assay to measure deaminase activity on a 3’-fluorescein (FAM) labeled oligonucleotide substrate. UDG, uracil DNA glycosylase. AP, abasic site. Middle—a representative denaturing gel (100 nM DNA, variable enzyme concentration) is shown, along with unreacted substrate and product controls (C and U, respectively). Right—product formation was quantified as a function of enzyme concentration (n = 3) and fit to a sigmoidal dose-response curve to determine the amount of enzyme needed to convert half of the substrate (EC50) under these fixed reaction conditions with 95% confidence interval shown. Each data point represents the mean and standard deviation across three biological replicates. (c) Construct schematics for mammalian expression of A3A-INS2, A3A(E72A)-INS2, and A3A-SPL2 variants used to determine the impact of optGFP insertion on the DNA damage response in HEK293T cells (d) HEK293T cells were transfected with catalytically inactive mutant A3A(E72A)-INS2, A3A-INS2, or co-transfected with A3A-SPL2N and A3A-SPL2C. After transfection, cells were stained for γH2AX and sorted for both GFP and γH2AX expression. The bar plot depicts frequency of GFP+ or GFP+/γH2AX+ cells after transfection of HEK293T cells with the indicated constructs, corresponding to the representative histogram shown in Fig. 1e. The mean, standard deviation, and individual observations from independent biological replicated are shown. (e) Representative immunofluorescent images of transfected U2OS cells are shown, corresponding to Fig. 1e. DAPI stain highlights the nucleus, GFP staining shows insert expression or split reconstitution, and γH2AX serves as a marker of active A3A-mediated DNA damage. A 10 µm scale bar is provided. Representative experiments were repeated independently three times and the results were reproducible.
(a) Parent construct schematics for intact BE4max scaffold editors with AID’, evoA1, and A3A. (b) Construct schematics for split-engineered seBE-T2A editors with AID’, evoA1, and A3A. Constructs were created by insertion of a cassette that splits the intact deaminase into two fragments, separated by a self-cleaving T2A peptide. At the bottom is the alternative seBE construct strategy, where the T2A fragment is replaced with an internal ribosome entry sequence (IRES) that leads to expression of two independently translated split protein fragments (denoted as seBEN and seBEC) with no need for protease processing.
Extended Data Fig. 4 Split-engineered base editors with A3A enable small-molecule-controlled editing.
Editing efficiency is evaluated in a d2gfp-HEK293T cell line. The presence of d2gfp-targeting sgRNA can introduce a stop codon (Q158*) and abrogate fluorescence to generate GFPoff cells, which can be tracked by either flow cytometry or deep-sequencing. Two-sided Mann–Whitney test was performed on all comparisons between means in this figure (n.s., not significant; *p ≤ 0.05, exact p-values provided as statistical source data files). (a) Representative flow cytometry histograms associated with transfection of intact or seBE-T2A constructs with or without rapamycin. (b) Mean, standard deviation and individual data points shown for quantification of GFPoff cells by flow cytometry (c) Left—deep sequencing results demonstrating C to T conversion efficiency at the target cytosine. The mean, standard deviation and three biological replicates are shown. Fold-change (FC) is the ratio of mean values for the higher versus lower condition. Right—editing footprints across the d2gfp locus for A3A-BE4max and A3A-seBE-T2A in the absence or presence of rapamycin. The numbers mark the distance from the protospacer adjacent motif site. The target cytosine base is noted with a blue arrow. Data represent position-wise averages of three biological replicates, with individual replicate data provided in Supplementary Table 1. (d) sgRNA-independent off-target genomic editing in cells transfected with intact A3A-BE4 or A3A-seBE-IRES and sgRNA targeting EXM1, along with dSaCas9-sgRNA targeting a different locus. Left—EMX1 on-target editing. Right—off-target editing at the locus opened by dSaCas9. The mean, standard deviation, and three biological replicates are shown. Editing footprints for each locus and replicates are provided in Supplementary Fig. 4. (e) Quantification of γH2AX upon base editor expression with or without rapamycin. HEK293T cells were transfected with an empty vector (pcDNA-EV), catalytically inactive mutant A3A(E72A)-BE4max, A3A-seBE-T2A, A3A-seBE-IRES, intact A3A-BE4max, or isolated A3A domain (pcDNA-A3A) and an EMX1-targeting sgRNA expressing GFP. Cells were either maintained with or without rapamycin and then stained for γH2AX. Shown are the percentage of γH2AX positive cells in the GFP positive population. The mean, standard deviation, and three biological replicates are shown. individual observations are shown (n = 3). Two-sided Mann-Whitney test was performed (n.s., not significant; *p ≤ 0.05, exact p-values provided as statistical source data files).
HEK293T cells were untreated or transfected with intact BE4max or seBE constructs in the absence or presence of rapamycin. C or G describes whether the coding (C) or non-coding (G) strand cytosine is targeted, respectively, with the subscript denoting the position of the sgRNA targeted by the base editor. Shown are experiments with control constructs and (a) evoA1-seBE-T2A (corresponding to Fig. 3b and Fig. 4b), (b) evoA1-seBE-IRES (corresponding to Fig. 3c and Fig. 4b), and (c) AID’-seBE-IRES (corresponding to Fig. 3c and Fig. 4b). Bars indicate mean and error bars indicate standard deviations of n = 3 biological replicates. Complete editing footprints for each locus and replicate in (a–c) are provided in Supplementary Figs. 1–3. (d) Western blot of cells transfected with evoA1-based constructs in the absence and presence of rapamycin. The Cas9 antibody probes the intact editor and the seBEC fragment (which are of similar size); Hsp90 antibody serves as a loading control; c-myc antibody probe the N-terminal tag of the seBEN fragment. Representative experiment was repeated independently two times and the results were reproducible.
Total RNA was analyzed using the RADAR pipeline (RNA-editing Analysis-pipeline to Decode All twelve-types of RNA-editing events). The unique edits for each sample were cataloged by removing edits contained in either of the sgRNA-only samples. Left—shown the pie charts indicating the type of edit in each of the three independent replicates. Right—mean fractions of specific edits across the three replicates are provided.
Extended Data Fig. 7 Lentiviral constructs for intact and split-engineered base editor editing in K562 cells.
(a) Lentiviral construct schematics for intact BE4 max, sgRNA-only, seBEC and seBEN + sgRNA are shown. (b) Quantification of GFPoff cells by flow cytometry for cells with EMX1-targeting sgRNA along with intact BE4max or seBE-IRES. Cells were either treated with no rapamycin or with rapamycin (25 nM) added at either day 3 or day 5 (marked with arrow) and then maintained continuously. Mean and standard deviation are noted, with individual data points shown (n = 3).
For all samples, viable cells were selected based on a forward scatter vs side scatter plot. Viable cells were then analyzed by the gating strategies shown. (a) Gating strategy for intact, inserted, and split A3A constructs shown in Fig. 1e and Extended Data Fig. 2d. (b) Gating strategy for base editing constructs generating GFPOFF cells shown in Fig. 2c and Extended Data Fig. 4a. Left - After sorting for live cells, a second gate for efficiently transfected cells was applied. Right – the GFPOFF population was then quantified. (c) Gating strategy for AID’- and A3A-based BE4max and seBE4max constructs shown in Fig. 4d and Extended Data Fig. 4e.
Unprocessed gels from Fig. 1d.
Statistical source data.
Statistical source data.
Statistical source data.
Unprocessed gels from Extended Data Fig. 1c.
Unprocessed gels from Extended Data Fig. 2b.
Statistical source data.
Unprocessed western blots from Extended Data Fig. 5d.
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Berríos, K.N., Evitt, N.H., DeWeerd, R.A. et al. Controllable genome editing with split-engineered base editors. Nat Chem Biol 17, 1262–1270 (2021). https://doi.org/10.1038/s41589-021-00880-w
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