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PreprintCited by 9CRISPR and Genetic EngineeringRNA regulation and diseaseVirus-based gene therapy research

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Engineering TadA ortholog-derived cytosine base editor without motif preference and adenosine activity limitation

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Zhou, Yingsi, Li, Guoling, Dong, Xue, Luo, Jiamin, Li, Tong, Zhao, Guoli, Zhang, Hainan, Zhou, Jingxing, Cui, Shuna, Zeng, Zhenhai, Wang, Haoqiang, Wang, Yin, Yu, Yuyang, Yuan, Yuan, Xu, Chunlong, Huang, Jinhai·2024·Nature Communications
DOI 10.21203/rs.3.rs-3876111/v1PMC PMC11405849

Preprint — published as 10.1038/s41467-024-52485-1 · Paper page

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Abstract

The engineered TadA variants used in cytosine base editors (CBEs) present unique advantages, including a smaller size and fewer off-target effects compared to cytosine base editors that rely on natural deaminases. However, the current TadA variants demonstrate a preference for base editing in DNA with specific motif sequences and possess dual deaminase activity, acting on both cytosine and adenosine in adjacent positions, limiting their application scope. To address these issues, we employed TadA orthologs screening and multi sequence alignment (MSA)-guided protein engineering techniques to create a highly effective cytosine base editor (aTdCBE) without motif and adenosine deaminase activity limitations. Notably, the delivery of aTdCBE to a humanized mouse model of Duchenne muscular dystrophy (DMD) mice achieved robust exon 55 skipping and restoration of dystrophin expression. Our advancement in engineering TadA ortholog for cytosine editing enrich the base editing toolkits for gene-editing therapy and other potential applications.

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Fig. 1 Screening for TadA orthologs with cytosine deaminase activity in mammalian cells. a The experimental workflow for detecting base editing activity with fluorescence reporter system in HEK293T cells. b The Phylogenetic Tree of TadA orthologs. c Screening for TadA orthologs using fluorescence reporter system. Data are presented as means ± S.D. Values and error bars represent mean and S.D., n = 3 independent biological replicates. All of the above base editors do not contain UGI. PAM stands for protospacer adjacent motif. NC represents negative control. Source data are provided as a Source Data file.
Fig. 1 Screening for TadA orthologs with cytosine deaminase activity in mammalian cells. a The experimental workflow for detecting base editing activity with fluorescence reporter system in HEK293T cells. b The Phylogenetic Tree of TadA orthologs. c Screening for TadA orthologs using fluorescence reporter system. Data are presented as means ± S.D. Values and error bars represent mean and S.D., n = 3 independent biological replicates. All of the above base editors do not contain UGI. PAM stands for protospacer adjacent motif. NC represents negative control. Source data are provided as a Source Data file.CC BY-NC-ND · PubMed Central
Fig. 2 Engineering of AjTadA to improve base editing efficiency in mammalian cells. a The experimental workflow for engineering of AjTadA. b Substitutions of non-positively charged amino acids of AjTadA at the AHC sites. Each dot represents activity for a single variant. c Substitutions of 25E and 46 P of AjTadA.v1 with seven amino acid residues. Values represent mean, n = 2 independent biological replicates. d Combination of mutations in 25E and 46 P of AjTadA.v1. Values and error bars represent means ± S.D., n = 3 independent biological replicates. e Combination of single amino acid mutation with AjTadA.v2 at three endogenous loci. Values and error bars represent means ± S.D., n = 3 independent biological replicates. All of the above base editors do not contain UGI. Source data are provided as a Source Data file.
Fig. 2 Engineering of AjTadA to improve base editing efficiency in mammalian cells. a The experimental workflow for engineering of AjTadA. b Substitutions of non-positively charged amino acids of AjTadA at the AHC sites. Each dot represents activity for a single variant. c Substitutions of 25E and 46 P of AjTadA.v1 with seven amino acid residues. Values represent mean, n = 2 independent biological replicates. d Combination of mutations in 25E and 46 P of AjTadA.v1. Values and error bars represent means ± S.D., n = 3 independent biological replicates. e Combination of single amino acid mutation with AjTadA.v2 at three endogenous loci. Values and error bars represent means ± S.D., n = 3 independent biological replicates. All of the above base editors do not contain UGI. Source data are provided as a Source Data file.CC BY-NC-ND · PubMed Central
Fig. 3 ATdCBE enables robust genomic base editing in mammalian cells. a Comparison of C-to-T conversion efficiency for endogenous loci by base editors derived from aTdCBE, Td-CBEmax, TadCBEd, B3PCY2-CBE, hA3A*-CBE and YE1-BE4max in HEK293T cells. b Base editing activity window plots showing mean C-to-T editing at all tested target positions. c Base editing activity window plots showing mean A-to-G editing at all tested target positions. d Comparison of motif preference for 25 endogenous loci by base editors derived from aTdCBE, Td-CBEmax, TadCBEd, B3PCY2-CBE, hA3A*-CBE and YE1-BE4max in HEK293T cells. The editing window of CBEs was primarily concentrated between bases 4–7. Consequently, the analysis focused on preference motifs exhibiting higher editing efficiency within this range. e High-throughput library experiments to evaluate the motif preference of aTdCBE, Td-CBEmax, TadCBEd and B3PCY2-CBE. f Introducing premature termination codons into the PCSK9 coding region using CBEs. The numbers in the grid represent the average editing efficiency of cytosines with corresponding motifs at positions 4–7 for each sgRNA. Data are presented as means ± S.D. Values represent n = 3 independent biological replicates. P-values determined by one-sided Mann-Whitney U-test and adjusted by Benjamini-Hochberg procedure. All of the above base editors contain UGI. NC represents negative control. Source data are provided as a Source Data file.
Fig. 3 ATdCBE enables robust genomic base editing in mammalian cells. a Comparison of C-to-T conversion efficiency for endogenous loci by base editors derived from aTdCBE, Td-CBEmax, TadCBEd, B3PCY2-CBE, hA3A*-CBE and YE1-BE4max in HEK293T cells. b Base editing activity window plots showing mean C-to-T editing at all tested target positions. c Base editing activity window plots showing mean A-to-G editing at all tested target positions. d Comparison of motif preference for 25 endogenous loci by base editors derived from aTdCBE, Td-CBEmax, TadCBEd, B3PCY2-CBE, hA3A*-CBE and YE1-BE4max in HEK293T cells. The editing window of CBEs was primarily concentrated between bases 4–7. Consequently, the analysis focused on preference motifs exhibiting higher editing efficiency within this range. e High-throughput library experiments to evaluate the motif preference of aTdCBE, Td-CBEmax, TadCBEd and B3PCY2-CBE. f Introducing premature termination codons into the PCSK9 coding region using CBEs. The numbers in the grid represent the average editing efficiency of cytosines with corresponding motifs at positions 4–7 for each sgRNA. Data are presented as means ± S.D. Values represent n = 3 independent biological replicates. P-values determined by one-sided Mann-Whitney U-test and adjusted by Benjamini-Hochberg procedure. All of the above base editors contain UGI. NC represents negative control. Source data are provided as a Source Data file.CC BY-NC-ND · PubMed Central
Fig. 4 ATdCBE treatment robustly rescued dystrophin expression in TA six weeks after AAV injection. a Overview for the in vivo intramuscular (IM) injection of the AAV9-aTdCBE construct into the tibialis anterior (TA) of the right leg of 3-week-old DMDΔ54 mdx mice. The DMDΔE54 mdx mice were derived from the mating of the humanized DMD∆E54 mice with mdx mice. Left leg was injected with saline as a control. Black arrows indicate time points for tissue collection after injection. AjTadA.v2-Y146R, UGIs, Cas9n split by Rma intein and gRNA were packaged into 2 separated AAV particles. A muscle-specific promoter Spc5.12 was used to drive AjTadA-Cas9n-N or Cas9n-C. b Schematic illustrating exon skipping strategies to restore the correct open reading frame (ORF) of the DMD transcript. The shape and color of the boxes representing DMD exons indicate the reading frame. Specifically, the deletion of exon 54 in the DMD gene results in a premature stop codon in exon 55 (depicted in red). The in-frame ORF can be achieved by editing exon 55 splice acceptor sites (SAS) (ag base pair). c The DNA editing efficiency was analyzed after 6-week treatment. d RT-PCR products from muscle of DMDΔE54 mdx mice were analyzed by gel electrophoresis. e Dystrophin (Abcam, ab15277) is shown in green. Scale bar, 100 μm. f Quantification of Dys+ fibers in cross sections of TA muscles. g Western blot analysis of dystrophin (Sigma, D8168) and vinculin (CST, 13901S) expression in TA muscles 6 weeks after injection with AAV-aTdCBE or saline. For comparative analyses, wild-type (WT) mice were derived from crosses between STOCK Tg (DMD) 72Thoen/J mice (#018900) and mdx mice, which carry a c.2977 C > T, p.Gln993* mutation in exon 23 on Chr.X. Dilutions of protein extract from WT mice were used to standardize dystrophin expression (25%, 50% and 75%). Each band (#1-4) represents an individual mouse sample. Vinculin was used as the loading control. Data are represented as mean ± S.E.M (n = 4 independent biological replicates). Each dot represents an individual mouse. Source data are provided as a Source Data file.
Fig. 4 ATdCBE treatment robustly rescued dystrophin expression in TA six weeks after AAV injection. a Overview for the in vivo intramuscular (IM) injection of the AAV9-aTdCBE construct into the tibialis anterior (TA) of the right leg of 3-week-old DMDΔ54 mdx mice. The DMDΔE54 mdx mice were derived from the mating of the humanized DMD∆E54 mice with mdx mice. Left leg was injected with saline as a control. Black arrows indicate time points for tissue collection after injection. AjTadA.v2-Y146R, UGIs, Cas9n split by Rma intein and gRNA were packaged into 2 separated AAV particles. A muscle-specific promoter Spc5.12 was used to drive AjTadA-Cas9n-N or Cas9n-C. b Schematic illustrating exon skipping strategies to restore the correct open reading frame (ORF) of the DMD transcript. The shape and color of the boxes representing DMD exons indicate the reading frame. Specifically, the deletion of exon 54 in the DMD gene results in a premature stop codon in exon 55 (depicted in red). The in-frame ORF can be achieved by editing exon 55 splice acceptor sites (SAS) (ag base pair). c The DNA editing efficiency was analyzed after 6-week treatment. d RT-PCR products from muscle of DMDΔE54 mdx mice were analyzed by gel electrophoresis. e Dystrophin (Abcam, ab15277) is shown in green. Scale bar, 100 μm. f Quantification of Dys+ fibers in cross sections of TA muscles. g Western blot analysis of dystrophin (Sigma, D8168) and vinculin (CST, 13901S) expression in TA muscles 6 weeks after injection with AAV-aTdCBE or saline. For comparative analyses, wild-type (WT) mice were derived from crosses between STOCK Tg (DMD) 72Thoen/J mice (#018900) and mdx mice, which carry a c.2977 C > T, p.Gln993* mutation in exon 23 on Chr.X. Dilutions of protein extract from WT mice were used to standardize dystrophin expression (25%, 50% and 75%). Each band (#1-4) represents an individual mouse sample. Vinculin was used as the loading control. Data are represented as mean ± S.E.M (n = 4 independent biological replicates). Each dot represents an individual mouse. Source data are provided as a Source Data file.CC BY-NC-ND · PubMed Central

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Citations

Cites

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  31. Flati T et al (2020) HPC-REDItools: a novel HPC-aware tool for improved large scale RNA-editing analysis. BMC. Bioinformatics. 21, 353, doi:10.1186/s12859-020-03562-x Methods Computational analysis of TadA orthologs Firstly, we downloaded 15167 TadA protein sequences from NCBI database. We further used BLASTP to remove redundant proteins with identity over than 90%24. Then, we performed multiple sequence alignment using MAFFT25. MEGA11 were used to construct phylogenetic tree26. Calculate_AHC.pl were used to identify highly conservative residues and AHC residues. Plasmid constructions Human Codon-optimized orthologous TadAs were synthesized commercially (GenScript Co., Ltd) and cloned to generate pT7_NLS-TadA-Cas9-NLS_pA_pCBH_mCherry_pA plasmid by NEBuilder (New England Biolabs). All sequences are listed in Supplementary Table 1. Mammalian cell culture, transfection and flow cytometry analysis The HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, 11965-092) supplemented with 10% fetal bovine serum (Gibco, 10099-141C), and 1% Pen-Strep-Glutamine (100×) (Gibco, 10378-016) at 37°C with 5% CO2 in a cell incubator. For TadA variants screening, HEK293T cells cultured in 24-well plates were co-transfected with 1.0 µg of tagBFP-*EGFP reporter plasmid and TadA-mCherry plasmid in a molar ratio of 1:1 with Polyetherimide (PEI). After 48 hours, mCherry, BFP and EGFP fluorescence were analyzed by Beckman CytoFlex flow-cytometer. To evaluate genome editing in endogenous sites, cells were harvested at 48 hours after transfection and sorted by BD FACS Aria III flow cytometer. FACS data were analyzed with FlowJo X (v10.0.7). Detection of gene editing frequency · DOI
  32. To evaluate the specificity of TadA base editors, the Cas-OFFinder was employed to predict the potential off-target sites28. Search queries covered both Cas9 spacer sequence and PAM of the on-target site. The PAM of research was set to NGG and the mismatches were set to less than 5. All other parameters were left as default. The potential off-target sites were amplified and deep sequenced for analysis
  33. All cell experimental results are presented as mean ± s.d, while all animal experimental results are presented as mean ± s.e.m. Unpaired two-tailed Student’s t-test was used for comparisons and P-value < 0.05 was considered to be statistically significant. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. GraphPad Prism (v 9.0.0) was used for statistics (www.graphpad.com/).

Metadata

Authors
Zhou, Y., Li, G., Dong, X., Luo, J., Li, T., Zhao, G., Zhang, H., Zhou, J., Cui, S., Zeng, Z., Wang, H., Wang, Y., Yu, Y., Yuan, Y., Xu, C., & Huang, J.
Affiliations
  • HuidaGene Therapeutics Inc., Shanghai, China.
  • Huigene Therapeutics Inc.
  • Chinese Academy of Medical Sciences & Peking Union Medical College (CN)
  • HuidaGene Therapeutics Inc.
  • HUIDAGENE Therapeutics Co., Ltd.
  • Lingang Laboratory, Shanghai Center for Brain Science and Brain-Inspired Technology
Corresponding author
  • Zhou, Yingsi
  • Xu, Chunlong
  • Huang, Jinhai
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2024-02-27
Volume
15
Issue
1
Pages
8090-8090
ISSN
2041-1723 (ISSN-L)
DOI
10.21203/rs.3.rs-3876111/v1
PMC
PMC11405849
OpenAlex
W4402581033
Cited by
9
References
1
Citations by year
2026: 2 · 2025: 7
FWCI
1.22
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by-nc-nd)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China ([2021], 82271048, 22S11900200, 82301215)
  • Government of Jiangsu Province
  • Science and Technology Commission of Shanghai Municipality (22S11900200, 23XD1420500)
  • Chinese Academy of Agricultural Sciences
  • Jiangsu Provincial Key Research and Development Program (BE2021372)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
Keywords
TADA, cytosine base editors, adenosine deaminase activity, protein engineering, exon skipping, Duchenne muscular dystrophy, dystrophin expression, gene editing therapy, off-target effects
MeSH terms
Gene Editing, Adenosine, Adenosine Deaminase, Animals, Cytosine, Disease Models, Animal, Exons, Humans, Protein Engineering, Dystrophin, Muscular Dystrophy, Duchenne, Escherichia coli Proteins, Mice, HEK293 Cells, CRISPR-Cas Systems

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