A-to-G/C/T and C-to-T/G/A dual-function base editor for creating multi-nucleotide variants

Bingxiu Ma; Han Wu; Shixue Gou; Meng Lian; Cong Xia; Kaiming Yang; Long Jin; Junyuan Liu; Yunlin Wu; Yahai Shu; Haizhao Yan; Zhanjun Li; Liangxue Lai; Yong Fan

doi:10.1016/j.jgg.2024.10.001

Volume 51 Issue 12

Dec. 2024

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Article Navigation > Journal of Genetics and Genomics > 2024 > 51(12): 1494-1504

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A-to-G/C/T and C-to-T/G/A dual-function base editor for creating multi-nucleotide variants

doi: 10.1016/j.jgg.2024.10.001

a. Department of Obstetrics and Gynecology, Guangdong Provincial Key Laboratory of Major Obstetric Diseases, Guangdong Provincial Clinical Research Center for Obstetrics and Gynecology, Guangdong-Hong Kong-Macao Greater Bay Area Higher Education Joint Laboratory of Maternal-Fetal Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510150, China;
b. China-New Zealand Joint Laboratory on Biomedicine and Health, CAS Key Laboratory of Regenerative Biology, Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China;
c. Sanya Institute of Swine Resource, Hainan Provincial Research Centre of Laboratory Animals, Sanya, Hainan 572000, China;
d. Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019RU015), Guangzhou, Guangdong 510530, China;
e. Guangzhou National Laboratory, Guangzhou, Guangdong 510005, China;
f. Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100020, China;
g. Key Laboratory of Zoonosis Research, Ministry of Education, College of Animal Science, Jilin University, Changchun, Jilin 130062, China

Funds:

This work was supported by the National Key Research and Development Program of China (2021YFC2700904, 2021YFF0702601, 2021YFA0805300), the National Natural Science Foundation of China (82071723, 82100482, 82101733), Guangdong Basic and Applied Basic Research Foundation (2021B1515020069), Guangzhou Science and Technology Planning Project (2023A03J0377), and Research Unit of Generation of Large Animal Disease Models, Chinese Academy of Medical Sciences (2019-I2M-5-025).

Received Date: 2024-05-01
Accepted Date: 2024-10-04
Rev Recd Date: 2024-10-02

Available Online: 2025-06-05

Publish Date: 2024-10-25

Abstract

Abstract

Multi-nucleotide variants (MNVs) are critical genetic variants associated with various genetic diseases. However, tools for precisely installing MNVs are limited. In this study, we present the development of a dual-base editor, BDBE, by integrating TadA-dual and engineered human N-methylpurine DNA glycosylase (eMPG) into nCas9 (D10A). Our results demonstrate that BDBE effectively converts A-to-G/C/T (referred to as A-to-B) and C-to-T/G/A (referred to as C-to-D) simultaneously, yielding nine types of dinucleotides from adjacent CA nucleotides while maintaining minimal off-target effects. Notably, BDBE4 exhibits exceptional performance across multiple human cell lines and successfully simulated all nine dinucleotide MNVs from the gnomAD database. These findings indicate that BDBE significantly expands the product range of base editors and offers a valuable resource for advancing MNV research.
- CRISPR,
- Gene editing,
- Dual-base editor,
- A-to-G/C/T and C-to-T/G/A,
- MNV,
- Genetic diseases

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References(61)

References

Anzalone, A.V., Randolph, P.B., Davis, J.R., Sousa, A.A., Koblan, L.W., Levy, J.M., Chen, P.J., Wilson, C., Newby, G.A., Raguram, A., et al., 2019. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157.

Bae, S., Park, J., Kim, J.S., 2014. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475.

Besenbacher, S., Sulem, P., Helgason, A., Helgason, H., Kristjansson, H., Jonasdottir, A., Jonasdottir, A., Magnusson, O.T., Thorsteinsdottir, U., Masson, G., et al., 2016. Multi-nucleotide de novo mutations in humans. PLoS Genet. 12, e1006315.

Bock, D., Rothgangl, T., Villiger, L., Schmidheini, L., Matsushita, M., Mathis, N., Ioannidi, E., Rimann, N., Grisch-Chan, H.M., Kreutzer, S., et al., 2022. In vivo prime editing of a metabolic liver disease in mice. Sci. Transl. Med. 14, eabl9238.

Bollen, Y., Post, J., Koo, B.K., Snippert, H.J.G., 2018. How to create state-of-the-art genetic model systems: strategies for optimal CRISPR-mediated genome editing. Nucleic Acids Res. 46, 6435-6454.

Chaim, I.A., Nagel, Z.D., Jordan, J.J., Mazzucato, P., Ngo, L.P., Samson, L.D., 2017. In vivo measurements of interindividual differences in DNA glycosylases and APE1 activities. Proc. Natl. Acad. Sci. U. S. A. 114, E10379-E10388.

Chen, H., Liu, S., Padula, S., Lesman, D., Griswold, K., Lin, A., Zhao, T., Marshall, J.L., Chen, F., 2020. Efficient, continuous mutagenesis in human cells using a pseudo-random DNA editor. Nat. Biotechnol. 38, 165-168.

Chen, L., Park, J.E., Paa, P., Rajakumar, P.D., Prekop, H.T., Chew, Y.T., Manivannan, S.N., Chew, W.L., 2021a. Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins. Nat. Commun. 12, 1384.

Chen, T., Chen, X., Zhang, S., Zhu, J., Tang, B., Wang, A., Dong, L., Zhang, Z., Yu, C., Sun, Y., et al., 2021b. The genome sequence archive family: toward explosive data growth and diverse data types. Genomics Proteomics Bioinformatics 19, 578-583.

Clement, K., Rees, H., Canver, M.C., Gehrke, J.M., Farouni, R., Hsu, J.Y., Cole, M.A., Liu, D.R., Joung, J.K., Bauer, D.E., et al., 2019. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224-226.

Members, C.-N., Partners, 2022. Database resources of the National Genomics Data Center, China National Center for Bioinformation in 2022. Nucleic Acids Res. 50, D27-D38.

Degalez, F., Jehl, F., Muret, K., Bernard, M., Lecerf, F., Lagoutte, L., Desert, C., Pitel, F., Klopp, C., Lagarrigue, S., 2021. Watch out for a second SNP: focus on multi-nucleotide variants in coding regions and rescued stop-gained. Front. Genet. 12, 659287.

Ding, X., Seebeck, T., Feng, Y., Jiang, Y., Davis, G.D., Chen, F., 2019. Improving CRISPR-Cas9 genome editing efficiency by fusion with chromatin-modulating peptides. CRISPR J. 2, 51-63.

Doman, J.L., Raguram, A., Newby, G.A., Liu, D.R., 2020. Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors. Nat. Biotechnol. 38, 620-628.

Gaudelli, N.M., Komor, A.C., Rees, H.A., Packer, M.S., Badran, A.H., Bryson, D.I., Liu, D.R., 2017. Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage. Nature 551, 464-471.

Ghosh, D., Raghavan, S.C., 2021. Nonhomologous end joining: new accessory factors fine tune the machinery. Trends Genet. 37, 582-599.

He, Y., Zhou, X., Chang, C., Chen, G., Liu, W., Li, G., Fan, X., Sun, M., Miao, C., Huang, Q., et al., 2024. Protein language models-assisted optimization of a uracil-N-glycosylase variant enables programmable T-to-G and T-to-C base editing. Mol. Cell 84, 1257-1270 e1256.

Hess, G.T., Fresard, L., Han, K., Lee, C.H., Li, A., Cimprich, K.A., Montgomery, S.B., Bassik, M.C., 2016. Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat. Methods 13, 1036-1042.

Hindi, N.N., Elsakrmy, N., Ramotar, D., 2021. The base excision repair process: comparison between higher and lower eukaryotes. Cell Mol. Life. Sci. 78, 7943-7965.

Huang, M.E., Qin, Y., Shang, Y., Hao, Q., Zhan, C., Lian, C., Luo, S., Liu, L.D., Zhang, S., Zhang, Y., et al., 2024. C-to-G editing generates double-strand breaks causing deletion, transversion and translocation. Nat. Cell Biol. 26, 294-304.

Kaplanis, J., Akawi, N., Gallone, G., Mcrae, J.F., Prigmore, E., Wright, C.F., Fitzpatrick, D.R., Firth, H.V., Barrett, J.C., Hurles, M.E., et al., 2019. Exome-wide assessment of the functional impact and pathogenicity of multinucleotide mutations. Genome Res. 29, 1047-1056.

Karczewski, K.J., Francioli, L.C., Tiao, G., Cummings, B.B., Alfoldi, J., Wang, Q., Collins, R.L., Laricchia, K.M., Ganna, A., Birnbaum, D.P., et al., 2020. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature 581, 434-443.

Kim, K., Ryu, S.M., Kim, S.T., Baek, G., Kim, D., Lim, K., Chung, E., Kim, S., Kim, J.S., 2017. Highly efficient RNA-guided base editing in mouse embryos. Nat. Biotechnol. 35, 435-437.

Koblan, L.W., Arbab, M., Shen, M.W., Hussmann, J.A., Anzalone, A.V., Doman, J.L., Newby, G.A., Yang, D., Mok, B., Replogle, J.M., et al., 2021. Efficient C∗G-to-G∗C base editors developed using CRISPRi screens, target-library analysis, and machine learning. Nat. Biotechnol. 39, 1414-1425.

Koblan, L.W., Doman, J.L., Wilson, C., Levy, J.M., Tay, T., Newby, G.A., Maianti, J.P., Raguram, A., Liu, D.R., 2018. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat. Biotechnol. 36, 843-846.

Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A., Liu, D.R., 2016. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420-424.

Kurt, I.C., Zhou, R., Iyer, S., Garcia, S.P., Miller, B.R., Langner, L.M., Grunewald, J., Joung, J.K., 2021. CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat. Biotechnol. 39, 41-46.

Lam, D.K., Feliciano, P.R., Arif, A., Bohnuud, T., Fernandez, T.P., Gehrke, J.M., Grayson, P., Lee, K.D., Ortega, M.A., Sawyer, C., et al., 2023. Improved cytosine base editors generated from TadA variants. Nat. Biotechnol. 41, 686-697.

Lau, A.Y., Wyatt, M.D., Glassner, B.J., Samson, L.D., Ellenberger, T., 2000. Molecular basis for discriminating between normal and damaged bases by the human alkyladenine glycosylase, AAG. Proc. Natl. Acad. Sci. U. S. A. 97, 13573-13578.

Lek, M., Karczewski, K.J., Minikel, E.V., Samocha, K.E., Banks, E., Fennell, T., O'donnell-Luria, A.H., Ware, J.S., Hill, A.J., Cummings, B.B., et al., 2016. Analysis of protein-coding genetic variation in 60,706 humans. Nature 536, 285-291.

Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., Genome Project Data Processing, S., 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078-2079.

Liang, Y., Xie, J., Zhang, Q., Wang, X., Gou, S., Lin, L., Chen, T., Ge, W., Zhuang, Z., Lian, M., et al., 2022. AGBE: a dual deaminase-mediated base editor by fusing CGBE with ABE for creating a saturated mutant population with multiple editing patterns. Nucleic Acids Res. 50, 5384-5399.

Liu, Y., Li, X., He, S., Huang, S., Li, C., Chen, Y., Liu, Z., Huang, X., Wang, X., 2020. Efficient generation of mouse models with the prime editing system. Cell Discov. 6, 27.

Liu, Z., Chen, M., Chen, S., Deng, J., Song, Y., Lai, L., Li, Z., 2018a. Highly efficient RNA-guided base editing in rabbit. Nat. Commun. 9, 2717.

Liu, Z., Lu, Z., Yang, G., Huang, S., Li, G., Feng, S., Liu, Y., Li, J., Yu, W., Zhang, Y., et al., 2018b. Efficient generation of mouse models of human diseases via ABE- and BE-mediated base editing. Nat. Commun. 9, 2338.

Ma, Y., Zhang, J., Yin, W., Zhang, Z., Song, Y., Chang, X., 2016. Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods 13, 1029-1035.

Madhusudan, S., Smart, F., Shrimpton, P., Parsons, J.L., Gardiner, L., Houlbrook, S., Talbot, D.C., Hammonds, T., Freemont, P.A., Sternberg, M.J., et al., 2005. Isolation of a small molecule inhibitor of DNA base excision repair. Nucleic Acids Res. 33, 4711-4724.

Mckenna, A., Hanna, M., Banks, E., Sivachenko, A., Cibulskis, K., Kernytsky, A., Garimella, K., Altshuler, D., Gabriel, S., Daly, M., et al., 2010. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297-1303.

Mehta, K.P.M., Lovejoy, C.A., Zhao, R., Heintzman, D.R., Cortez, D., 2020. HMCES maintains replication fork progression and prevents double-strand breaks in response to APOBEC deamination and abasic site formation. Cell Rep. 31, 107705.

Mohni, K.N., Wessel, S.R., Zhao, R., Wojciechowski, A.C., Luzwick, J.W., Layden, H., Eichman, B.F., Thompson, P.S., Mehta, K.P.M., Cortez, D., 2019. HMCES maintains genome integrity by shielding abasic sites in single-strand DNA. Cell 176, 144-153 e113.

Neugebauer, M.E., Hsu, A., Arbab, M., Krasnow, N.A., Mcelroy, A.N., Pandey, S., Doman, J.L., Huang, T.P., Raguram, A., Banskota, S., et al., 2023. Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nat. Biotechnol. 41, 673-685.

Priestley, P., Baber, J., Lolkema, M.P., Steeghs, N., De Bruijn, E., Shale, C., Duyvesteyn, K., Haidari, S., Van Hoeck, A., Onstenk, W., et al., 2019. Pan-cancer whole-genome analyses of metastatic solid tumours. Nature 575, 210-216.

Rattray, A.J., Strathern, J.N., 2003. Error-prone DNA polymerases: when making a mistake is the only way to get ahead. Annu. Rev. Genet. 37, 31-66.

Richter, M.F., Zhao, K.T., Eton, E., Lapinaite, A., Newby, G.A., Thuronyi, B.W., Wilson, C., Koblan, L.W., Zeng, J., Bauer, D.E., et al., 2020. Phage-assisted evolution of an adenine base editor with improved Cas domain compatibility and activity. Nat. Biotechnol. 38, 883-891.

Saparbaev, M., Laval, J., 1994. Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases. Proc. Natl. Acad. Sci. U. S. A. 91, 5873-5877.

Song, F., Stieger, K., 2017. Optimizing the DNA donor template for homology-directed repair of double-strand breaks. Mol. Ther. Nucleic Acids 7, 53-60.

Srinivasan, S., Kalinava, N., Aldana, R., Li, Z., Van Hagen, S., Rodenburg, S.Y.A., Wind-Rotolo, M., Qian, X., Sasson, A.S., Tang, H., et al., 2021. Misannotated multi-nucleotide variants in public cancer genomics datasets lead to inaccurate mutation calls with significant implications. Cancer Res. 81, 282-288.

Tang, S., Stokasimov, E., Cui, Y., Pellman, D., 2022. Breakage of cytoplasmic chromosomes by pathological DNA base excision repair. Nature 606, 930-936.

Tao, W., Liu, Q., Huang, S., Wang, X., Qu, S., Guo, J., Ou, D., Li, G., Zhang, Y., Xu, X., et al., 2021. CABE-RY: A PAM-flexible dual-mutation base editor for reliable modeling of multi-nucleotide variants. Mol. Ther. Nucleic Acids 26, 114-121.

Tong, H., Liu, N., Wei, Y., Zhou, Y., Li, Y., Wu, D., Jin, M., Cui, S., Li, H., Li, G., et al., 2023a. Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase. Natl. Sci. Rev. 10, nwad143.

Tong, H., Wang, X., Liu, Y., Liu, N., Li, Y., Luo, J., Ma, Q., Wu, D., Li, J., Xu, C., et al., 2023b. Programmable A-to-Y base editing by fusing an adenine base editor with an N-methylpurine DNA glycosylase. Nat. Biotechnol. 41, 1080-1084.

Wang, D., Cao, W., Yang, W., Jin, W., Luo, H., Niu, X., Gong, J., 2022. Pancan-MNVQTLdb: systematic identification of multi-nucleotide variant quantitative trait loci in 33 cancer types. NAR Cancer 4, zcac043.

Wang, Q., Pierce-Hoffman, E., Cummings, B.B., Alfoldi, J., Francioli, L.C., Gauthier, L.D., Hill, A.J., O'donnell-Luria, A.H., Genome Aggregation Database Production, T., Genome Aggregation Database, C., et al., 2020. Landscape of multi-nucleotide variants in 125,748 human exomes and 15,708 genomes. Nat. Commun. 11, 2539.

Yang, C., Ma, Z., Wang, K., Dong, X., Huang, M., Li, Y., Zhu, X., Li, J., Cheng, Z., Bi, C., et al., 2023. HMGN1 enhances CRISPR-directed dual-function A-to-G and C-to-G base editing. Nat. Commun. 14, 2430.

Yang, W., Gao, Y., 2018. Translesion and Repair DNA Polymerases: Diverse Structure and Mechanism. Annu. Rev. Biochem. 87, 239-261.

Ye, L., Zhao, D., Li, J., Wang, Y., Li, B., Yang, Y., Hou, X., Wang, H., Wei, Z., Liu, X., et al., 2024. Glycosylase-based base editors for efficient T-to-G and C-to-G editing in mammalian cells. Nat. Biotechnol.

Ye, N., Ding, Y., Wild, C., Shen, Q., Zhou, J., 2014. Small molecule inhibitors targeting activator protein 1 (AP-1). J. Med. Chem. 57, 6930-6948.

Zawahir, Z., Dayam, R., Deng, J., Pereira, C., Neamati, N., 2009. Pharmacophore guided discovery of small-molecule human apurinic/apyrimidinic endonuclease 1 inhibitors. J. Med. Chem. 52, 20-32.

Zhang, X., Zhu, B., Chen, L., Xie, L., Yu, W., Wang, Y., Li, L., Yin, S., Yang, L., Hu, H., et al., 2020. Dual base editor catalyzes both cytosine and adenine base conversions in human cells. Nat. Biotechnol. 38, 856-860.

Zhao, Y., Li, M., Liu, J., Xue, X., Zhong, J., Lin, J., Ye, B., Chen, J., Qiao, Y., 2023. Dual guide RNA-mediated concurrent C&G-to-T&A and A&T-to-G&C conversions using CRISPR base editors. Comput. Struct. Biotechnol. J. 21, 856-868.

Zuo, E., Sun, Y., Yuan, T., He, B., Zhou, C., Ying, W., Liu, J., Wei, W., Zeng, R., Li, Y., et al., 2020. A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects. Nat. Methods 17, 600-604.

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