Improving precision base editing of the zebrafish genome by Rad51DBD-incorporated single-base editors

Zhilin Zhong; Xueli Hu; Renjie Zhang; Xu Liu; Wenqi Chen; Shubin Zhang; Jianjian Sun; Tao P. Zhong

doi:10.1016/j.jgg.2024.10.006

Volume 52 Issue 1

Jan. 2025

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Improving precision base editing of the zebrafish genome by Rad51DBD-incorporated single-base editors

doi: 10.1016/j.jgg.2024.10.006

a. Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China;
b. Guangdong Cardiovascular Institute, Guangdong Provincial People's Hospital, Southern Medical University, Guangzhou, Guangdong 510100, China

Funds:

We acknowledge Dr. Dali Li from East China Normal University, Dr. Rongjia Zhou from Wuhan University, and Dr. Wei Qin from South China Normal University for providing plasmids

Dr. Dan Zhang from East China Normal University for assistance with data analyses. We thank ECNU Multifunctional Platform for Innovation (004) for image acquisition and the Instruments Sharing Platform of School of Life Sciences at ECNU for supporting research. We are grateful to members of TPZ for comments on the manuscript and helpful discussions. This work was supported by grants from Ministry of Science and Technology of the People's Republic of China (2018YFA0801004 and 2018YFA0800103) and the National Natural Science Foundation of China (NSFC31530044, NSFC31970780, NSFC82202056).

Received Date: 2024-05-26
Accepted Date: 2024-10-11
Rev Recd Date: 2024-10-09

Available Online: 2025-07-11

Publish Date: 2024-10-18

Abstract

Abstract

Single-base editors, including cytosine base editors (CBEs) and adenine base editors (ABEs), facilitate accurate C·G to T·A and A·T to G·C, respectively, holding promise for the precise modeling and treatment of human hereditary disorders. Efficient base editing and expanded base conversion range have been achieved in human cells through base editors fusing with Rad51 DNA binding domain (Rad51DBD), such as hyA3A-BE4max. Here, we show that hyA3A-BE4max catalyzes C-to-T substitution in the zebrafish genome and extends editing positions (C₁₂–C₁₆) proximal to the protospacer adjacent motif. We develop a codon-optimized counterpart zhyA3A-CBE5, which exhibits substantially high C-to-T conversion with 1.59- to 3.50-fold improvement compared with the original hyA3A-BE4max. With these tools, disease-relevant hereditary mutations can be more efficaciously generated in zebrafish. We introduce human genetic mutation rpl11^Q42∗ and abcc6a^R1463C by zhyA3A-CBE5 in zebrafish, mirroring Diamond-Blackfan anemia and Pseudoxanthoma Elasticum, respectively. Our study expands the base editing platform targeting the zebrafish genomic landscape and the application of single-base editors for disease modeling and gene function study.
- Zebrafish,
- Base editor,
- Rad51DBD,
- zhyA3A-CBE5,
- PXE syndrome

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References(49)

References

Arbab, M., Matuszek, Z., Kray, K.M., Du, A., Newby, G.A., Blatnik, A.J., Raguram, A., Richter, M.F., Zhao, K.T., Levy, J.M., et al., 2023. Base editing rescue of spinal muscular atrophy in cells and in mice. Science 380, eadg6518.

Bagi, C.M., Zakur, D.E., Berryman, E., Andresen, C.J., Wilkie, D., 2015. Correlation between μCT imaging, histology and functional capacity of the osteoarthritic knee in the rat model of osteoarthritis. J. Transl. Med. 13, 276.

Brampton, C., Aherrahrou, Z., Chen, L.H., Martin, L., Bergen, A.A., Gorgels, T.G., Erdmann, J., Schunkert, H., Szabo, Z., Varadi, A., et al., 2014. The level of hepatic ABCC6 expression determines the severity of calcification after cardiac injury. Am. J. Pathol. 184, 159-170.

Campens, L., Vanakker, O.M., Trachet, B., Segers, P., Leroy, B.P., De Zaeytijd, J., Voet, D., De Paepe, A., De Backer, T., De Backer, J., 2013. Characterization of cardiovascular involvement in pseudoxanthoma elasticum families. Arterioscler. Thromb. Vasc. Biol. 33, 2646-2652.

Chen, L., Hong, M., Luan, C., Gao, H., Ru, G., Guo, X., Zhang, D., Zhang, S., Li, C., Wu, J., et al., 2024. Adenine transversion editors enable precise, efficient A·T-to-C·G base editing in mammalian cells and embryos. Nat. Biotechnol. 42, 638-650.

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.

Da Costa, L.M., Marie, I., Leblanc, T.M., 2021. Diamond-Blackfan anemia. Hematology Am. Soc. Hematol. Educ. Program. 2021, 353-360.

Dickey, T.H., Altschuler, S.E., Wuttke, D.S., 2013. Single-stranded DNA-binding proteins: multiple domains for multiple functions. Structure 21, 1074-1084.

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.

Gehrke, J.M., Cervantes, O., Clement, M.K., Wu, Y., Zeng, J., Bauer, D.E., Pinello, L., Joung, J.K., 2018. An APOBEC3A-Cas9 base editor with minimized bystander and off-target activities. Nat. Biotechnol. 36, 977-982.

Germain, D.P., 2017. Pseudoxanthoma elasticum. Orphanet J. Rare Dis. 12, 85.

Geurts, M.H., Gandhi, S., Boretto, M.G., Akkerman, N., Derks, L.L.M., van Son, G., Celotti, M., Harshuk-Shabso, S., Peci, F., Begthel, H., et al., 2023. One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids. Nat. Commun. 14, 4998.

Horos, R., von Lindern, M., 2012. Molecular mechanisms of pathology and treatment in Diamond blackfan anaemia. Br. J. Haematol. 159, 514-527.

Hu, X., Plomp, A., Wijnholds, J., ten Brink, J., van Soest, S., van den Born, L.I., Leys, A., Peek, R., de Jong, P.T.V.M., Bergen, A.A.B., 2003. ABCC6/MRP6 mutations: further insight into the molecular pathology of pseudoxanthoma elasticum. Eur. J. Hum. Genet. 11, 215-224.

Hwang, B., Lee, W., Yum, S.-Y., Jeon, Y., Cho, N., Jang, G., Bang, D., 2019. Lineage tracing using a Cas9-deaminase barcoding system targeting endogenous L1 elements. Nat. Commun. 10, 1234.

Kim, Y.B., Komor, A.C., Levy, J.M., Packer, M.S., Zhao, K.T., Liu, D.R., 2017. Increasing the genome-targeting scope and precision of base editing with engineered Cas9-cytidine deaminase fusions. Nat. Biotechnol. 35, 371-376.

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.

Lee, S., Ding, N., Sun, Y., Yuan, T., Li, J., Yuan, Q., Liu, L., Yang, J., Wang, Q., Kolomeisky, A.B., et al., 2020. Single C-to-T substitution using engineered APOBEC3G-nCas9 base editors with minimum genome- and transcriptome-wide off-target effects. Sci. Adv. 6, eaba1773.

Li, C., Zhang, R., Meng, X., Chen, S., Zong, Y., Lu, C., Qiu, J.-L., Chen, Y.-H., Li, J., Gao, C., 2020. Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors. Nat. Biotechnol. 38, 875-882.

Liang, F., Zhang, Y., Li, L., Yang, Y., Fei, J.-F., Liu, Y., Qin, W., 2022. SpG and SpRY variants expand the CRISPR toolbox for genome editing in zebrafish. Nat. Commun. 13, 3421.

Liao, J., Chen, S., Hsiao, S., Jiang, Y., Yang, Y., Zhang, Y., Wang, X., Lai, Y., Bauer, D.E., Wu, Y., 2023. Therapeutic adenine base editing of human hematopoietic stem cells. Nat. Commun. 14, 207.

Lipton, J.M., Ellis, S.R., 2009. Diamond-Blackfan anemia: diagnosis, treatment, and molecular pathogenesis. Hematol. Oncol. Clin. N. Am. 23, 261-282.

Lu, X., Liu, Y., Yan, G., Li, S., Qin, W., Lin, S., 2018. Optimized Target-AID system efficiently induces single base changes in zebrafish. J. Genet. Genomics 45, 215-217.

Nolte, K.B., 2000. Sudden cardiac death owing to pseudoxanthoma elasticum: a case report. Hum. Pathol. 31, 1002-1004.

Omarjee, L., Roy, C., Leboeuf, C., Favre, J., Henrion, D., Mahe, G., Leftheriotis, G., Martin, L., Janin, A., Kauffenstein, G., 2019. Evidence of cardiovascular calcification and fibrosis in pseudoxanthoma elasticum mouse models subjected to DOCA-salt hypertension. Sci. Rep. 9, 16327.

Pingel, S., Pausewang, K.S., Passon, S.G., Blatzheim, A.K., Gliem, M., Charbel Issa, P., Hendig, D., Horlbeck, F., Tuleta, I., Nickenig, G., et al., 2017. Increased vascular occlusion in patients with pseudoxanthoma elasticum. Vasa 46, 47-52.

Qin, W., Lu, X., Liu, Y., Bai, H., Li, S., Lin, S., 2018. Precise A∗T to G∗C base editing in the zebrafish genome. BMC Biol. 16, 139.

Randall, L.B., Sretenovic, S., Wu, Y., Yin, D., Zhang, T., Eck, J.V., Qi, Y., 2021. Genome- and transcriptome-wide off-target analyses of an improved cytosine base editor. Plant Physiol. 187, 73-87.

Rees, H.A., Liu, D.R., 2018. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770-788.

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.

Shimada, B.K., Pomozi, V., Zoll, J., Kuo, S., Martin, L., Le Saux, O., 2021. ABCC6, pyrophosphate and ectopic calcification: therapeutic solutions. Int. J. Mol. Sci. 22, 4555.

Sun, J., She, P., Liu, X., Gao, B., Jin, D., Zhong, T.P., 2020. Disruption of Abcc6 transporter in zebrafish causes ocular calcification and cardiac fibrosis. Int. J. Mol. Sci. 22, 278.

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

Ulirsch, J.C., Verboon, J.M., Kazerounian, S., Guo, M.H., Yuan, D., Ludwig, L.S., Handsaker, R.E., Abdulhay, N.J., Fiorini, C., Genovese, G., et al., 2018. The genetic landscape of Diamond-Blackfan anemia. Am. J. Hum. Genet. 103, 930-947.

Vlachos, A., Ball, S., Dahl, N., Alter, B.P., Sheth, S., Ramenghi, U., Meerpohl, J., Karlsson, S., Liu, J.M., Leblanc, T., et al., 2008. Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference. Br. J. Haematol. 142, 859-876.

Wang, X., Pan, W., Sun, C., Yang, H., Cheng, Z., Yan, F., Ma, G., Shang, Y., Zhang, R., Gao, C., et al., 2024. Creating large-scale genetic diversity in Arabidopsis via base editing-mediated deep artificial evolution. Genome Biol. 25, 215.

Wei, C., Liu, H., Wang, W., Luo, P., Chen, Q., Li, R., Wang, C., Botella, J.R., Zhang, H., 2022. Expanding the editing window of cytidine base editors with the Rad51 DNA-binding domain in rice. Front. Plant Sci. 13, 865848.

Xiong, X., Li, Z., Liang, J., Liu, K., Li, C., Li, J.F., 2022. A cytosine base editor toolkit with varying activity windows and target scopes for versatile gene manipulation in plants. Nucleic Acids Res. 50, 3565-3580.

Xue, N., Liu, X., Zhang, D., Wu, Y., Zhong, Y., Wang, J., Fan, W., Jiang, H., Zhu, B., Ge, X., et al., 2023. Improving adenine and dual base editors through introduction of TadA-8e and Rad51DBD. Nat. Commun. 14, 1224.

Yang, L., Huo, Y., Wang, M., Zhang, D., Zhang, T., Wu, H., Rao, X., Meng, H., Yin, S., Mei, J., et al., 2024. Engineering APOBEC3A deaminase for highly accurate and efficient base editing. Nat. Chem. Biol. 20, 1176-1187.

Zhang, X., Chen, L., Zhu, B., Wang, L., Chen, C., Hong, M., Huang, Y., Li, H., Han, H., Cai, B., et al., 2020. Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain. Nat. Cell Biol. 22, 740-750.

Zhang, Y., Qin, W., Lu, X., Xu, J., Huang, H., Bai, H., Li, S., Lin, S., 2017. Programmable base editing of zebrafish genome using a modified CRISPR-Cas9 system. Nat. Commun. 8, 118.

Zhang, Z., Jia, H., Zhang, Q., Wan, Y., Zhou, Y., Jia, Q., Zhang, W., Yuan, W., Cheng, T., Zhu, X., et al., 2013. Assessment of hematopoietic failure due to Rpl11 deficiency in a zebrafish model of Diamond-Blackfan anemia by deep sequencing. BMC Genom. 14, 896.

Zhao, D., Li, J., Li, S., Xin, X., Hu, M., Price, M.A., Rosser, S.J., Bi, C., Zhang, X., 2021. Glycosylase base editors enable C-to-A and C-to-G base changes. Nat. Biotechnol. 39, 35-40.

Zhao, Y., Shang, D., Ying, R., Cheng, H., Zhou, R., 2020. An optimized base editor with efficient C-to-T base editing in zebrafish. BMC Biol. 18, 190.

Ziegler, S.G., Ferreira, C.R., MacFarlane, E.G., Riddle, R.C., Tomlinson, R.E., Chew, E.Y., Martin, L., Ma, C.T., Sergienko, E., Pinkerton, A.B., et al., 2017. Ectopic calcification in pseudoxanthoma elasticum responds to inhibition of tissue-nonspecific alkaline phosphatase. Sci. Transl. Med. 9, eaal1669.

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