Abstract
The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293 T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.
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Acknowledgements
We are very grateful to Dr. Cuiying Chen from Shantou University for professional advice in this study.
Funding
This work was supported by the National Natural Science Foundation of China (32373108), Natural Science Foundation of Guandong Province (2022A1515010778), Special Fund for Science and Technology of Guangdong Province (STKJ202209036), Department of Education of Guangdong Province (2022ZDZX4006), and Special Fundation for Rural Revitalization of Guangdong Province (Dzxny018).
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Xiaokang Ye: investigation, formal analysis, writing—original draft preparation; Jiali Lin: investigation, data curation; Qiuji Chen: resources; Jiehuan Lv: resources; Chunsheng Liu: resources; Yuping Wang: resources; Shuqi Wang: writing—review and editing; Xiaobo Wen: writing—review and editing; Fan Lin: conception, supervision, writing—review and editing, funding acquirement.
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All animal procedures were carried out in accordance with the Guideline for the Care and Use of Laboratory Animals of Shantou University.
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Ye, X., Lin, J., Chen, Q. et al. An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line. Mar Biotechnol (2024). https://doi.org/10.1007/s10126-024-10320-0
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DOI: https://doi.org/10.1007/s10126-024-10320-0