Guangdong Provincial Key Laboratory of Marine Biotechnology, Institute of Marine Sciences, Shantou University, 243 Daxue Road, Shantou, 515063, China.
College of Marine Sciences, South China Agricultural University, Guangzhou, 510642, China.
Mar Biotechnol (NY). 2024 Jun;26(3):588-598. doi: 10.1007/s10126-024-10320-0. Epub 2024 Apr 23.
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.
簇状规律间隔短回文重复/CRISPR 相关蛋白 9（CRISPR/Cas9）系统已被广泛应用于动物中，作为一种有效的基因组编辑工具。然而，该技术在鱼类细胞系中难以实施，部分原因是缺乏有效的启动子，无法在单个载体中同时驱动 sgRNA 和 Cas9 蛋白的表达。在这项研究中，表明斑马鱼 U6 RNA 聚合酶 III（ZFU6）启动子可以有效地诱导酪氨酸酶（tyr）基因编辑，并在与 Cas9 mRNA 共注射到斑马鱼胚胎中时导致视网膜色素丧失。此外，通过用 ZFU6 启动子替换人 U6 启动子，基于广泛应用于哺乳动物细胞的 lentiCRISPRV2 系统，构建了用于在斑马鱼成纤维细胞系（PAC2）中表达 CRISPR/Cas9 系统的优化的一体化载体。这个新载体可以成功靶向细胞通讯网络因子 2a（ctgfa）基因，并在 PAC2 细胞中证明了其功能。值得注意的是，该载体还可以用于编辑哺乳动物 293T 细胞系中的内源性 EMX1 基因，这意味着它具有广泛的应用潜力。总之，我们建立了一种用于斑马鱼细胞系的新基因编辑工具，它可以成为鱼类基因功能高通量分析的有用体外平台。
