Hunan Provincial Key Laboratory for Health Aquaculture and Product Processing in Dongting Lake Area, Hunan Engineering Research Center of Aquatic Organism Resources and Environmental Ecology, Zoology Key Laboratory of Hunan Higher Education, Changde Research Center for Agricultural Biomacromolecule, Innovation Team of Microbial Technology, College of Life and Environmental Sciences, Hunan University of Arts and Science, Changde, Hunan, China.
Changde Fishery Administration Station, Changde, China.
Mar Biotechnol (NY). 2024 Aug;26(4):639-648. doi: 10.1007/s10126-024-10328-6. Epub 2024 Jun 4.
Specific cell depletion is a common means to study the physiological function of cell lineages and tissue regeneration. However, 100% depletion is difficult to achieve with existing cell depletion strategies. With the increasing maturity of CRISPR/Cas9 technology, it is increasingly used for the depletion of various cells. However, even with this technology, it is difficult to complete the depletion of specific gene knockout cells. For this reason, cell depletion with the use of repetitive sequences as the target of CRISPR/Cas9 was explored using zebrafish. All cells were used as the target cells for the first set of experiments. The results showed that injection of a mixture of DANA-gRNA and Cas9 mRNA into zygotes resulted in substantial cell apoptosis. Cells are almost invisible in the embryonic animal pole during the dome stage. The activities of the caspase-3 and caspase-9 proteins and the mRNA level of the P53 gene were significantly increased. Then, primordial germ cells (PGCs) in embryos were used as the target cells in subsequent experiments. To specifically knock out PGCs, we injected the mix of DANA-gRNA, pkop: Cas9 plasmid (the kop promotor allows Cas9 expression only in PGCs), and eGFP-nos3'UTR mRNA into zebrafish fertilized eggs. The results revealed that the activity of the caspase-3 protein was significantly increased, and the mRNA levels of P53, ku70, and ku80 were significantly upregulated, while the number of PGCs decreased gradually. Few PGCs labeled with GFP could be seen 20 h post-fertilization (hpf), and no PGCs could be seen at the germinal ridge 24 hpf. Therefore, the combination of CRISPR/Cas9 technology and repetitive sequences can achieve efficient cell depletion regardless of whether there is generalized expression or expression in specific cells. These results indicate that it is feasible to eliminate cells by using repeat sequences as CRISPR/Cas9 system target sites.
特异性细胞耗竭是研究细胞谱系生理功能和组织再生的常用方法。然而，现有的细胞耗竭策略很难达到 100%的耗竭。随着 CRISPR/Cas9 技术的日益成熟，它越来越多地被用于各种细胞的耗竭。然而，即使使用这项技术，也很难完成特定基因敲除细胞的耗竭。为此，研究人员利用 CRISPR/Cas9 以重复序列为靶点来研究斑马鱼的细胞耗竭。在第一组实验中，所有细胞均作为靶细胞。结果表明，将 DANA-gRNA 和 Cas9 mRNA 的混合物注射到受精卵中会导致大量细胞凋亡。在盘状期，胚胎动物极中的细胞几乎看不见。Caspase-3 和 Caspase-9 蛋白的活性以及 P53 基因的 mRNA 水平显著增加。然后，在后续实验中，以胚胎中的原始生殖细胞（PGCs）作为靶细胞。为了特异性敲除 PGCs，我们将 DANA-gRNA、pkop: Cas9 质粒（kop 启动子允许 Cas9 仅在 PGCs 中表达）和 eGFP-nos3'UTR mRNA 的混合物注射到斑马鱼受精卵中。结果表明，Caspase-3 蛋白的活性显著增加，P53、ku70 和 ku80 的 mRNA 水平显著上调，而 PGCs 的数量逐渐减少。受精后 20 小时（hpf）几乎看不到 GFP 标记的 PGCs，受精后 24 小时（hpf）时看不到生殖嵴中的 PGCs。因此，CRISPR/Cas9 技术与重复序列的结合可以实现高效的细胞耗竭，无论是否存在普遍表达或在特定细胞中表达。这些结果表明，使用重复序列作为 CRISPR/Cas9 系统的靶位点来消除细胞是可行的。
