特异性细胞耗竭是研究细胞谱系和组织再生的生理功能的常用手段。然而,用现有的细胞耗尽策略难以实现100%的耗尽。随着CRISPR/Cas9技术的日益成熟,它越来越多地用于各种细胞的消耗。然而,即使有了这项技术,很难完成特定基因敲除细胞的耗竭。出于这个原因,使用斑马鱼探索使用重复序列作为CRISPR/Cas9靶标的细胞耗竭。所有细胞用作第一组实验的靶细胞。结果表明,将DANA-gRNA和Cas9mRNA的混合物注射到受精卵中导致大量细胞凋亡。在圆顶阶段,细胞在胚胎动物极中几乎是看不见的。caspase-3和caspase-9蛋白的活性和P53基因的mRNA水平显着增加。然后,胚胎中的原始生殖细胞(PGCs)在随后的实验中用作靶细胞。为了特别敲除PGCs,我们注射了DANA-gRNA的混合物,pkop:Cas9质粒(kop启动子仅允许Cas9在PGCs中表达),和eGFP-nos3UTRmRNA进入斑马鱼受精卵。结果表明,caspase-3蛋白的活性显着增加,P53,ku70和ku80的mRNA水平显着上调,PGCs的数量逐渐减少。受精后20小时(hpf)很少可以看到GFP标记的PGCs,在24hpf的生发脊没有看到PGCs。因此,CRISPR/Cas9技术和重复序列的组合可以实现有效的细胞耗竭,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,通过使用重复序列作为CRISPR/Cas9系统靶位点消除细胞是可行的。
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.