Department of Pharmacology, Faculty of Medicine and Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland.
BioMediTech, Faculty of Medicine and Health Technology, Tampere University, Tampere, Finland.
Sci Rep. 2020 Dec 3;10(1):21023. doi: 10.1038/s41598-020-77677-9.
CRISPR/Cas9 driven mutagenesis in zygotes is a popular tool for introducing targeted mutations in model organisms. Compared to mouse, mutagenesis in zebrafish is relatively inefficient and results in somatic mosaicism most likely due to a short single-cell stage of about 40 min. Here we explored two options to improve CRISPR/Cas9 mutagenesis in zebrafish-extending the single-cell stage and defining conditions for carrying out mutagenesis in oocytes prior to in vitro fertilization. Previous work has shown that ovarian fluid from North American salmon species (coho and chinook salmon) prolong oocyte survival ex vivo so that they are viable for hours instead of dying within minutes if left untreated. We found that commonly farmed rainbow trout (Oncorhynchus mykiss) ovarian fluid (RTOF) has similar effect on zebrafish oocyte viability. In order to prolong single-cell stage, we incubated zebrafish zygotes in hydrogen sulfide (HS) and RTOF but failed to see any effect. However, the reduction of temperature from standard 28 to 12 °C postponed the first cell division by about an hour. In addition, the reduction in temperature was associated with increased CRISPR/Cas9 mutagenesis rate. These results suggest that the easily applicable reduction in temperature facilitates CRISPR/Cas9 mutagenesis in zebrafish.
CRISPR/Cas9 驱动的胚胎突变是在模式生物中引入靶向突变的一种流行工具。与小鼠相比，斑马鱼中的突变相对效率较低，并且由于大约 40 分钟的单细胞阶段较短，最有可能导致体细胞核移植嵌合体。在这里，我们探索了两种改进斑马鱼 CRISPR/Cas9 突变的方法——延长单细胞阶段和定义在体外受精前对卵母细胞进行突变的条件。先前的工作表明，来自北美的鲑鱼物种（银鲑和虹鳟）的卵巢液（coho 和 chinook 鲑鱼）延长了卵母细胞的体外存活时间，因此如果未经处理，它们可以存活数小时而不是在数分钟内死亡。我们发现，常见的养殖虹鳟（Oncorhynchus mykiss）卵巢液（RTOF）对斑马鱼卵母细胞活力具有相似的影响。为了延长单细胞阶段，我们将斑马鱼胚胎在硫化氢（HS）和 RTOF 中孵育，但未观察到任何效果。然而，将温度从标准的 28°C 降低到 12°C 将第一次细胞分裂推迟了大约一个小时。此外，温度降低与 CRISPR/Cas9 突变率的增加有关。这些结果表明，易于应用的降低温度有助于斑马鱼中的 CRISPR/Cas9 突变。
