CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos
CRISPR-Cas13d Induces Efficient mRNA Knockdown in Animal Embryos
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DOI：
10.1016/j.devcel.2020.07.013
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发表时间：
2020-09-28
影响因子：
11.8
通讯作者：
Moreno-Mateos, Miguel A.
中科院分区：
文献类型：
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作者：
Kushawah, Gopal;Hernandez-Huertas, Luis;Moreno-Mateos, Miguel A.
早期胚胎发育完全由储存在卵母细胞中的母体基因产物驱动。虽然在建立早期发育计划的关键，母亲的基因功能仍然难以捉摸，由于缺乏系统的破坏和评估的技术。CRISPR-Cas 13系统最近已被用于降解酵母、植物和哺乳动物细胞系中的RNA。然而，尚未在动物系统中对Cas 13的潜力进行系统研究。在这里，我们表明CRISPR-RfxCas 13 d（CasRx）是一种有效而精确的系统，可以消除斑马鱼胚胎中的特定mRNA转录物。我们证明，合子表达和母系提供的转录本是有效的目标，导致转录水平平均下降76%，并重演众所周知的胚胎表型。此外，我们表明，该系统可用于青鳉，鳉鱼和小鼠胚胎。总而言之，我们的研究结果表明，CRISPR-RfxCas 13 d是一种有效的敲除平台，可以在动物胚胎中查询基因功能。
Early embryonic development is driven exclusively by maternal gene products deposited into the oocyte. Although critical in establishing early developmental programs, maternal gene functions have remained elusive due to a paucity of techniques for their systematic disruption and assessment. CRISPR-Cas13 systems have recently been employed to degrade RNA in yeast, plants, and mammalian cell lines. However, no systematic study of the potential of Cas13 has been carried out in an animal system. Here, we show that CRISPR-RfxCas13d (CasRx) is an effective and precise system to deplete specific mRNA transcripts in zebrafish embryos. We demonstrate that zygotically expressed and maternally provided transcripts are efficiently targeted, resulting in a 76% average decrease in transcript levels and recapitulation of well-known embryonic phenotypes. Moreover, we show that this system can be used in medaka, killifish, and mouse embryos. Altogether, our results demonstrate that CRISPR-RfxCas13d is an efficient knockdown platform to interrogate gene function in animal embryos.