Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA.
Department of Neuroscience, University of Copenhagen, Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark.
Development. 2021 Apr 1;148(7). doi: 10.1242/dev.193565. Epub 2021 Apr 15.
An important strategy for establishing mechanisms of gene function during development is through mutation of individual genes and analysis of subsequent effects on cell behavior. Here, we present a single-plasmid approach for genome editing in chick embryos to study experimentally perturbed cells in an otherwise normal embryonic environment. To achieve this, we have engineered a plasmid that encodes Cas9 protein, gene-specific guide RNA (gRNA), and a fluorescent marker within the same construct. Using transfection- and electroporation-based approaches, we show that this construct can be used to perturb gene function in early embryos as well as human cell lines. Importantly, insertion of this cistronic construct into replication-incompetent avian retroviruses allowed us to couple gene knockouts with long-term lineage analysis. We demonstrate the application of our newly engineered constructs and viruses by perturbing β-catenin in vitro and Sox10, Pax6 and Pax7 in the neural crest, retina, and neural tube and segmental plate in vivo, respectively. Together, this approach enables genes of interest to be knocked out in identifiable cells in living embryos and can be broadly applied to numerous genes in different embryonic tissues.
在发育过程中建立基因功能机制的一个重要策略是通过单个基因的突变和分析随后对细胞行为的影响。在这里，我们提出了一种在鸡胚中进行基因组编辑的单质粒方法，以研究在正常胚胎环境中受到实验干扰的细胞。为了实现这一目标，我们设计了一种质粒，该质粒在同一构建体中编码 Cas9 蛋白、基因特异性向导 RNA（gRNA）和荧光标记。使用转染和电穿孔方法，我们表明该构建体可用于干扰早期胚胎和人细胞系中的基因功能。重要的是，将这种顺式结构构建体插入非复制性禽逆转录病毒中，使我们能够将基因敲除与长期谱系分析结合起来。我们通过在体外干扰β-连环蛋白以及在体内分别干扰神经嵴、视网膜和神经管以及节段盘中的 Sox10、Pax6 和 Pax7，展示了我们新设计的构建体和病毒的应用。总之，这种方法能够在活体胚胎中的可识别细胞中敲除感兴趣的基因，并可广泛应用于不同胚胎组织中的许多基因。
