A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos.
A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos.
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DOI：
10.1242/dev.193565
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发表时间：
2021-04-01
期刊：
影响因子：
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通讯作者：
Bronner ME
中科院分区：
文献类型：
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作者：
Gandhi S;Li Y;Tang W;Christensen JB;Urrutia HA;Vieceli FM;Piacentino ML;Bronner ME
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对细胞行为的后续影响的分析。在这里，我们提出了一种用于鸡胚基因组编辑的单质粒方法，以研究在正常胚胎环境中实验扰动的细胞。为了实现这一目标，我们已经在同一构建体中设计了编码Cas9蛋白、基因特异性指导RNA（gRNA）和荧光标记物的质粒。使用转染和电穿孔为基础的方法，我们表明，这种结构可以用来扰乱早期胚胎以及人类细胞系的基因功能。重要的是，将这种顺反子构建体插入到复制缺陷型禽类逆转录病毒中，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-catenin和在体内分别干扰神经嵴、视网膜、神经管和节段板中的Sox 10、Pax 6和Pax 7来展示我们新工程化的构建体和病毒的应用。总之，这种方法使感兴趣的基因能够在活胚胎中的可识别细胞中被敲除，并且可以广泛应用于不同胚胎组织中的许多基因。总结：使用自切割核酶的单质粒方法能够通过电穿孔递送CRISPR组分，并且可以整合到病毒递送系统中用于长期跟踪突变细胞。
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. Summary: A single-plasmid approach using self-cleaving ribozymes enables delivery of CRISPR components via electroporation and can be integrated into viral delivery systems for long-term tracking of mutant cells.