Precise genome engineering in Drosophila using prime editing
Precise genome engineering in Drosophila using prime editing
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DOI：
10.1101/2020.08.05.232348
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发表时间：
2020-08
期刊：
影响因子：
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通讯作者：
Justin A. Bosch;Gabriel Birchak;N. Perrimon
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文献类型：
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作者：
Justin A. Bosch;Gabriel Birchak;N. Perrimon
精确的基因组工程使研究人员能够修改基因功能，标记内源性蛋白质或模拟人类疾病突变。在这里，我们采用了prime editing，这是一种基于CRISPR的新技术，它使用逆转录将精确的变化写入目标基因组位置，用于模式生物果蝇。我们创建并优化了遗传工具，以在培养细胞和体内编辑三个基因（乌木，白色和分叉）。重要的是，我们证明了在乌木中精确编辑的有效种系传递。由于果蝇是第一个使用这种方法进行测试的非哺乳动物，因此这项研究证明了prime editing在其他动物物种中的潜在广泛影响和可翻译性。精确的基因组编辑是研究模式生物基因功能的重要工具。引物编辑是在哺乳动物细胞中开发的精确编辑系统，不需要双链断裂或供体DNA，并且具有低脱靶效应。在这里，我们对模式生物黑腹果蝇（Drosophila melanogaster）进行了主要编辑，并开发了最佳编辑的条件。通过在转基因果蝇的培养细胞或体细胞中表达主要编辑组分，我们精确地在三个经典的可见标记基因乌木、白色和分叉中引入过早终止密码子。此外，通过将编辑限制在生殖细胞中，我们证明了乌木中精确编辑的有效种系传递到36%的后代。我们的研究结果表明，引物编辑是一个有用的系统，在果蝇研究基因功能，如工程精确的点突变，缺失，或表位标签。
Significance Precise genome engineering allows researchers to modify gene function, tag endogenous proteins, or model human disease mutations. Here, we adapt prime editing, a new CRISPR-based technology that uses reverse transcription to write precise changes into a target genomic location, for the model organism Drosophila melanogaster. We created and optimized genetic tools to edit three genes (ebony, white, and forked) in cultured cells and in vivo. Importantly, we demonstrate efficient germ-line transmission of a precise edit in ebony. As Drosophila is the first nonmammalian animal to be tested using this method, this study demonstrates the potential wide impact and translatability of prime editing in other animal species. Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism Drosophila melanogaster and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes, ebony, white, and forked. Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit in ebony to 36% of progeny. Our results suggest that prime editing is a useful system in Drosophila to study gene function, such as engineering precise point mutations, deletions, or epitope tags.