A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in Drosophila.

IF 2.4 4区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Fly Pub Date : 2020-03-01 Epub Date: 2020-10-21 DOI:10.1080/19336934.2020.1832416

Kevin G Nyberg, Joseph Q Nguyen, Yong-Jae Kwon, Shelby Blythe, Greg J Beitel, Richard Carthew

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Abstract

Genome editing via homology-directed repair (HDR) has made possible precise and deliberate modifications to gene sequences. CRISPR/Cas9-mediated HDR is the simplest means to carry this out. However, technical challenges remain to improve efficiency and broaden applicability to any genetic background of Drosophila melanogaster as well as to other Drosophila species. To address these issues, we developed a two-stage marker-assisted strategy in which embryos are injected with RNPs and pre-screened using T7EI. Using sgRNA in complex with recombinant Cas9 protein, we assayed each sgRNA for genome-cutting efficiency. We then conducted HDR using sgRNAs that efficiently cut target genes and the application of a transformation marker that generates RNAi against eyes absent. This allows for screening based on eye morphology rather than colour. These new tools can be used to make a single change or a series of allelic substitutions in a region of interest, or to create additional genetic tools such as balancer chromosomes.

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利用 sgRNA-Cas9 RNPs 对果蝇进行精确高效基因组编辑的管道。

通过同源定向修复（HDR）进行基因组编辑，可以对基因序列进行精确和有意的修改。CRISPR/Cas9 介导的 HDR 是最简单的方法。然而，要提高效率并扩大对任何遗传背景的黑腹果蝇以及其他果蝇物种的适用性，仍然存在技术挑战。为了解决这些问题，我们开发了一种两阶段标记辅助策略，即给胚胎注射 RNPs 并使用 T7EI 进行预筛选。使用 sgRNA 与重组 Cas9 蛋白结合，我们检测了每个 sgRNA 的基因组切割效率。然后，我们使用能有效切割目标基因的 sgRNA 进行 HDR，并应用转化标记产生针对眼睛缺失的 RNAi。这样就可以根据眼睛的形态而不是颜色进行筛选。这些新工具可用于在感兴趣的区域进行单个或一系列等位基因置换，或创建额外的遗传工具，如平衡染色体。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Fly 生物-生化与分子生物学

CiteScore

2.90

自引率

0.00%

发文量

审稿时长

>12 weeks

期刊介绍： Fly is the first international peer-reviewed journal to focus on Drosophila research. Fly covers a broad range of biological sub-disciplines, ranging from developmental biology and organogenesis to sensory neurobiology, circadian rhythm and learning and memory, to sex determination, evolutionary biology and speciation. We strive to become the “to go” resource for every researcher working with Drosophila by providing a forum where the specific interests of the Drosophila community can be discussed. With the advance of molecular technologies that enable researchers to manipulate genes and their functions in many other organisms, Fly is now also publishing papers that use other insect model systems used to investigate important biological questions. Fly offers a variety of papers, including Original Research Articles, Methods and Technical Advances, Brief Communications, Reviews and Meeting Reports. In addition, Fly also features two unconventional types of contributions, Counterpoints and Extra View articles. Counterpoints are opinion pieces that critically discuss controversial papers questioning current paradigms, whether justified or not. Extra View articles, which generally are solicited by Fly editors, provide authors of important forthcoming papers published elsewhere an opportunity to expand on their original findings and discuss the broader impact of their discovery. Extra View authors are strongly encouraged to complement their published observations with additional data not included in the original paper or acquired subsequently.