利用 CRISPR/Cas9 在果蝇中实现无痕等位基因替换的工具和策略。

IF 2.4 4区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Fly Pub Date : 2017-01-02 Epub Date: 2016-08-05 DOI:10.1080/19336934.2016.1220463
Abigail M Lamb, Elizabeth A Walker, Patricia J Wittkopp
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引用次数: 0

摘要

通过 CRISPR/Cas9 RNA 引导的核酸酶系统进行基因组编辑,为遗传分析开辟了令人兴奋的可能性。然而,与同源定向修复相关的技术挑战已被证明是在没有不想要的、二次突变(俗称 "疤痕")的情况下产生变化的障碍。为了解决这些问题,我们开发了一种两阶段标记辅助策略,以促进果蝇进行精确的 "无痕 "编辑,并将分子筛选的要求降到最低。利用这种方法,我们在不改变 CRISPR 剪切位点最终序列的情况下,修改了感兴趣基因中的 2 个碱基对。我们使用一种新型转化标记来执行这种两阶段等位基因交换,这种标记能驱动在蛹翅中的表达,可以在常见的眼睛表达报告物存在的情况下对其进行筛选。我们开发的工具可用于在任何黑腹果蝇遗传背景以及其他果蝇物种中对感兴趣的区域进行单个或一系列等位基因置换。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9.

Tools and strategies for scarless allele replacement in Drosophila using CRISPR/Cas9.

Genome editing via the CRISPR/Cas9 RNA-guided nuclease system has opened up exciting possibilities for genetic analysis. However, technical challenges associated with homology-directed repair have proven to be roadblocks for producing changes in the absence of unwanted, secondary mutations commonly known as "scars." To address these issues, we developed a 2-stage, marker-assisted strategy to facilitate precise, "scarless" edits in Drosophila with a minimal requirement for molecular screening. Using this method, we modified 2 base pairs in a gene of interest without altering the final sequence of the CRISPR cut sites. We executed this 2-stage allele swap using a novel transformation marker that drives expression in the pupal wings, which can be screened for in the presence of common eye-expressing reporters. The tools we developed can be used to make a single change or a series of allelic substitutions in a region of interest in any D. melanogaster genetic background as well as in other Drosophila species.

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来源期刊
Fly
Fly 生物-生化与分子生物学
CiteScore
2.90
自引率
0.00%
发文量
17
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.
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