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A pipeline for precise and efficient genome editing by sgRNA-Cas9 RNPs in Drosophila
FLY ( IF 1.2 ) Pub Date : 2020-10-21 , DOI: 10.1080/19336934.2020.1832416
Kevin G Nyberg 1 , Joseph Q Nguyen 1 , Yong-Jae Kwon 1 , Shelby Blythe 1 , Greg J Beitel 1 , Richard Carthew 1, 2
Affiliation  

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.



中文翻译:

通过 sgRNA-Cas9 RNPs 在果蝇中进行精确高效的基因组编辑的管道

摘要

通过同源定向修复 (HDR) 进行的基因组编辑使得对基因序列进行精确和有意的修改成为可能。CRISPR/Cas9 介导的 HDR 是执行此操作的最简单方法。然而,技术挑战仍然存在,以提高效率并扩大对黑腹果蝇以及其他果蝇物种的任何遗传背景的适用性。为了解决这些问题,我们开发了一种两阶段标记辅助策略,其中胚胎注射 RNP 并使用 T7EI 进行预筛选。使用与重组 Cas9 蛋白复合的 sgRNA,我们检测了每个 sgRNA 的基因组切割效率。然后,我们使用可有效切割目标基因的 sgRNA 进行 HDR,并应用可产生 RNAi 的转化标记来对抗缺失的眼睛. 这允许根据眼睛形态而不是颜色进行筛查。这些新工具可用于在感兴趣的区域进行单个更改或一系列等位基因替换,或创建额外的遗传工具,例如平衡染色体。

更新日期:2020-11-25
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