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简化的同样性辅助CRISPR用于Drosophila中的基因编辑

Anne E Rankin¹, Elizabeth Fox², Townley Chisholm¹

¹Phillips Exeter Academy, Exeter, NH 03833, USA.

G3 (Bethesda, Md.)

|December 7, 2023

概括

研究人员优化了CRISPR基因组编辑,将Drosophila GAL4线转换为LexA线. 这种改进的方法使用具有可见标记的通用敲进供体菌株,简化了用于基因调节研究的新LexA线的生成.

科学领域:

分子生物学分子生物学
遗传学遗传学是一个
发展生物学发展生物学

背景情况:

在研究基因功能和调节方面,聚类定期间隔的短平行体重复 (CRISPR) /Cas9基因组编辑至关重要.
现有的同质性辅助CRISPR敲门方法需要优化,以便高效地将GAL4线路转换为LexA线路.

研究的目的:

修改同质性辅助的CRISPR敲门方法,将Drosophila GAL4线路转换为LexA线路.
开发一种具有改进查标记的通用 knock-in 供体菌株.
为基因表达研究生成新型LexA线的集合.

主要方法:

使用了经过修订的同类辅助CRISPR敲门方法.
开发并使用了一个平衡器染色体链接的通用敲进供体菌株.
嵌入体颜色 (黄色) 和红色光蛋白 (RFP) 表达标记器用于敲进识别.
从现有的GAL4线路中生成和特征化新的LexA线路.

主要成果:

通过使用修订的CRISPR方法,成功将GAL4线路转换为LexA线路.
在LexA敲进后代中证明了黄色转基因的一致和强大的表达.
在双转基因磁带中观察到3xP3-RFP转基因的显著增加表达.
产生了16个新的LexA线,其组织表达模式与其原始的GAL4线相似.

结论:

修订后的CRISPR敲门方法和通用供体菌株为产生Drosophila中的LexA线提供了一种有效的方法.
开发的选标记简化了成功敲入的识别和验证.
生成的LexA线路适合研究基因调节和功能,反映了GAL4线路表达模式.

关键词:

这种植物是Drosophila.这就是CRISPR/Cas9的作用.这就是HACKy.莱克萨斯 (LexA) 是一个

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