Genome-Wide CRISPR Screening to Identify Mammalian Factors that Regulate Intron Retention.
Genome-Wide CRISPR Screening to Identify Mammalian Factors that Regulate Intron Retention.
复制标题
全基因组 CRISPR 筛选以鉴定调节内含子保留的哺乳动物因素。
DOI：
10.1007/978-1-0716-2521-7_16
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Conrad,NicholasK
中科院分区：
文献类型：
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作者：
Scarborough,AnnaM;Govindan,Ashwin;Conrad,NicholasK
内含子保留 (IR) 调节基因表达，以控制新陈代谢、分化和细胞周期等基本生物过程。尽管 IR 控制的基因种类繁多，但很少有技术能够以公正的方式识别 IR 的调节因子。在这里，我们描述了一种 CRISPR 敲除筛选方法，可用于发现 IR 的调节因子。该方法使用含有来自感兴趣基因的保留内含子的 GFP 报告构建体，使得 GFP 信号以与内源基因相同的方式受 IR 调节。然后将 GFP 水平用作全基因组 CRISPR 筛选的读数。我们已经成功地使用这种方法来识别 MAT2A 转录本的 IR 的新调节因子，并提出类似的筛选将广泛适用于识别控制特定转录本的 IR 的新因子。
Intron retention (IR) regulates gene expression to control fundamental biological processes like metabolism, differentiation, and cell cycle. Despite a wide variety of genes controlled by IR, few techniques are available to identify regulators of IR in an unbiased manner. Here, we describe a CRISPR knockout screening method that can be applied to uncover regulators of IR. This method uses GFP reporter constructs containing a retained intron from a gene of interest such that GFP signal is regulated by IR in the same fashion as the endogenous gene. The GFP levels are then used as a readout for genome-wide CRISPR screening. We have successfully used this approach to identify novel regulator of IR of the MAT2A transcript and propose that similar screens will be broadly applicable for the identification of novel factors that control IR of specific transcripts.