Genome-wide screening for functional long noncocing RNAs in human cells by Cas9 targeting of splice sites
Genome-wide screening for functional long noncocing RNAs in human cells by Cas9 targeting of splice sites
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通过 Cas9 靶向剪接位点对人类细胞中的功能性长非编码 RNA 进行全基因组筛选。
DOI：
10.1038/nbt.4283
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发表时间：
2018-12-01
影响因子：
46.9
通讯作者：
Wei, Wensheng
中科院分区：
文献类型：
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作者：
Liu, Ying;Cao, Zhongzheng;Wei, Wensheng
由于缺乏可扩展的功能丧失筛选工具，人类基因组中许多长非编码RNA（lncRNA）的功能仍然未知。我们之前使用了CRISPR-Cas9对（参考文献10）。1-3)用于lncRNA的小规模功能筛选的单向导RNA（sgRNA）（4）。在这里，我们展示了使用sgRNA靶向剪接位点并实现外显子跳跃或内含子保留的lncRNA功能的全基因组筛选。剪接位点靶向在靶向79个核糖体基因的阴性选择筛选中优于常规CRISPR文库。使用基因组规模的剪接靶向sgRNA文库，我们进行了覆盖10，996个lncRNA的筛选，并鉴定了230个对慢性髓性白血病K562细胞的细胞生长至关重要的lncRNA。用相同的文库筛选GM 12878淋巴母细胞样细胞和HeLa细胞鉴定了lncRNA必要性的细胞类型特异性差异。广泛的验证证实了我们的方法的鲁棒性。
The functions of many long noncoding RNAs (lncRNAs) in the human genome remain unknown owing to the lack of scalable loss-of-function screening tools. We previously used pairs of CRISPR-Cas9 (refs. 1-3) single guide RNAs (sgRNAs) for small-scale functional screening of lncRNAs(4). Here we demonstrate genome-wide screening of lncRNA function using sgRNAs to target splice sites and achieve exon skipping or intron retention. Splice-site targeting outperformed a conventional CRISPR library in a negative selection screen targeting 79 ribosomal genes. Using a genome-scale library of splicing-targeting sgRNAs, we performed a screen covering 10,996 lncRNAs and identified 230 that are essential for cellular growth of chronic myeloid leukemia K562 cells. Screening GM12878 lymphoblastoid cells and HeLa cells with the same library identified cell-type-specific differences in lncRNA essentiality. Extensive validation confirmed the robustness of our approach.