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, 2, 3) single guide RNAs (sgRNAs) for small-scale functional screening of lncRNAs4. 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.
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We acknowledge the staff of the BIOPIC High-Throughput Sequencing Center (Peking University) for their assistance in next-generation sequencing analysis, the National Center for Protein Sciences Beijing (Peking University), and the core facilities at School of Life Sciences (Peking University) for help in fluorescence-activated cell sorting. We also acknowledge the High-performance Computing Platform of Peking University. This project was supported by funds from the National Science Foundation of China (NSFC31430025), the Beijing Advanced Innovation Center for Genomics at Peking University, and the Peking-Tsinghua Center for Life Sciences (W.W.).
A patent has been filed relating to the data presented. W.W. is a founder and scientific advisor for EdiGene.
Supplementary Figures 1–14 (PDF 12615 kb)
sgRNAs of splicing-targeting library on essential ribosomal genes (XLSX 287 kb)
sgRNA read counts and phenotypes in ribosomal gene screening (XLSX 635 kb)
sgRNAs of whole-genome human lncRNA library (XLSX 6458 kb)
sgRNA read counts in splicing-targeting screen for lncRNAs in multiple cell lines (XLSX 18380 kb)
Screen scores of lncRNAs by splicing-targeting screen in multiple cell lines (XLSX 2586 kb)
Screen scores of lncRNAs by splicing-targeting screen in multiple cell lines (generated after filtering sgRNAs with potential off-target effects) (XLSX 2655 kb)
Individually cloned sgRNAs and pgRNAs for validation (XLSX 20 kb)
Source code for the computational analysis of lncRNA screens described in this paper. (ZIP 2285 kb)
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Liu, Y., Cao, Z., Wang, Y. et al. Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites. Nat Biotechnol 36, 1203–1210 (2018). https://doi.org/10.1038/nbt.4283
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