Letter | Published:

Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites

Nature Biotechnology volume 36, pages 12031210 (2018) | Download Citation

Abstract

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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Acknowledgements

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.).

Author information

Author notes

    • Yu Guo
    •  & Pengfei Yuan

    Present address: EdiGene Inc, Beijing, China.

    • Ying Liu
    • , Zhongzheng Cao
    • , Yinan Wang
    •  & Yu Guo

    These authors contributed equally to this work.

Affiliations

  1. Biomedical Pioneering Innovation Center (BIOPIC), Beijing Advanced Innovation Center for Genomics, Peking-Tsinghua Center for Life Sciences, Peking University Genome Editing Research Center, State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China.

    • Ying Liu
    • , Zhongzheng Cao
    • , Yinan Wang
    • , Yu Guo
    • , Ping Xu
    • , Pengfei Yuan
    • , Zhiheng Liu
    • , Yuan He
    •  & Wensheng Wei
  2. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.

    • Ying Liu
    • , Zhongzheng Cao
    • , Yinan Wang
    •  & Zhiheng Liu

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Contributions

W.W. conceived and supervised the project. W.W., Y.L. and Z.C. designed the experiments. Y.L., Z.C., P.X. and Y.H. performed the experiments. Y.G. designed the oligonucleotides used for ribosomal gene mutagenesis and genome-wide lncRNA library, and Z.L. designed the pgRNAs used for individual validation. Y.W., Y.G. and P.Y. performed the bioinformatics analysis. Y.L., Z.C., Y.W. and W.W. wrote the manuscript with the help of all other authors.

Competing interests

A patent has been filed relating to the data presented. W.W. is a founder and scientific advisor for EdiGene.

Corresponding author

Correspondence to Wensheng Wei.

Supplementary information

PDF files

  1. 1.

    Supplementary Figures

    Supplementary Figures 1–14

  2. 2.

    Life Sciences Reporting Summary

Excel files

  1. 1.

    Supplementary Table 1

    sgRNAs of splicing-targeting library on essential ribosomal genes

  2. 2.

    Supplementary Table 2

    sgRNA read counts and phenotypes in ribosomal gene screening

  3. 3.

    Supplementary Table 3

    sgRNAs of whole-genome human lncRNA library

  4. 4.

    Supplementary Table 4

    sgRNA read counts in splicing-targeting screen for lncRNAs in multiple cell lines

  5. 5.

    Supplementary Table 5

    Screen scores of lncRNAs by splicing-targeting screen in multiple cell lines

  6. 6.

    Supplementary Table 6

    Screen scores of lncRNAs by splicing-targeting screen in multiple cell lines (generated after filtering sgRNAs with potential off-target effects)

  7. 7.

    Supplementary Table 7

    Individually cloned sgRNAs and pgRNAs for validation

Zip files

  1. 1.

    Supplementary Code

    Source code for the computational analysis of lncRNA screens described in this paper.

About this article

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DOI

https://doi.org/10.1038/nbt.4283