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

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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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Figure 1: Lentivirally delivered sgRNAs generate intron retention or exon skipping by disrupting splice sites.
Figure 2: Splicing-targeting enables genome-scale screening for the identification of lncRNAs essential for cell growth and proliferation.
Figure 3: Validation of candidate lncRNAs.
Figure 4: Cell type specificity of lncRNA function across multiple cell lines.

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Change history

  • 16 January 2019

    In the supplementary information originally posted, there were incorrect values for the GM12878 cell line in Supplementary Table 5. The error has been corrected online.

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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.).

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Authors

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.

Corresponding author

Correspondence to Wensheng Wei.

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Competing interests

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

Supplementary information

Supplementary Figures

Supplementary Figures 1–14 (PDF 12615 kb)

Life Sciences Reporting Summary (PDF 130 kb)

Supplementary Table 1

sgRNAs of splicing-targeting library on essential ribosomal genes (XLSX 287 kb)

Supplementary Table 2

sgRNA read counts and phenotypes in ribosomal gene screening (XLSX 635 kb)

Supplementary Table 3

sgRNAs of whole-genome human lncRNA library (XLSX 6458 kb)

Supplementary Table 4

sgRNA read counts in splicing-targeting screen for lncRNAs in multiple cell lines (XLSX 18380 kb)

Supplementary Table 5

Screen scores of lncRNAs by splicing-targeting screen in multiple cell lines (XLSX 2586 kb)

Supplementary Table 6

Screen scores of lncRNAs by splicing-targeting screen in multiple cell lines (generated after filtering sgRNAs with potential off-target effects) (XLSX 2655 kb)

Supplementary Table 7

Individually cloned sgRNAs and pgRNAs for validation (XLSX 20 kb)

Supplementary Code

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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