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Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library

Nature Biotechnology volume 34, pages 12791286 (2016) | Download Citation

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Abstract

CRISPR–Cas9 screens have been widely adopted to analyze coding-gene functions, but high-throughput screening of non-coding elements using this method is more challenging because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. We report a high-throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We validated 9 of 51 lncRNA hits using CRISPR–Cas9-mediated genomic deletion, functional rescue, CRISPR activation or inhibition and gene-expression profiling. Our high-throughput pgRNA genome deletion method will enable rapid identification of functional mammalian non-coding elements.

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Change history

  • 09 November 2016

    In the version of this article initially published online, genomic deletion values (listed in base pairs) in Figure 1e were incorrect: in the second line, the value given as 3,476 should have been 3,511; third line, 3,479 should have been 3,514; fourth line, 3,475 should have been 3,510; fifth line, 3,484 should have been 3,519. The errors have been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We acknowledge the staff of the BIOPIC sequencing facility (Peking University) for their assistance, and National Center for Protein Sciences Beijing (Peking University) for help in Fluorescence Activated Cell Sorting. The project was supported by funds from the National Science Foundation of China (NSFC31430025, NSFC31170126, NSFC81471909), Beijing Advanced Innovation Center for Genomics at Peking University, and the Peking-Tsinghua Center for Life Sciences (to W.W.), the NIH grant U01 CA180980 (to X.S.L.), R01 HG008728 (to M.B. and X.S.L.), and the Claudia Adams Barr Award in Innovative Basic Cancer Research from the Dana-Farber Cancer Institute.

Author information

Author notes

    • Shiyou Zhu
    •  & Wei Li

    These authors contributed equally to this work.

    • Xiaole Shirley Liu
    •  & Wensheng Wei

    These authors jointly directed this work.

Affiliations

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

    • Shiyou Zhu
    • , Jingze Liu
    • , Ping Xu
    • , Zhongzheng Cao
    • , Jingyu Peng
    • , Pengfei Yuan
    •  & Wensheng Wei
  2. Peking University–Tsinghua University–National Institute of Biological Sciences Joint Graduate Program (PTN), Peking University, China.

    • Shiyou Zhu
    •  & Jingze Liu
  3. Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USA.

    • Wei Li
    • , Chen-Hao Chen
    •  & Xiaole Shirley Liu
  4. Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Wei Li
    • , Chen-Hao Chen
    • , Tengfei Xiao
    • , Myles Brown
    •  & Xiaole Shirley Liu
  5. Department of Prevention Medicine, School of Medicine, Ningbo University, Ningbo, Zhejiang, China.

    • Qi Liao
  6. Broad Institute of MIT and Harvard, Cambridge Center, Cambridge, Massachusetts, USA.

    • Han Xu
  7. Division of Molecular and Cellular Oncology, Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA.

    • Tengfei Xiao
    •  & Myles Brown
  8. Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China.

    • Zhongzheng Cao
  9. Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

    • Myles Brown

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Contributions

X.S.L. and W.W. conceived and supervised the project. W.W., S.Z., J.P., and P.Y. designed the experiments. S.Z., J.L., P.X. and Z.C. performed the experiments with help from W.L., T.X. and M.B. and T.X. W.L., C.-H.C. and H.X. designed the oligos used for pgRNA library construction. W.L. performed the data analysis, with the help of Q.L. on the functional expression analysis of candidate lncRNAs. S.Z., W.L., X.S.L. and W.W. wrote the manuscript with the help of all other authors.

Competing interests

A patent application has been filed to the State Intellectual Property Office of the PR China, which mainly covers the methodology for the construction of the pgRNA library and its application in functional screening studies.

Corresponding authors

Correspondence to Xiaole Shirley Liu or Wensheng Wei.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–15, Supplementary Tables 1–4, and Supplementary Text 1 and 2

Excel files

  1. 1.

    Supplementary Table 5

    MAGeCK results of negatively and positively selected lncRNAs in Huh7.5 cell line.

  2. 2.

    Supplementary Table 6

    pgRNA design for functional validation of selected lncRNAs.

  3. 3.

    Supplementary Table 7

    sgRNA design of CRISPR-inhibitor and CRISPR-activator for functional validation of selected lncRNAs.

  4. 4.

    Supplementary Table 8

    The Gene Ontology (GO) enrichment results of genes that are correlated with top lncRNA hits in liver cancer cell lines.

  5. 5.

    Supplementary Table 9

    MAGeCK results of negatively and positively selected lncRNAs in HeLa cell line.

  6. 6.

    Supplementary Table 10

    Oligos for the pgRNA library construction, the read counts of control and treatment samples, and the locations of targeting lncRNAs.

  7. 7.

    Supplementary Table 11

    Primers used for PCR amplification of the genomic DNAs and library construction.

  8. 8.

    Supplementary Table 12

    Primers for Quantitative PCR.

Zip files

  1. 1.

    Supplementary Code

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DOI

https://doi.org/10.1038/nbt.3715

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