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

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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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Figure 1: Lentivirally delivered paired-guide RNAs create large-fragment deletion with high efficiency in human cells stably expressing Cas9.
Figure 2: pgRNA library design, cloning and screening.
Figure 3: Identification of negatively and positively selected lncRNAs.
Figure 4: Validation of candidate lncRNAs.
Figure 5: Functional expression analysis of selected selection of identified lncRNAs.

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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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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




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.

Corresponding authors

Correspondence to Xiaole Shirley Liu or Wensheng Wei.

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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.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15, Supplementary Tables 1–4, and Supplementary Text 1 and 2 (PDF 6841 kb)

Supplementary Table 5

MAGeCK results of negatively and positively selected lncRNAs in Huh7.5 cell line. (XLSX 212 kb)

Supplementary Table 6

pgRNA design for functional validation of selected lncRNAs. (XLSX 45 kb)

Supplementary Table 7

sgRNA design of CRISPR-inhibitor and CRISPR-activator for functional validation of selected lncRNAs. (XLSX 45 kb)

Supplementary Table 8

The Gene Ontology (GO) enrichment results of genes that are correlated with top lncRNA hits in liver cancer cell lines. (XLSX 563 kb)

Supplementary Table 9

MAGeCK results of negatively and positively selected lncRNAs in HeLa cell line. (XLSX 202 kb)

Supplementary Table 10

Oligos for the pgRNA library construction, the read counts of control and treatment samples, and the locations of targeting lncRNAs. (XLSX 1487 kb)

Supplementary Table 11

Primers used for PCR amplification of the genomic DNAs and library construction. (XLSX 35 kb)

Supplementary Table 12

Primers for Quantitative PCR. (XLSX 37 kb)

Supplementary Code (ZIP 4338 kb)

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Zhu, S., Li, W., Liu, J. et al. Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR–Cas9 library. Nat Biotechnol 34, 1279–1286 (2016).

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