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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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.
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 Figures 1–15, Supplementary Tables 1–4, and Supplementary Text 1 and 2 (PDF 6841 kb)
MAGeCK results of negatively and positively selected lncRNAs in Huh7.5 cell line. (XLSX 212 kb)
pgRNA design for functional validation of selected lncRNAs. (XLSX 45 kb)
sgRNA design of CRISPR-inhibitor and CRISPR-activator for functional validation of selected lncRNAs. (XLSX 45 kb)
The Gene Ontology (GO) enrichment results of genes that are correlated with top lncRNA hits in liver cancer cell lines. (XLSX 563 kb)
MAGeCK results of negatively and positively selected lncRNAs in HeLa cell line. (XLSX 202 kb)
Oligos for the pgRNA library construction, the read counts of control and treatment samples, and the locations of targeting lncRNAs. (XLSX 1487 kb)
Primers used for PCR amplification of the genomic DNAs and library construction. (XLSX 35 kb)
Primers for Quantitative PCR. (XLSX 37 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). https://doi.org/10.1038/nbt.3715
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