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.
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Shalem, O. et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).
Wang, T. et al. Identification and characterization of essential genes in the human genome. Science 350, 1096–1101 (2015).
Koike-Yusa, H., Li, Y., Tan, E.P., Velasco-Herrera, Mdel C. & Yusa, K. Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library. Nat. Biotechnol. 32, 267–273 (2014).
Zhou, Y. et al. High-throughput screening of a CRISPR/Cas9 library for functional genomics in human cells. Nature 509, 487–491 (2014).
Rajagopal, N. et al. High-throughput mapping of regulatory DNA. Nat. Biotechnol. 34, 167–174 (2016).
Korkmaz, G. et al. Functional genetic screens for enhancer elements in the human genome using CRISPR-Cas9. Nat. Biotechnol. 34, 192–198 (2016).
Shalem, O., Sanjana, N.E. & Zhang, F. High-throughput functional genomics using CRISPR-Cas9. Nat. Rev. Genet. 16, 299–311 (2015).
Peng, J., Zhou, Y., Zhu, S. & Wei, W. High-throughput screens in mammalian cells using the CRISPR-Cas9 system. FEBS J. 282, 2089–2096 (2015).
Canver, M.C. et al. BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. Nature 527, 192–197 (2015).
Han, J. et al. Efficient in vivo deletion of a large imprinted lncRNA by CRISPR/Cas9. RNA Biol. 11, 829–835 (2014).
Yin, Y. et al. Opposing roles for the lncRNA haunt and its genomic locus in regulating HOXA gene activation during embryonic stem cell differentiation. Cell Stem Cell 16, 504–516 (2015).
Ren, Q. et al. A dual-reporter system for real-time monitoring and high-throughput CRISPR/Cas9 library screening of the hepatitis C virus. Sci. Rep. 5, 8865 (2015).
Zheng, Q. et al. Precise gene deletion and replacement using the CRISPR/Cas9 system in human cells. Biotechniques 57, 115–124 (2014).
Du, Z. et al. Integrative genomic analyses reveal clinically relevant long noncoding RNAs in human cancer. Nat. Struct. Mol. Biol. 20, 908–913 (2013).
Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).
Xu, H. et al. Sequence determinants of improved CRISPR sgRNA design. Genome Res. 25, 1147–1157 (2015).
Li, W. et al. MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens. Genome Biol. 15, 554 (2014).
Subramanian, A. et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc. Natl. Acad. Sci. USA 102, 15545–15550 (2005).
Cheng, A.S. et al. EZH2-mediated concordant repression of Wnt antagonists promotes β-catenin-dependent hepatocarcinogenesis. Cancer Res. 71, 4028–4039 (2011).
Gillies, S.D., Morrison, S.L., Oi, V.T. & Tonegawa, S. A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell 33, 717–728 (1983).
Xiao, X. et al. Splice site strength-dependent activity and genetic buffering by poly-G runs. Nat. Struct. Mol. Biol. 16, 1094–1100 (2009).
Gilbert, L.A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).
Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).
Eferl, R. & Wagner, E.F. AP-1: a double-edged sword in tumorigenesis. Nat. Rev. Cancer 3, 859–868 (2003).
Hatziapostolou, M. et al. An HNF4α-miRNA inflammatory feedback circuit regulates hepatocellular oncogenesis. Cell 147, 1233–1247 (2011).
Rinn, J.L. & Chang, H.Y. Genome regulation by long noncoding RNAs. Annu. Rev. Biochem. 81, 145–166 (2012).
Barretina, J. et al. The cancer cell line encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603–607 (2012).
Sramkoski, R.M. et al. A new human prostate carcinoma cell line, 22Rv1. In Vitro Cell. Dev. Biol. Anim. 35, 403–409 (1999).
Louro, R., Smirnova, A.S. & Verjovski-Almeida, S. Long intronic noncoding RNA transcription: expression noise or expression choice? Genomics 93, 291–298 (2009).
Vidigal, J.A. & Ventura, A. Rapid and efficient one-step generation of paired gRNA CRISPR-Cas9 libraries. Nat. Commun. 6, 8083 (2015).
Yates, A. et al. Ensembl 2016. Nucleic Acids Res. 44, D710–D716 (2016).
Smyth, G.K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article3 (2004).
Wang, T., Wei, J.J., Sabatini, D.M. & Lander, E.S. Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).
Sanjana, N.E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).
Gibson, D.G. Enzymatic assembly of overlapping DNA fragments. Methods Enzymol. 498, 349–361 (2011).
Anders, S. & Huber, W. Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010).
Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).
Anders, S., Pyl, P.T. & Huber, W. HTSeq--a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).
Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Taylor, B.S. et al. Integrative genomic profiling of human prostate cancer. Cancer Cell 18, 11–22 (2010).
Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455, 1061–1068 (2008).
Partensky, F. & Garczarek, L. Microbiology: arms race in a drop of sea water. Nature 474, 582–583 (2011).
Cancer Genome Atlas Research Network. Comprehensive genomic characterization of squamous cell lung cancers. Nature 489, 519–525 (2012).
Li, J. et al. TANRIC: an interactive open platform to explore the function of lncRNAs in cancer. Cancer Res. 75, 3728–3737 (2015).
Wilks, C. et al. The cancer genomics hub (CGHub): overcoming cancer through the power of torrential data. Database (Oxford) 2014, bau093 (2014).
Trapnell, C. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012).
Alexa, A., Rahnenführer, J. & Lengauer, T. Improved scoring of functional groups from gene expression data by decorrelating GO graph structure. Bioinformatics 22, 1600–1607 (2006).
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)
About this article
Cite this article
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
Optimized RNA-targeting CRISPR/Cas13d technology outperforms shRNA in identifying functional circRNAs
Genome Biology (2021)
Nature Reviews Cancer (2021)
Nature Aging (2021)
Nature Cell Biology (2021)
Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening
Nature Protocols (2021)