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

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.

Figure 1

Lentivirally-delivered paired-guide RNAs create large-fragment deletion with high efficiency in Cas9 stably expressing human cells. (a) Structures of the lentiviral plasmids expressing paired guide RNAs (pgRNAs). The U6 promoter(s) and gRNA coding sequences were cloned into an LL3.7 lentiviral backbone. Amplified DNA fragments encoding customized pgRNAs were ligated into the lentiviral backbone with U6 promoters (U6₂) or only one mutual U6 promoter (U6₁) using the Golden Gate method. (b,c) pgRNA vectors were delivered into human cells that express Cas9 through lentivirus. Large-fragment deletions induced by pgRNAs targeting the CSPG4 gene were identified by PCR. Six pairs of gRNAs that produced large-fragment deletions from 2–4.5 kb were chosen (b) and primers L1/R1 were used for the genomic PCR reactions (b,c). All infected Huh7.5_OC cells were enriched by FACS and incubated for 6 days. The letter U6₂ or U6₁ represents two tandem ways in Fig. 1a and the control is a pair of gRNAs with one targeting the CSPG4 locus and the other targeting AAVS1 region. (d) Quantification for the efficiency of large-fragment deletions using genomic PCR over the course of time post transduction. The pgRNAs (3+3′ in Fig. 1c, generating 3.5-kb deletion by design) were delivered into Huh7.5_OC cells through lentiviral infection, and genomic DNA was extracted from different time points as indicated (upper). The primers L2/R2 corresponding to sequences flanking pgRNA targeting sites (b) were used for quantification, and primers L3/R3 corresponding to sequences farther away from the targeting sites (b) were used for normalization. Primer sequences are listed in Supplementary Table 11. Images were analysed using ImageJ software and data are presented as the mean ± s.d. (n = 3) (below). (e) DNA sequencing analysis of large-fragment deletions in the human CSPG4 locus targeted by pgRNAs (3+3′) from pooled cells 3 weeks post infection (Fig. 1d). Partial sequences of targeted genes containing the two gRNAs’ targeting regions are labelled in red and the shaded nucleotides represent the PAM sequences. Dashes indicate deletions.

Figure 2

pgRNA library design, cloning and screening. (a) pgRNA library design. 671 candidate lncRNAs were identified from ENSEMBL. For each candidate lncRNA, pgRNAs targeting promoter or gene bodies were designed with one unique gRNA for each pair serving as decoding barcode using the pgRNADesign algorithm (Online Methods and Supplementary Code). (b) pgRNA plasmid library construction. Each array-synthesized 137-nt DNA oligo contains two gRNAs (represented in red and purple). Oligos were amplified to produce dsDNA molecules, and cloned into a lentiviral backbone using a Gibson reaction. The final constructs were obtained after the insertion of a linker segment by BsmBI digestion and ligation (Online Methods and Supplementary Fig. 1. (c) The pgRNA library was delivered into Huh7.5_OC cells by lentiviral infection with a MOI of about 0.3. Infected cells were harvested by FACS for green fluorescence 3 days post infection. For screening, library cells were cultured for 30 days before genome DNA extraction and high-throughput sequencing analysis of the barcode gRNA regions.

Figure 3

Identification of negatively and positively selected lncRNAs. (a) The Pearson correlation coefficient between the replicates of control samples (Ctrl) and 30-day enrichment samples (Exp). (b) The log fold change distribution of pgRNAs targeting negative controls, positive controls and lncRNAs. *P < 0.05; **P < 0.01; Wilcox rank sum test. Center lines represent median values; box limits represent the interquartile range; whiskers extend each 1.5 times the interquartile range; dots represent outliers. (c) Essential genes served as positive controls are enriched in negative selections using Gene Set Enrichment Analysis (GSEA). The degree of enrichment is measured as Enrichment Score (ES), a measurement of over-representation of a gene set commonly used in the Gene-Set Enrichment Analysis (GSEA). (d) The log fold change values and genomic locations of all pgRNAs targeting EZH2, a positive control gene. A zoom-in view of pgRNAs near the promoter of EZH2 is also shown. The majority of pgRNAs are depleted, including pgRNAs targeting the promoter, promoter + exons and introns of EZH2 (table on the right). (e) The mean read counts of pgRNAs targeting the promoter and promoter + exon of a positive control ribosomal gene, RPL18A. (f,g) The Robust Rank Aggregation (RRA) scores of top ranked negatively selected lncRNAs (f) and positively selected lncRNAs (g) calculated by MAGeCK. Some positive control genes that are negatively selected are also shown as black triangles. A smaller RRA score indicates a stronger selection of the corresponding lncRNAs.

Figure 4

Validation of candidate lncRNAs. (a–c) Effects of large-fragment deletions of RPL18A (a), negatively selected lncRNAs (b) and positively selected lncRNAs (c) on cell proliferation in Huh7.5_OC cells. 3~5 pairs of gRNAs of each lncRNA targeting promoter or promoter + exon were chosen for validation. The pgRNAs, expressing from a backbone carrying CMV promoter-driven EGFP, were delivered into cells by lentiviral infection, and percentage of EGFP⁺ cells was quantified by FACS. The first quantification started from three days post viral infection, labelled as Day 0 in this and the rest of figures. Cell proliferation was determined by normalizing EGFP⁺ percentages at indicated time points with control (Day 0). Newly designed pgRNAs different to those used in the original library are marked with an asterisk (*). The arrows indicate the transcriptional start sites. Open and shaded boxes refer to exons of non-coding and coding genes respectively. (d) Effects of transcriptional repression of negatively selected lncRNAs on cell proliferation. RPL18A, AC004463.6, RP11-439K3.1 and AC095067.1 mRNA levels (normalized to GAPDH) were quantified. All primers used for quantitative PCR are listed in Supplementary Table 12. (e) Effects of sgRNA and pgRNAs targeting negatively-selected lncRNAs on cell proliferation in Huh7.5 cells. Data are presented as the mean ± s.d. (n = 3). P values were calculated using Student’s t test and corrected for multiple comparison using Benjamini Hochberg procedure, *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Figure 5

Functional expression analysis of selected selection of identified lncRNAs. (a–c) RNA-seq profiling of cells that have a LINC01087 (positively selected) knockout. (a) RNA-seq gene expression clustering of cells with a LINC01087 knockout using three different sets of pgRNAs, and control cells in which the AAVS1 locus is deleted. Clustering is based on the Euclid distances between samples, using the expressions of all genes. (b) Two Fos family genes, FOS and FOSB, were up-regulated in the LINC01087 knockout. (c) Enriched signatures of genes that are up-regulated upon deletion of LINC01087 using GSEA analysis. These signatures include genes that are up-regulated in liver cancer (MSigDB ID: LEE_LIVER_CANCER_SURVIVAL_UP), HNF4α target genes (SUMI_HNF4A_TARGETS), and genes in KEGG retinol metabolism pathway (KEGG_RETINOL_METABOLISM). All signatures are statistically significant (q = 0; permutation test). (d,e) The Gene Ontology (GO) terms of genes that are co-expressed with top negatively- (d) or positively- (e) selected lncRNAs in liver cancer. (f) One negatively selected lncRNA, AC004463.6 is significantly over-expressed in liver cancer tumours and metastasis prostate tumours. (g) Comparison of lncRNA screens in Huh7.5 and HeLa cells (a cervical cancer cell line). Two lncRNAs are essential in both cell lines (red dots), and three lncRNAs are only essential in Huh7.5 cells (blue dots). Similar to Fig. 3f–g, a smaller RRA score indicates stronger selection of the corresponding lncRNAs. (h) Effects of deletion of selected lncRNAs on HeLa cell proliferation. Data are presented as the mean ± s.d. (n = 2). P values were calculated using Student’s t test and corrected for multiple comparisons using the Benjamini Hochberg procedure, *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.