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from GenomeWeb by Andrew Han –
A new study published today in Nature Biotechnology turns high-throughput CRISPR/Cas9-based knockout screening towards long non-coding RNAs to find functional elements.
A Chinese-American collaboration, led by first author Shiyou Zhu and senior author Wei Li of Peking University and first author Wensheng Wei and senior author Xiaole Shirley Liu of the Dana-Farber Cancer Institute, designed a paired guide RNA (gRNA) screening method that identified 51 lncRNAs positively or negatively regulating cell growth in Huh7.5 OC cells.
It’s a deletion-based strategy, Neville Sanjana of the New York Genome Center and New York University told GenomeWeb, where two gRNAs simultaneously direct cleavage to cut out a chunk of DNA. “I think this is a nice preview of how we can scale up to cover larger intervals of DNA to tackle the non-coding genome.
Identification of negatively and positively selected lncRNAs
(a) The Pearson correlation coefficient between the replicates (Rep.) of control samples (Ctrl) and 30-d enrichment samples (Exp). (b) The log fold-change distribution of pgRNAs targeting negative controls, positive controls and lncRNAs. *P < 0.05; **P < 0.01; Wilcoxon rank–sum test. Center lines represent median values; box limits represent the interquartile range; whiskers each extend 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 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 at 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.
