CRISPRi-based genome-scale identification of functional long non-coding RNA loci in human cells

By S. Liu, Max A. Horlbeck, S. Cho, H. Birk, M. Malatesta, Daniel He, F. Attenello, Jacqueline E. Villalta, M. Cho, Yuwen Chen and 7 more. Science, 2016

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A very focused function for lncRNAs The human genome generates many thousands of long noncoding RNAs (lncRNAs). A very small number of lncRNAs have been shown to be functional. Liu et al. carried out a large-scale CRISPR-based screen to assess the function of ∼17,000 lncRNAs in seven different human cell lines. A considerable number (∼500) of the tested lncRNAs influenced cell growth, suggesting biological function. In almost all cases, though, the function was highly cell type—specific, often limited to just one cell type. Science, this issue p. 10.1126/science.aah7111 A considerable fraction of long noncoding RNAs have highly cell type–specific biological functions. INTRODUCTION The human genome contains tens of thousands of loci that produce long noncoding RNAs (lncRNAs), transcripts that have no apparent protein-coding potential. A subset of lncRNAs have been found to play critical roles in cellular processes, organismal development, and disease. Although these examples are suggestive of the importance and diversity of lncRNAs, the vast majority of lncRNA genes have not been functionally tested. RATIONALE Because it is currently not possible to predict which lncRNA loci are functional or what function they perform, there is a need for large-scale, systematic approaches to interrogating the functional contribution of lncRNA loci. We therefore developed a genome-scale screening platform based on CRISPR-mediated interference (CRISPRi), which uses a catalytically inactive CRISPR effector protein, (d)Cas9, fused to a repressive KRAB domain and targeted by a single guide RNA (sgRNA), to inhibit gene expression. By catalyzing repressive chromatin modifications around the transcription start site (TSS) and serving as a transcriptional roadblock, CRISPRi tests a broad range of lncRNA gene functions, including the production of cis- and trans-acting RNA transcripts, cis-mediated regulation related to lncRNA transcription itself, and enhancer-like function of some lncRNA loci. RESULTS We designed a CRISPRi Non-Coding Library (CRiNCL), which targets 16,401 lncRNA genes each with 10 sgRNAs per TSS, and applied this pooled screening approach to identify lncRNA genes that modify robust cell growth. We screened seven human cell lines, including six transformed cell lines and induced pluripotent stem cells (iPSCs), and identified 499 lncRNA loci that modified cell growth upon CRISPRi targeting; 372 and 299 of these loci were distal from any protein coding gene or mapped enhancer, respectively. Extensive validation confirmed the screen results and demonstrated the robust and specific performance of CRISPRi for repressing lncRNA transcription. Remarkably, 89% of the lncRNA gene hits modified growth in just one of the cell lines tested, and no hits were common to all seven cell lines. Although nearly all of the hit genes were expressed in the cell line in which they exhibited a growth phenotype, expression alone was insufficient to explain the cell type specificity of their function. Transcriptional profiling revealed extensive gene expression changes upon CRISPRi targeting of lncRNA loci in the cells in which they modified growth, whereas targeting the same lncRNA locus in other cell lines resulted in minimal changes to the transcriptome beyond depletion of the targeted lncRNA transcript itself. CONCLUSION Our study considerably increases the number of known functional lncRNA loci. More broadly, our CRISPRi approach enables mechanistic studies of specific lncRNA functions and, when applied systematically, supports the global exploration of the complex biology contained in the lncRNA-expressing genome. Finally, in contrast to recent studies that found that essential protein-coding genes typically are required across a broad range of cell types, we show that lncRNA function is highly cell type–specific, a finding that has important implications for their involvement in both normal biology and disease. CRISPRi screening of lncRNAs in human cells. CRISPRi can precisely repress transcription of lncRNAs. The CRISPRi Non-Coding Library (CRiNCL) was generated to interrogate the function of thousands of long noncoding RNAs in seven different cell lines. Validation studies confirmed the exquisite cell type–specific function of lncRNAs. The human genome produces thousands of long noncoding RNAs (lncRNAs)—transcripts >200 nucleotides long that do not encode proteins. Although critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. We developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in seven diverse cell lines, including six transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth-modifying function exclusively in one cell type. We further found that lncRNA knockdown can perturb complex transcriptional networks in a cell type–specific manner. These data underscore the functional importance and cell type specificity of many lncRNAs.

Subfields: Cancer Research, Endocrinology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/3642c2f0a040b0170fd889b96bfb3eead48967e5

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