Transcriptome-scale RNA-targeting CRISPR screens reveal essential lncRNAs in human cells

Mammalian genomes host a diverse array of RNA that includes protein-coding and noncoding transcripts. However, the functional roles of most long noncoding RNAs (lncRNAs) remain elusive. Using massively parallel RNA-targeting CRISPR Cas13 screens, we probed how loss of ~6,200 lncRNAs impacts cell fitness across five human cell lines and identified 778 lncRNAs with either context-specific or broad essentiality. We observe a high level of consistency between distinct guide RNAs targeting the same lncRNA in the pooled screens and confirm their essentiality with individual perturbations. We find that the overwhelming majority of essential lncRNAs operate independently of their nearest protein-coding genes. Using transcriptome profiling in single cells, we discover that essential lncRNAs modulate key cellular pathways for proliferation and that their loss can impair cell cycle progression and drive apoptosis. Many essential lncRNAs demonstrate dynamic expression across tissues during development and, using ~9,000 primary tumors, we pinpoint those lncRNAs whose expression in tumors correlates with survival, yielding new biomarkers and potential therapeutic targets. This transcriptome-wide survey of functional lncRNAs advances our understanding of noncoding transcripts and demonstrates the potential of transcriptome-scale noncoding screens with Cas13. To identify essential lncRNAs, we engineered five human cell lines, HAP1, HEK293FT, K562, MDA-MB-231 and THP1, to express the nuclear-localized RfxCas13d effector under doxycycline-inducible control. Next, we transduced each cell line with the lentiviral gRNA library at a low multiplicity-of-infection to ensure each cell received only a single perturbation and induced Cas13 expression via doxycycline addition. We harvested genomic DNA from these cells at 0, 7, and 14 days post-Cas13 induction and computed changes in gRNA abundance via amplicon sequencing.

哺乳动物基因组承载着种类繁多的RNA分子，涵盖编码蛋白的转录本与非编码转录本。然而，绝大多数长链非编码RNA（long noncoding RNAs，lncRNAs）的功能角色仍未明确。本研究借助大规模并行RNA靶向CRISPR Cas13筛选技术，探究了约6200个长链非编码RNA的敲除对5种人类细胞系细胞适配性的影响，最终鉴定出778个具有情境特异性或广谱必需性的长链非编码RNA。在混合筛选中，靶向同一长链非编码RNA的不同向导RNA（guide RNAs，gRNAs）之间呈现高度一致性，本研究通过单基因扰动实验验证了这些长链非编码RNA的必需性。研究发现，绝大多数必需长链非编码RNA的功能独立于其邻近的蛋白编码基因。借助单细胞转录组分析，本研究发现必需长链非编码RNA可调控增殖相关的关键细胞通路，其敲除会损害细胞周期进程并诱导细胞凋亡。诸多必需长链非编码RNA在发育过程中的不同组织间呈现动态表达模式；本研究借助约9000例原发性肿瘤样本，筛选出了肿瘤中表达水平与患者生存率相关的长链非编码RNA，为其提供了新型生物标志物与潜在治疗靶点。这项针对功能性长链非编码RNA的全转录组研究加深了我们对非编码转录本的认知，同时证实了基于Cas13的全转录组非编码RNA筛选技术的应用潜力。为鉴定必需长链非编码RNA，本研究构建了5种可在多西环素诱导下表达核定位RfxCas13d效应蛋白的人类细胞系，分别为HAP1、HEK293FT、K562、MDA-MB-231与THP1。随后，本研究以低感染复数将慢病毒介导的向导RNA文库转入各细胞系，确保每个细胞仅接受单次基因扰动，并通过添加多西环素诱导Cas13的表达。本研究在Cas13诱导后的第0、7、14天收集这些细胞的基因组DNA，并通过扩增子测序计算向导RNA的丰度变化。