Dissecting long non-coding RNAs derived from microRNA genes in hematopoiesis (scRNA sequencing)

Albeit majority of eukaryotic genome can be pervasively transcribed to a diverse population of lncRNAs and various subtypes of lncRNA have been discovered. However, the genome-wide study of miRNA-derived lncRNAs is still lacking. Here, we report that over 800 miRNA gene-originated lncRNAs (molncRNAs) generated from miRNA loci. One of them, molnc-301b from miR-301b and miR-130b, functions as an "RNA decoy" to facilitate dissociation of the chromatin remodeling protein SMARCA5 from chromatin and thereby sequester transcription and mRNA translation. Specifically, molnc-301b attenuates erythropoiesis via mitigating the transcription of erythropoietic and translation-associated genes, such as GATA1 and FOS. In addition, we have established a useful and powerful CRISPR screen platform to characterize the biological functions of molncRNAs at large-scale and single-cell level, and identified 26 functional molncRNAs in hematopoietic cells. Collectively, we focused on miRNA-derived lncRNAs, deciphered their landscape during normal hematopoiesis, and comprehensively evaluated their potential roles. Overall design: We designed 3 pooled gRNA libraries: library 1 (molncRNA(-) and internal control) contained more than 2 pairs of gRNAs for each of 12 internal controls (ICs) and the 361 miRNA loci, which were targeted by D1 and D2 gRNAs recognizing the 1-3 kb or 0.5-1 kb downstream region of miRNA precursor to specifically perturb molncRNAs' expression. Library 2 (molncRNA(-/-) and internal control) contained gRNAs for the same target genes as library 1, but recognized the 1-2 kb upstream or 1-3 kb downstream region of the miRNA precursors (U1 and D1 gRNAs) to knock out both molncRNAs and the cognate miRNAs). Library 3 (non-targeting control library) consisted of 2,456 pairs of non-targeting control (NTC) gRNAs. We have applied three replicates for library1 and library2. K562 cells were infected with a mix of lentiviruses expressing Cas9-mCherry at a multiplicity of infection (MOI) ~50 and gRNA-EGFP at a MOI ~4. Infected EGFP+/mCherry+ cells were sorted for 10x Genomics 3' scRNA-seq poly(A)-primed platform, and their corresponding gRNAs were further amplified using a specific amplification protocol(Hill et al., 2018).