A genome–wide CRISPR activation screen identifies SCREEM a novel SNAI1 super-enhancer demarcated by eRNAs in monocytes [gRNA_SCREEN]

The genome is pervasively transcribed to produce a vast array of non-coding RNAs (ncRNAs). Long noncoding RNAs (lncRNAs) are transcripts of > 200 nucleotides and are best known for their ability to regulate gene expression. Enhancer RNAs (eRNAs) are subclass of lncRNAs that are synthesized from enhancer regions and have also been shown to coordinate gene expression. The biological function and significance of most lncRNAs and eRNAs remain to be determined. Epithelial to mesenchymal transition (EMT) is a ubiquitous cellular process that occurs during cellular migration, homeostasis, fibrosis, and cancer-cell metastasis. EMT-transcription factors, such as SNAI1 induce a complex transcriptional program that coordinates the morphological and molecular changes associated with EMT. Such complex transcriptional programs are often subject to coordination by networks of ncRNAs and thus can be leveraged to identify novel functional ncRNA loci. Here, using a genome-wide CRISPR activation (CRISPRa) screen targeting ~10,000 lncRNA loci we identified ncRNA loci that could either promote or attenuate EMT. We discovered a novel locus that we named SCREEM (SNAI1 cis-regulatory eRNAs expressed in monocytes). The SCREEM locus contained a cluster of eRNAs that when activated using CRISPRa induced expression of the neighboring gene SNAI1, driving concomitant EMT. However, the SCREEM eRNA transcripts themselves appeared dispensable for the induction of SNAI1 expression. Interestingly, the SCREEM eRNAs and SNAI1 were co-expressed in activated monocytes, where the SCREEM locus demarcated a monocyte-specific super-enhancer. These findings suggest an unexpected role for SNAI1 in monocytes. Exploration of the SCREEM-SNAI axis could reveal novel aspects of monocyte biology. HBEC3-KT--human bronchial immortalized epithelial cell line-- 330million cells were transduced (stably expressing dcas9-vp64 and P65) with pooled sgRNA contained lentiviral particles at a MOI of 0.3 (1000X representation of the library in surviving cells). Th transduced cells were selected with Zeocin (50ug/ml) for 14-days (maintaining a 500X per passage i.e. 50 million cells). The SAM expressing HBEC3-KT cells (Zeocin [sgRNA-hsf1-lib; (50ug/ml)], Hygromycin [p65(10ug/ml)], and blasticidin [dcas9-vp64(10ug/ml)] were then treated with 10ng/ml of TGFβ1 (R&D) for 72hrs. Subsequently, the cells were harvested and processed for CD44+ sorting. Note, for cell sorting using BD FACSAria II sorter, 120-micron tip was used to let free flow of large mesenchymal-like cells. We sorted ~50X representation of the library from CD44-low (bottom 10% of the peak) and CD44-high (top 10% of the peak) cells. Genomic DNA were harvested from sorted populations using the Quick-DNA plus (Zymo Research) according to the manufacturer’s protocol. To determine distribution of the sgRNA, the spectrophotometer quantified gDNA product was sequenced using primers (listed in Supplementary file 1 of this paper) in Illumina MiSeq (80 cycles of read 1 (forward) and 8 cycles of index 1 with a 5% PhiX control to improve library diversity in order to cover >50 reads per sgRNA in the library).