Functional assessement of putative myometrial enhancers by the CRISPR activation system

Timely control of parturition is crucial for maternal and fetal health. Failures on this biological process often result in pregnancy complications including preterm birth, labor dystocia, and health disorders on newborn babies. The myometrium is the muscular structure of the uterus maintaining uterine structural integrity and providing contractile force for parturition. The myometrial structure changes in adaptation to the pregnancy via stage-specific transcriptomic profiles. Data from the mouse model indicate that changes of myometrial epigenomic landscape precedes the adoption of stage-specific gene expression pattern at term. The present study documents the transcriptomic profile and epigenomic landscape of term pregnant myometrial tissues and functionally characterize a subset of putative enhancers to further understand the enhancer-gene interaction in human the myometrium. Guide RNAs (gRNA) were uitilized to recruite the dCas9-VPR CRSIPR activators to putative enhancers of interest for promoting gene expression in the immortalized human myometrial cell line hTERT-HM cells. Cells that carried gRNA-expressing and dCas9-VPR-expressing lentiviral vectors were isolated by cell sorters. The single cell RNAseq assay was performed on the sorted cells to detect gRNA expression and profile the single cell transcriptome in the same cell (Perturb-Seq).