Bioinformatics and single-cell CRISPRi-based screen reveals effector genes and implicates multi-tissue etiology for BMD [scCRISPR screen]
To nominate identify tissues relevant to the etiology of bone mineral density we generated ATAC-seq in pediatric hMSC-osteoblasts, hFOB 1.19 cells (hFOBs), osteoclasts, and primary chondrocytes. Leveraging the results of this experiment, we designed a CRISPRi screen in hFOBs with scRNA-seq expression read out. The targets selected for the screen were informed by newly generated Capture-C and bulk RNA-seq from hFOBs. Overall design: hFOBs constitutitively exprressing dCas9-CRISPRi-KRAB were transfected at a low MOI with a pool of 296 sgRNAs targeted to 89 sites. Cells with sgRNAs were selected for 9 days by puromycin and then differentiated for 5 days at 39.5°C prior to sequencing.