Monitoring of Dual CRISPR/Cas9-Mediated Steroidogenic Acute Regulatory Protein Gene Deletion and Cholesterol Accumulation Using High-Resolution Fluorescence In Situ Hybridization in a Single Cell.

摘要: Recent advances in fluorescence microscopy, coupled with CRISPR/Cas9 gene editing technology, provide opportunities for understanding regulation at the single-cell level. The application of direct imaging shown here provides an situ side-by-side comparison CRISPR/Cas9-edited cells and adjacent unedited cells. We apply his methodology to StAR Y-1 adrenal MA-10 testis is a gatekeeper protein that controls access cholesterol from cytoplasm inner mitochondria. loss this mitochondrial transfer mediator rapidly increases lipid droplets cells, as seen StAR-/- mice. Here, we describe dual strategy marked by GFP/mCherry expression deletes activity within 12 h. used single-molecule hybridization (sm-FISH) directly monitor time course single achieved deletion high efficiency gRNA targeting proximal promoter exon 2. Seventy percent transfected showed slow DNA measured PCR, Br-cAMP stimulated transcription. This was sm-FISH both loci individual relative non-target Cyp11a1 exon7. also distinguishes two effects on without deletion. stimulation primary spliced RNA were removed 4 h before any effect cytoplasmic mRNA occurred. disappeared between 24 parallel deletion, while ester increased fourfold. These alternative changes match distinct processes. approach facilitates rapid testing strategies immediate assessment adaptation responses perturbation clonal expansion procedures.