Abstract
Here, we present a protocol for iterative enrichment of integrated single guide RNA (sgRNA) via derivative CRISPR-Cas9 from genomic DNA (gDNA) of phenotypically sorted fixed cells. We describe steps for high-scale lentiviral production, genome-wide screening, extracting gDNA from fixed cells, cloning of integrated sgRNAs, and high-scale transformation. This protocol introduces three key advantages: (1) applicability to fixed cells, (2) bypassing epigenetic drift, and (3) pause points lowering the contamination risk. We believe this approach will benefit researchers applying somatic cell genetics in cell biology. For complete details on the use and execution of this protocol, please refer to Ordoñez et al.1.
Keywords:
CRISPR; Cell Biology; High-Throughput Screening; Microscopy; Molecular Biology.