Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: CRISPRi/a Screening with Human iPSCs

doi: 10.1007/978-1-0716-1484-6_23

Figure Lengend Snippet: (A) CRISPR screening can detect causative genes in a phenotype of interest by comparing the distributions of sgRNAs. After each cell is labelled by a sgRNA using pooled lentiviral library, the cells with a phenotype of interest are selected. Then, the distribution of sgRNAs in each population can be measured by next-generation sequencing. (B) We show an example schedule of CRISPRi screening with iPSC-CMs that identifies essential genes for CM differentiation. The CRISPRi iPSCs were infected with a sgRNA library and underwent puromycin selection. Then, iPSCs were differentiated to CMs. To screen essential genes for CM differentiation, CMs were stained by TNNT2 and sorted by flow cytometry. The volcano plot shows multiple hit genes and a positive control (TNNT2) as essential genes for CM differentiation.

Article Snippet: CRISPRi/a library 1 Human Genome-wide CRISPRi-v2 Libraries or your custom library (Addgene, #83969) or Your custom sgRNA library: follow the protocols in Addgene #83969.

Techniques: CRISPR, Next-Generation Sequencing, Infection, Selection, Staining, Flow Cytometry, Positive Control