Abstract
Using nuclease-deficient dead (d)Cas9 without enzymatic activity fused to transcriptional inhibitors (CRISPRi) allows for transcriptional interference and results in a powerful tool for the elucidation of developmental, homeostatic and disease mechanisms. We inserted dCas9KRAB (CRISPRi) cassette into the AAVS1 locus of hiPSC lines, which resulted in homozygous knock-in with an otherwise unaltered genome. Expression of dCas9KRAB protein, pluripotency and the ability to differentiate into all three embryonic germ layers were validated. Furthermore, functional cardiomyocyte generation was tested. The hiPSC-CRISPRi cell lines offer a valuable tool for studying endogenous transcriptional repression with single and multiplexed possibilities in all human cell types.