Extended Data Fig. 5: Generation and characterization of NDF and Spt16 single- and double-Knockin human iPSC cells.
From: Phase-separated NDF−FACT condensates facilitate transcription elongation on chromatin
a, Design of donor vectors for CRISPR-Cas9-mediated homologous recombination to generate single- and double-knockin (KI) cells targeting NDF and Spt16 genes. b-c, Agarose gel images displaying genotyping results for homozygous single (b) and double (c) KI iPSC clones. Genotyping primers: NDF-genotyping-F2 & R2 for NDF, Spt16-genotyping-F2 & R2 for Spt16. d, Western blot analysis comparing endogenous expression of NDF and Spt16 in wild-type (WT) and KI iPSC cells. Left Panel: WT and NDF single KI; Middle panel: WT and Spt16 single KI; Right panel: NDF and Spt16 double KI. NDF proteins were stained with anti-NDF antibody, e, Immunofluorescence staining for Oct4 showing its expression pre- and post-genome editing in iPSC cells. Scale bar=5 μm. f, Western blot demonstrating Nestin expression in neuron stem cells derived from WT and genome-edited iPSC cells. This confirms that the edited cells retain pluripotency and physiological relevance to human stem cells. The star indicates non-specific band.
