Fig. 6
From: Rewiring of SINE-MIR enhancer topology and Esrrb modulation in expanded and naive pluripotency
Inactivation or knockout of a MIR enhancer affects target gene expression and cell proliferation. A Schematic of CRISPR-based inactivation of Klf5-MIR. dCas9 fused with KRAB and MeCP2 effectors were guided to Klf5-MIR by 2 sgRNAs (sg1 and sg2). Two primer pairs (EK ChIP1 and EK ChIP2), annealing near the sgRNA-targeted site, were designed for ChIP-qPCR assay. B, C Expression of Klf5 and Nanog after CRISPRi of Klf5-MIR. D ESRRB binding on Klf5-MIR region after CRISPRi of Klf5-MIR, measured by ChIP-qPCR. As comparison, ESRRB-bound region outside Klf5-MIR (Positive control) and non-ESRRB-bound region (Negative control) were shown. E Schematic of CRISPR-based knockout of Klf5-MIR. Three sgRNAs (2 flanking and 1 targets internally) were designed to knockout Klf5-MIR. F Expression of Klf5 and Nanog in Klf5-MIR homozygous and heterozygous KO ESCs. G Cell proliferation of Klf5-MIR homozygous and heterozygous KO ESCs. H Expression of Klf5 and Nanog in Klf5-MIR homozygous and heterozygous KO EPSCs. I Cell proliferation of Klf5-MIR homozygous and heterozygous KO EPSCs