Fig. 5.

CRISPR/Cas9-mediated knockout (KO) of pMEI at the RPL17 locus. A) Validation of the pMEI: PCR amplicons from genomic DNA, indicating that the AluYa5 insertion is heterozygous in four of five tested cell lines. B UCSC Genome Browser track illustrating the genomic sequence context of the AluYa5 pMEI. Alignment of Sanger sequence reads of PCR amplicons from C) are shown in dark red. Blue arrows indicate target sites for the three CRISPR gRNA pairs. Light blue & orange horizontal lines indicate gDNA regions used in reporter gene (luciferase) assays. C) PCR amplicons of wild-type (WT) HEK293T cells and three independent pooled KOs (KO1, KO2 & KO3), indicating successful KO of the AluYa5 pMEI. D Relative mRNA levels of RPL17 in WT and KO cells, as measured by qRT-PCR (n = 4 biological replicates). E Firefly luciferase levels, indicating that the AluYa5 sequence drives substantially greater reporter gene expression than the two alleles of the size-matched flanking sequence containing the indel polymorphism rs111878775 (n = 5 biological replicates). Error bars indicate standard deviation