Fig. 5

A CRISPR-mediated partial deletion of a nimtRNA downregulates downstream exon inclusion of an endogenous host gene. a The nimtRNA^Lys gene, located within intron 28 of the human PPFIBP1 gene, was targeted by a CRISPR-mediated approach to analyze the influence of partial deletions within the nimtRNA^Lys gene on PPFIBP1 exon 29 inclusion. b Single clones of CRISPR-targeted cells were cultured and analyzed by Sanger sequencing for nimtRNA^Lys deletions; based on this analysis, three single clones, designated as 638-5, 638-9, and 638-10, indicated in orange, blue, and red, respectively, were selected. c Subsequently, by RT-PCR analysis comparing wt to bulk and single clone cells, respectively, the abundance of the PPFIBP1 mRNA transcript lacking exon 29 was assessed, employing primers as indicated. d The abundance of PPFIBP1 mRNA transcript harboring exon 29 was determined by RT-qPCR in wt cells and compared to bulk as well as single clone CRISPR-targeted cells employing primers as indicated. Cells targeted by a guideRNA not binding to the PPFIBP1 gene (gRNA mock) were employed as an additional control. Normalization was performed to GAPDH. e In addition to the specific inclusion of exon 29, the general abundance of PPFIBP1 mRNA levels was determined by employing primers binding to exon 21 and 22 upstream of the nimtRNA locus, as indicated. Cells targeted by a guideRNA not binding to the PPFIBP1 gene (gRNA mock) were employed as an additional control. Normalization was performed to GAPDH. Averages and standard deviations were determined from three independent sets of experiments