Fig. 2: Phenotypic characterization and OCT2 expression analysis after targeting LNROP using CRISPR-Cas9.

A Schematic representation of the sgRNAs library used to target LNROP in RAMOS cells stably expressing active Cas9. Bars correspond to individual guides, and they are represented according to their position over LNROP sequence where introns are colored in grey, exons in blue and MIR4323 in red. Bar height corresponds to the log₂ fold change in guide abundance as measured by sequencing. Red bars indicate significantly depleted sgRNAs (P < 0.05). Guides (#123, #133 and #161) selected for subsequent experiments are labeled at their positions. B Phenotype characterization of RAMOS after standard CRISPR-Cas9 using sgRNAs targeting the LNROP sequence and expressed along with GFP. The number of GFP-expressing cells present in cultures was measured after 3 and 49 days using flow cytometry. Results indicate the change in the presence of GFP-expressing cells, using day 3 as starting point. A non-targeting guide (green) and a guide targeting DNM2 (red) were used as negative and positive controls, respectively. Data are mean ± SD of three replicates. C RT-qPCR analysis of LNROP and OCT2 expression in RAMOS cells after standard CRISPR-Cas9 using three sgRNAs (#123, #133 and #161) targeting the LNROP sequence or a non-targeting guide (NT). Data are mean ± SD of three replicates. *P < 0.05; **P < 0.01; ***P < 0.001, unpaired t test.