Efficient development of platform cell lines using CRISPR-Cas9 and transcriptomics analysis

Antibody-drug conjugates offers many advantages as a drug delivery platform that allows for highly specific targeting of cell types and genes. Ideally, testing the efficacy of these systems requires two cell types to be different only in the gene targeted by the drug, with the rest of the cellular machinery unchanged, in order to minimize other potential differences from obscuring the effects of the drug. In this study, we created multiple variants of U87MG cells with targeted mutation in the TP53 gene using the CRISPR-Cas9 system, and determined that their major transcriptional differences stem from the loss of p53 function. Using the transcriptome data, we predicted which mutant clones would have less divergent phenotypes from the wild type and thereby serve as the best candidates to be used as drug delivery testing platforms. Further in vitro and in vivo assays of cell morphology, proliferation rate and antibody-mediated uptake supported our predictions. Based on the combined analysis results, we successfully selected two best qualifying mutant clones. This study serves as proof-of-principle of the approach and paves the way for extending to additional cell types and target genes. Overall design: Targeted TP53 knockout of U87MG cells was performed using CRISPR-Cas9 gene editing. 7 clones with mutated TP53 were selected for transcriptomics analysis based on CAGE technology. CAGE libraries of the mutant clones and the wild type were constructed (nAnT-iCAGE protocol) and sequenced on illumina HiSeq 2500 (50 nt single read). Each sample was prepared and sequenced in 3 replicates.