Abstract

During the past few years CRISPR-based genome-editing tools extended their application beyond conventional NHEJ and HDR, finding applications in various fields accelerating drug discovery. Here we present how CRISPR/Cas9 can be used for generating appropriate in vitro cellular models relevant for neurological diseases that could provide integrative platforms for high-throughput (HT) drug screening campaigns and find application in orthogonal (secondary/specificity) assay development. First, we developed a pathological model using gene knock down (both by siRNA and inducible shRNA) of a disease-related splice factor in a neuroblastoma cell line. Modulation of two dependent splice variants (downregulation of a normal and upregulation of an aberrant/pathological variant) was monitored by RT-qPCR using a triplex TaqMan assay in miniaturized 384-well plate format. By performing a TaqMan-based HT screen using cell lysates of the pathological neuroblastoma model and a 2,200 compound RNA-targeting library (SMSM =Small Molecule Splicing Modulator set; selected from most recent collections), we identified small molecules that could revert normal/aberrant splice variant expression. In addition to a TaqMan-based readout, we inserted a HiBiT marker via the CRISPR/Cas9 approach for protein quantification of the C-terminal tagged normal variant and selected clones that showed HiBiT signal modulation upon siRNA stimulus (splice factor silencing) in good correlation with transcript modulation. An inducible shRNA-based depletion assay was developed, transducing lentivirus in the HiBiT-tagged line that express Doxycycline-inducible shRNA against the disease-related splice factor in a dose-dependent manner. A Pilot screen testing 3,200 compounds confirmed good assay robustness, pharmacology, expected % activity of the hit reference compound and identified further candidates. Moreover, to build an assay platform to model neurodegeneration, we utilized CRISPR/Cas9-mediated engineering of iPSC lines and a gene trap approach, for efficient insertion of a biomarker into the AAVS1 Safe-Harbour locus, valuable for iPS-derived cellular models. We inserted a GCaMP6f Ca2+ reporter cassette to follow calcium fluctuations in iPS-derived neurons.