Background and objectives CRISPR/Cas9 is a novel technique for genomic inactivation of genes. In this study we assessed the feasibility of generating knock-out cell populations from human mesenchymal stem cells, without sub-cloning of cells.
Materials and methods CRISPR guide RNAs were designed and cloned into a lentiviral Cas9 expression system. HEK293 cells or MSC were transduced with lentivirus and selected with puromycin. CRISPR efficiency for genomic modification was determined by genomic PCR, surveyor nuclease digestion while protein expression was assessed by immunoblotting.
Results Transforming Growth Factor β (TGFβ) signalling is crucial for chondrogenic differentiation of MSC and the maintenance of the articular chondrocyte phenotype. Currently the exact role of individual downstream SMAD transcription factors is unknown. Efficient RNA interference mediated knock-down of SMAD proteins might still allow signal transduction via the remaining SMAD protein.
To generate knock-out cell populations, CRISPRs (guide RNAs) were designed to target the second coding exon of the five R-SMAD genes. The CRISPR efficiency was assessed using the surveyor nuclease assay on HEK293 cells and MSC. The evaluated CRISPRs had a variable efficiency in HEK293 cells: 20–90% genomic modification of the target gene was observed. Increasing the viral load (MOI) clearly improved this efficiency in MSC, while increased antibiotic selection pressure did not. To further improve the on-target efficiency we used a double guide RNA targeting strategy to excise a 100 base pair fragment. By regular PCR it was estimated that 25% of the total cell population carried the truncated form of the gene. The surveyor nuclease assay showed an even higher percentage of genomic modification. Importantly, this double targeting strategy decreased SMAD protein expression in HEK293T by 90%.
MSC were expected to be more vulnerable to CRISPR/Cas9 genome engineering and/or high viral loads. Following careful optimisation of lentiviral transduction and antibiotic selection protocols for MSC, we evaluated the effect of four control CRISPR/Cas9 constructs targeting GFP. Our preliminary findings indicate that MSC stably expressing CRISPR/Cas9 exhibit normal differentiation characteristics.
Conclusions We show that efficient targeting of genes using CRISPR/Cas9 in MSC cell populations, leading to strongly decreased protein expression, is feasible without clonal selection.