In vitro transformation assays for non-clinical safety assessment of CRISPR/Cas9 genome-edited cells

Off-target editing is one of the main safety concerns for the use of CRISPR/Cas9 genome editing in gene therapy. Although theoretically rare, these unwanted modifications could lead to malignant transformation, which renders tumorigenicity assessment of the cell therapy product indispensable. Here, we establish two in vitro assays, the soft agar colony forming assay (SACF) and growth in low attachment plates (GILA), as valid, quick and cost-efficient methods for tumorigenicity assessment of genome-edited cells. Using a CRISPR/Cas9 based approach to transform immortalized MCF10A cells, we identified PTPN12, a known tumor suppressor, as first true positive control in GILA and SACF. Next, we assessed the limit of detection for both assays and found that SACF is more sensitive than GILA (0.8% vs. 3.2% transformed cells). We further validated SACF and GILA by identifying a set of positive and negative controls. In contrast to SACF and GILA, an in vivo tumorigenicity study failed to detect the known tumorigenic potential of PTPN12-/- demonstrating the importance of including GILA and SACF in tumorigenicity testing. In conclusion, SACF and GILA are both attractive and valuable additions to non-clinical safety assessment of genome-edited cells.