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Updated: Aug 5, 2026
Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays
Published on: January 7, 2019
SACF and GILA for in vitro transformation assessment of CRISPR/Cas9-edited cell therapy candidates: a multi-site
Lena Dorsheimer1, Joana Rita Ferreira2, Bojing Wang3
1Research and Development, Translational Medicine Unit, Sanofi-Aventis Deutschland GmbH, Frankfurt am Main, Germany.
Abstract:
Tumorigenicity is a key safety concern for CRISPR/Cas9-based gene therapies, yet its assessment remains challenging due to the lack of relevant and sensitive in vivo models. This HESI Global multi-site study evaluated the Soft Agar Colony Formation (SACF) assay and the Growth in Low Attachment (GILA) assay for in vitro transformation testing of CRISPR/Cas9-edited cells as part of tumorigenicity risk assessment. Across four laboratories, MCF10A cells were spiked with varying concentrations of cells carrying a CRISPR/Cas9-mediated knockout of the known tumor suppressor PTPN12 to determine the assays' limit of detection (LOD), robustness and reproducibility. Both assays demonstrated a consistent LOD of 0.8% at most sites, with SACF showing a broader dynamic range and slightly stronger correlation across laboratories. GILA exhibited weaker correlation due to plateauing at high spike-in concentrations (>12.5%), and excluding these conditions improved inter-laboratory correlations. Additionally, the study identified key determinants of assay success, including the selection of appropriate cell lines and lots, and ensuring sufficient recovery time post-thaw. Together, these data establish multi-site reproducibility of SACF and GILA in the MCF10A-PTPN12 model evaluated here and support their use as animal-free, development-stage characterization tools contributing to tumorigenicity evidence generation.
Insights
Assessing the tumorigenicity risk of CRISPR/Cas9 gene therapies is crucial. This study validated the Soft Agar Colony Formation (SACF) and Growth in Low Attachment (GILA) assays, finding them reproducible and sensitive for detecting edited cells.
Area of Science:
- Biotechnology
- Gene Therapy Safety
- In Vitro Toxicology
Background:
- Tumorigenicity assessment is vital for CRISPR/Cas9 gene therapies.
- Existing in vivo models for safety evaluation are limited.
- In vitro methods are needed for early-stage risk assessment.
Purpose of the Study:
- Evaluate Soft Agar Colony Formation (SACF) and Growth in Low Attachment (GILA) assays.
- Determine the sensitivity and reproducibility of these assays for CRISPR/Cas9-edited cells.
- Support the development of animal-free tumorigenicity testing methods.
Main Methods:
- Multi-site study involving four laboratories.
- Used MCF10A cells spiked with CRISPR/Cas9-edited PTPN12 knockout cells.
- Assessed assay limit of detection (LOD), robustness, and reproducibility.
Main Results:
- Both SACF and GILA assays showed a consistent LOD of 0.8%.
- SACF demonstrated a broader dynamic range and stronger inter-laboratory correlation.
- GILA's correlation improved when high spike-in concentrations were excluded.
Conclusions:
- SACF and GILA assays are reproducible across multiple sites using the MCF10A-PTPN12 model.
- These assays serve as valuable animal-free tools for characterizing CRISPR/Cas9-edited cells.
- The findings support the use of these assays in early-stage tumorigenicity risk assessment.
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