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LATE-a novel sensitive cell-based assay for the study of CRISPR/Cas9-related long-term adverse treatment effects
Dawid Głów1, Simon Meyer1, Irene García Roldán1
1Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), Martinistr. 52, Hamburg 20246, Germany.
Abstract:
Since the introduction of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), genome editing has been broadly applied in basic research and applied biotechnology, whereas translation into clinical testing has raised safety concerns. Indeed, although frequencies and locations of off-target events have been widely addressed, little is known about their potential biological consequences in large-scale long-term settings. We have developed a long-term adverse treatment effect (LATE) in vitro assay that addresses potential toxicity of designer nucleases by assessing cell transformation events. In small-scale proof-of-principle experiments we reproducibly detected low-frequency (<0.5%) growth-promoting events in primary human newborn foreskin fibroblasts (NUFF cells) resulting from off-target cleavage in the TP53 gene. Importantly, the LATE assay detected not only off-target effects in TP53 not predicted by popular online tools but also growth-promoting mutations in other tumor suppressor genes, such as p21 and PLZF. It convincingly verified strongly reduced off-target activities of high fidelity compared with first-generation Cas9. Finally, the LATE assay was readily adapted to other cell types, namely clinically relevant human mesenchymal stromal cells (hMSCs) and retinal pigmented epithelial (RPE-1) cells. In conclusion, the LATE assay allows assessment of physiological adverse effects of the CRISPR/Cas system and might therefore be useful for preclinical safety studies.
Insights
A new assay detects adverse effects of CRISPR/Cas9 genome editing. This long-term adverse treatment effect (LATE) assay identifies off-target mutations in critical genes, ensuring safer clinical applications.
Area of Science:
- Genetics and Genomics
- Biotechnology
- Molecular Biology
Background:
- CRISPR/Cas9 genome editing offers powerful research and biotech tools.
- Clinical translation of CRISPR/Cas9 faces safety concerns regarding off-target effects.
- The biological consequences of long-term, large-scale off-target mutations remain largely unknown.
Purpose of the Study:
- To develop and validate a novel assay for assessing the potential toxicity of designer nucleases.
- To evaluate the biological consequences of CRISPR/Cas9 off-target events in long-term settings.
- To establish a method for preclinical safety assessment of genome editing technologies.
Main Methods:
- Development of a long-term adverse treatment effect (LATE) in vitro assay.
- Assessment of cell transformation events induced by designer nucleases.
- Utilized primary human newborn foreskin fibroblasts (NUFF cells), human mesenchymal stromal cells (hMSCs), and RPE-1 cells.
Main Results:
- The LATE assay detected low-frequency (<0.5%) growth-promoting events in NUFF cells due to off-target cleavage in the TP53 gene.
- Identified off-target mutations in TP53, p21, and PLZF not predicted by standard online tools.
- Demonstrated significantly reduced off-target activity with high-fidelity Cas9 variants compared to first-generation Cas9.
- Successfully adapted the LATE assay for use in clinically relevant hMSCs and RPE-1 cells.
Conclusions:
- The LATE assay effectively assesses physiological adverse effects of the CRISPR/Cas system.
- This assay can identify potentially harmful off-target mutations in tumor suppressor genes.
- The LATE assay is a valuable tool for preclinical safety studies of genome editing technologies.
