Physiologically Relevant 3D CRISPR Screening Enhances Mechanistic Insight into Chemical Toxicity Compared to 2D...

Area of Science:

• Toxicology
• Genomics
• Cell Biology

Background:

• Omics approaches in toxicology often lack causal links between genes and toxic effects.
• CRISPR screens offer a way to establish these causal connections for toxicogenomics.
• Existing in vitro models have limited physiological relevance compared to in vivo systems.

Purpose of the Study:

• To develop and validate a 3D CRISPR screening system for enhanced functional toxicogenomics.
• To improve the physiological relevance of in vitro toxicology assays.
• To identify genetic determinants and pathways involved in chemical toxicity using a 3D model.

Main Methods:

• Developed a 3D CRISPR screening system using HepG2/C3A spheroids in a rotating bioreactor (ClinoStar).
• Performed time-course screens to identify genes affecting spheroid development.
• Applied the 3D system to a doxorubicin toxicity study, comparing it with a 2D system.
• Utilized custom CRISPR sgRNA libraries targeting common human loss-of-function variants.

Main Results:

• The 3D system identified more candidate genetic determinants and pathways related to doxorubicin's DNA damage mechanism than the 2D system.
• The 3D CRISPR screen identified more known genes associated with doxorubicin response compared to the 2D system.
• The platform demonstrated improved identification of chemical-specific mechanisms.

Conclusions:

• The 3D CRISPR screening system is feasible and provides physiologically relevant functional toxicogenomics.
• This platform supports New Approach Methodologies (NAMs) in toxicology.
• It offers a scalable method to identify genetic factors influencing chemical-induced toxicity.