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

Abstract Many omics-based approaches in toxicology research primarily rely on correlative data, often lacking functional relationships or causal links between genotypes and phenotypes. CRISPR-based approaches can overcome this limitation by establishing direct causal connections between genes and toxicological phenotypes. Moreover, CRISPR screens enable scalable and systematic interrogation of gene function and associated mechanisms following chemical exposure, predominantly using in vitro models. In line with the paradigm of new approach methodologies (NAMs) in toxicology research, CRISPR screens hold promise to provide an in vitro cell-based functional toxicogenomics approach. One of the main limitations of conventional in vitro assays is their compromised physiological relevance to humans due to their inability to fully recapitulate in vivo physiology. To improve the functional and physiological relevance of the toxicogenomics approach, we developed a 3D CRISPR screening system using HepG2/C3A spheroids generated and cultivated in a continuously rotating bioreactor (ClinoStar). We first performed time-course 3D CRISPR screens to identify genes that confer growth disadvantage or advantage, influencing spheroid development compared to 2D cultures. We then applied this approach to a chemical toxicity study using doxorubicin, comparing the performance of the 3D and 2D systems in identifying chemical-specific mechanisms. The results showed that the 3D system captured more candidate genetic determinants and biological pathways related to DNA damage processes—a known toxicity mechanism of doxorubicin—demonstrating improved performance in identifying chemical-specific pathways over the 2D counterpart. In our screens, we employed custom CRISPR sgRNA libraries representing common human loss-of-function genetic variants (mean allele frequency > 0.1% in all individuals catalogued in the genome aggregation database), which potentially affect toxicity responses. By comparing our CRISPR screen results with previously reported genetic associations for doxorubicin response, we found that the 3D system identified more known associated genes than the 2D system. Together, the 3D CRISPR screening system demonstrated its feasibility and utility for physiologically relevant functional toxicogenomics. This platform enables in vitro NAMs, by providing a scalable and effective approach to identify causal genetic determinants and biological pathways that modulate chemical-induced toxicity. Competing Interest Statement The authors have declared no competing interest. Data availability The raw and processed CRISPR screen genomics data with the corresponding metadata in this study will be deposited into Gene Expression Omnibus (GEO) database repository as soon as it is available.