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Omics-aided design genome editing strategy for challenging human immortalized cell models
Patricia Mendoza-Garcia1, Benjamin Keith2, Markus Nordberg1
1Assays, Profiling & Cell Sciences, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
Plos One
|February 12, 2026
Summary
This study reveals that cell type significantly impacts CRISPR-Cas9 precise knock-in (KI) efficiency. Omics profiling identified cell-specific bottlenecks, like apoptotic priming, and found overexpressing proliferating cell nuclear antigen (PCNA) can enhance KI in difficult cells.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas9 is a powerful genome editing tool for biomedical research and drug development.
- Precise genome editing efficiency varies significantly across cell types, influenced by DNA repair mechanisms and cellular state.
- Understanding these cellular differences is crucial for optimizing gene editing outcomes.
Purpose of the Study:
- To identify and characterize molecular bottlenecks limiting precise genome editing, specifically knock-in (KI) efficiency, in hard-to-engineer cell lines.
- To compare the editability of HepG2 and MCF7 cell lines using multi-omics profiling.
- To pinpoint and validate molecular targets for enhancing precise KI efficiency.
Main Methods:
- Utilized multi-omics profiling (genomics, transcriptomics, proteomics, etc.) to analyze HepG2 and MCF7 cells.
- Investigated DNA repair pathways, including non-homologous end joining (NHEJ) and homologous recombination (HR).
- Assessed cellular states such as apoptotic priming and proliferation, identifying proliferating cell nuclear antigen (PCNA) as a key factor.
Main Results:
- Omics profiling revealed that limited homologous recombination (HR) and a high propensity for non-homologous end joining (NHEJ) are major barriers to precise KI.
- Apoptotic priming was identified as an additional, previously underappreciated, bottleneck affecting KI efficiency.
- Overexpression of proliferating cell nuclear antigen (PCNA) in MCF7 cells significantly enhanced precise KI efficiency, validating it as a therapeutic target.
Conclusions:
- Multi-omics approaches are essential for understanding the 'editability molecular signature' of cell models.
- Identifying and manipulating key pathways, such as those involving PCNA, can overcome cellular limitations and improve precise genome editing efficiency.
- This work provides a framework for optimizing CRISPR-Cas9 applications in diverse cell types for research and therapeutic development.
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