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Researchers developed a non-viral CRISPR/Cas9 platform for stable gene insertion in neuroblastoma cancer cells using two delivery methods: electroporation of Cas9 protein complexes and lipid nanoparticle delivery of Cas9 mRNA. The optimized system achieved up to 60% stable gene integration through electroporation without requiring additional enhancement chemicals, with the edited cells maintaining function in lab cultures, 3D bioprinted tumor models, and mouse xenografts. The study systematically compared donor DNA designs and delivery approaches to establish a practical workflow for genetic modification of solid tumor models without using viral vectors.
Why it matters
This biosafety level 1-compatible platform provides cancer researchers with a safer and more flexible alternative to viral methods for creating genetically modified tumor models. The approach could accelerate preclinical cancer research by enabling easier generation of stable cell lines for studying tumor biology and testing therapeutic interventions.
⚠️ Preprint – Noch nicht peer-reviewed
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Virus-free genome engineering provides a flexible alternative to viral vectors for generating genetically modified cell models. Here, we establish an integrated biosafety level 1-compatible CRISPR/Cas9 homology-directed repair (HDR) workflow for stable transgene knock-in in neuroblastoma cell lines using non-viral delivery approaches. We systematically evaluated donor cassette architecture and delivery conditions across electroporation-based Cas9 ribonucleoprotein (RNP) delivery and lipid nanoparticle (LNP)-mediated co-delivery of Cas9 mRNA, sgRNA, and donor DNA. Modular AAVS1-targeting donor constructs identified a compact EF1(s)-Donor-Q8-Tag-sPA cassette that consistently yielded the strongest HDR-associated knock-in readouts, achieving up to 60% stable reporter-positive cells following electroporation without HDR enhancers. While LNP-mediated delivery enabled efficient CRISPR cargo co-delivery and generation of genetically modified tumor cell populations, knock-in efficiencies remained lower than those observed with electroporation. Subsequent enrichment approaches enabled generation of highly pure edited cell populations following both delivery strategies. Functional validation demonstrated stable transgene expression in vitro, including in three-dimensional bioprinted tumor models, and in vivo in xenograft mice without impairing tumor growth or viability. Together, these findings establish a practical non-viral HDR platform for stable engineering of solid tumor models and provide a framework for further optimization of genome editing workflows across distinct delivery modalities.
Source: A Non-Viral CRISPR/Cas9 HDR Platform for Stable Engineering of Solid Tumor Models.