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<p>CRISPR/Cas9-mediated programmable gene editing has disrupted the biotechnology industry since it was first described in 2012. Safe in vivo delivery is a key bottleneck for its therapeutic use. Viral vector-mediated delivery raises concerns due to immunogenicity, long-term expression, and genomic disruption. Delivery of pre-complexed ribonucleoprotein (RNP) reduces off-target effects, and recombinant Cas9 production is more cost-effective than viral vector synthesis. CRISPR-Cas RNPs do not possess intrinsic cell entry mechanisms, and physical delivery methods are confined to ex vivo editing, necessitating non-viral delivery approaches. Nanogels (NG) are biocompatible polymeric nanoparticles capable of entrapping proteins. Here, we report the first proof of principle that NGs from thiol-functionalized polyglycidol can entrap active RNPs with high efficiency (60 ± 2%). We call these particles CRISPR-Gels. A commercially available <i>E. coli</i> lysate for cell-free transcription and translation (TXTL) was used to mimic the intracellular reductive degradation of NGs while providing a real-time fluorescence readout of RNP activity. Degradation and RNP activity were observed within 30–90&#xa0;min. The described TXTL assay can be utilized to evaluate the release of RNP in a cytosol-mimicking environment from redox-sensitive nanoparticles in a high-throughput and cost-effective way. Further studies are needed to assess the in vitro and in vivo performance of CRISPR-Gels.</p>

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Efficient encapsulation of CRISPR-Cas9 RNP in bioreducible nanogels and release in a cytosol-mimicking environment

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年7月26日

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  • Peter Westarp,
  • Thorsten Keller,
  • Jessica Brand,
  • Sonja Horvat,
  • Krystyna Albrecht,
  • Chase Beisel,
  • Juergen Groll

摘要

CRISPR/Cas9-mediated programmable gene editing has disrupted the biotechnology industry since it was first described in 2012. Safe in vivo delivery is a key bottleneck for its therapeutic use. Viral vector-mediated delivery raises concerns due to immunogenicity, long-term expression, and genomic disruption. Delivery of pre-complexed ribonucleoprotein (RNP) reduces off-target effects, and recombinant Cas9 production is more cost-effective than viral vector synthesis. CRISPR-Cas RNPs do not possess intrinsic cell entry mechanisms, and physical delivery methods are confined to ex vivo editing, necessitating non-viral delivery approaches. Nanogels (NG) are biocompatible polymeric nanoparticles capable of entrapping proteins. Here, we report the first proof of principle that NGs from thiol-functionalized polyglycidol can entrap active RNPs with high efficiency (60 ± 2%). We call these particles CRISPR-Gels. A commercially available E. coli lysate for cell-free transcription and translation (TXTL) was used to mimic the intracellular reductive degradation of NGs while providing a real-time fluorescence readout of RNP activity. Degradation and RNP activity were observed within 30–90 min. The described TXTL assay can be utilized to evaluate the release of RNP in a cytosol-mimicking environment from redox-sensitive nanoparticles in a high-throughput and cost-effective way. Further studies are needed to assess the in vitro and in vivo performance of CRISPR-Gels.