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Updated: Oct 1, 2025
Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles "Nanoblades"
Published on: March 31, 2021
Rationally designed nanoparticle delivery of Cas9 ribonucleoprotein for effective gene editing
Se-Youl Chae1, Euihwan Jeong2, Seounghun Kang1
1Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea.
Abstract:
Programmable endonucleases such as CRISPR/Cas9 system emerge as a promising tool to treat genetic and non-genetic diseases such as hypercholesterolemia, Duchenne muscular dystrophy, and cancer. However, the lack of safe and efficient vehicles that enable intracellular delivery of CRISPR/Cas9 endonuclease is a big hurdle for its therapeutic applications. Here, we employed porous nanoparticle for the Cas9 ribonucleoprotein (RNP) delivery and achieved efficient knockout of target genes in vitro and in vivo. The porous nanoparticle, called 'BALL', enabled safe and direct intracellular Cas9 RNP delivery by improving bioavailability and serum stability. The BALL-mediated delivery of Cas9 RNP showed superior indel efficiency of about 40% in vitro and 20% in vivo in a model system employing green fluorescent protein (GFP). More importantly, intramuscular injection of the Cas9 RNP-BALL complex targeting the myostatin (MSTN) gene which is known to suppress muscle growth achieved successful knockout of the MSTN gene, resulting in the increase of muscle and the improved motor functions. Thus, we believe that the BALL is a promising delivery system for CRISPR-based genome editing technology, which can be applied to the treatment of various genetic diseases.
Insights
Researchers developed a porous nanoparticle, BALL, for delivering CRISPR/Cas9 gene editing tools. This system efficiently delivered Cas9 ribonucleoprotein (RNP) for gene knockout in vitro and in vivo, showing promise for treating genetic diseases.
Area of Science:
- Biotechnology
- Molecular Biology
- Gene Therapy
Background:
- CRISPR/Cas9 technology offers therapeutic potential for genetic diseases.
- Efficient and safe intracellular delivery of CRISPR/Cas9 components remains a significant challenge for clinical applications.
Purpose of the Study:
- To develop and evaluate a novel porous nanoparticle system, termed 'BALL', for the intracellular delivery of Cas9 ribonucleoprotein (RNP).
- To assess the efficacy and safety of BALL-mediated Cas9 RNP delivery for gene editing in vitro and in vivo.
Main Methods:
- Porous nanoparticles (BALL) were engineered for Cas9 RNP encapsulation and delivery.
- In vitro gene knockout efficiency was assessed using a green fluorescent protein (GFP) model.
- In vivo studies involved delivering Cas9 RNP targeting the myostatin (MSTN) gene via intramuscular injection in a mouse model.
Main Results:
- BALL nanoparticles demonstrated improved bioavailability and serum stability for Cas9 RNP delivery.
- Efficient gene knockout was achieved with approximately 40% indel efficiency in vitro and 20% in vivo.
- MSTN gene knockout led to increased muscle mass and improved motor function in vivo.
Conclusions:
- The BALL nanoparticle system provides a safe and effective platform for intracellular delivery of CRISPR/Cas9 RNP.
- This delivery system holds significant promise for advancing CRISPR-based genome editing therapies for various genetic disorders.
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