Optimized protocols for efficient gene editing in mouse hepatocytes in vivo using CRISPR-Cas9 technology.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• CRISPR-Cas9 technology offers precise gene editing capabilities.
• Adeno-associated virus (AAV) is a versatile vector for delivering genetic material in vivo.
• Efficient gene editing in hepatocytes is crucial for studying liver function and disease.

Purpose of the Study:

• To establish a robust protocol for CRISPR-Cas9 mediated gene editing in mouse hepatocytes in vivo.
• To optimize AAV delivery for efficient gene knockout in the liver.
• To enable simultaneous targeting of multiple genes within hepatocytes.

Main Methods:

• Construction and packaging of AAV plasmids encoding CRISPR-Cas9 components.
• Intrahepatic administration of AAV vectors in mice.
• Assessment of gene knockout efficiency using molecular detection methods.
• Development of a protocol for dual-gene targeting.

Main Results:

• Achieved high titers of AAV (up to 10¹⁴).
• Demonstrated efficient gene knockout in hepatocytes within 15 days post-injection.
• Successfully implemented a protocol for simultaneous knockout of two target genes in the liver.

Conclusions:

• The developed protocol enables efficient and rapid in vivo gene editing in mouse hepatocytes.
• This method facilitates the study of gene function in the liver and holds potential for therapeutic applications.
• The protocol's adaptability for dual-gene targeting expands its utility in complex genetic studies.