Somatic Liver Knockout (SLiK): A Quick and Efficient Way to Generate Liver-Specific Knockout Mice Using Multiplex...

Area of Science:

• Molecular Biology
• Genetics
• Hepatology

Background:

• Developing efficient methods for targeted gene modification in specific organs is crucial for understanding gene function.
• Liver-specific gene knockout models are essential for studying hepatic biology and disease.
• Existing methods may lack efficiency or specificity for rapid, multi-gene targeting in vivo.

Purpose of the Study:

• To describe a protocol for Somatic liver knockout (SLiK), a novel method for rapid, liver-specific gene deletion.
• To detail the integration of CRISPR/Cas9 gene editing with hydrodynamic tail-vein injection for in vivo hepatocyte modification.
• To provide guidelines for sgRNA design, construct delivery, and validation of gene editing efficiency.

Main Methods:

• CRISPR/Cas9 gene editing system for targeted DNA modification.
• Hydrodynamic tail-vein injection for efficient delivery of targeting constructs into hepatocytes.
• Utilizing tyrosinemic liver selection pressure to promote replacement of host hepatocytes with edited cells.
• sgRNA design, cloning, and validation of CRISPR/Cas9 cutting in vivo.

Main Results:

• The SLiK protocol enables the generation of liver-specific knockouts for single or multiple genes.
• Efficient in vivo editing of hepatocytes is achieved through the combined use of CRISPR/Cas9 and hydrodynamic delivery.
• The protocol includes comprehensive steps for screening and validation of successful gene editing.

Conclusions:

• Somatic liver knockout (SLiK) provides a powerful and rapid approach for in vivo liver gene editing.
• This method facilitates the study of gene function in the liver by enabling targeted gene deletion.
• The described protocol offers a valuable tool for researchers in hepatology and genetic engineering.