Production of knockout mouse lines with Cas9

Area of Science:

• Genetics and Genomics
• Mammalian Genetics
• Molecular Biology

Background:

• Gene knockout mice are crucial for studying gene function and associated phenotypic effects in mammals.
• Traditional methods for generating knockout mice are time-consuming and resource-intensive.
• RNA-guided Cas9 nuclease technology offers a faster approach to creating knockout mouse lines.

Purpose of the Study:

• To present a standardized methodology for producing knockout mouse lines using Cas9 ribonucleoprotein (RNP) electroporation.
• To demonstrate the efficiency and high success rate of this protocol.

Main Methods:

• Utilizing Cas9 ribonucleoprotein (RNP) complexes for targeted gene editing.
• Employing electroporation for the delivery of RNPs into mouse zygotes.
• Standardized protocol for efficient generation of knockout founders and germline transmission.

Main Results:

• Achieved an 80% success rate in generating founder mice with null alleles.
• Demonstrated a high germline transmission rate of 93% for the generated knockout alleles.
• The protocol is adaptable to standard transgenic facility equipment.

Conclusions:

• The presented Cas9 RNP electroporation method provides an efficient and rapid approach for generating knockout mouse lines.
• This protocol significantly reduces the time and resources needed compared to traditional methods.
• The high success and transmission rates make this a valuable tool for mammalian genetics research.