Multistep allelic conversion in mouse pre-implantation embryos by AAV vectors.

Area of Science:

• Genetics and Genomics
• Molecular Biology
• Biotechnology

Background:

• Site-specific recombinases (SSRs) are essential for precise control of engineered alleles.
• Complex alleles often require multiple recombination events, which can be laborious and time-consuming.
• Existing methods for allele conversion can be inefficient and require significant animal resources.

Purpose of the Study:

• To develop an efficient and simplified method for ex vivo allele conversion using SSRs.
• To leverage adeno-associated viruses (AAVs) for delivering SSRs and facilitating genetic modifications.
• To reduce the number of animals required and accelerate the process of genome engineering.

Main Methods:

• Utilized adeno-associated viruses (AAVs) for the delivery of Cre, FLP (flippase), Dre, and Vika recombinases.
• Performed ex vivo allele conversion in a reporter mouse line.
• Investigated both simple and complex allele conversion scenarios, including sequential recombination events.

Main Results:

• AAV-mediated allele conversion demonstrated minimal toxicity and high efficiency.
• Simple allele conversions achieved a 100% efficiency rate.
• Complex, multi-site allele conversions consistently reached an 80% efficiency rate.

Conclusions:

• The developed AAV-based method significantly simplifies and accelerates ex vivo allele conversion.
• This strategy reduces the need for animal models in genetic engineering workflows.
• The technique offers a substantial improvement for genome engineering, particularly for complex alleles in conditional knockout models.