Site-specific recombinases (SSRs) are critical for achieving precise spatiotemporal control of engineered alleles. These enzymes play a key role in facilitating the deletion or inversion of loci flanked by recombination sites, resulting in the activation or repression of endogenous genes, selection markers or reporter elements. However, multiple recombination in complex alleles can be laborious. To address this, a new and efficient method using AAV vectors has been developed to simplify the conversion of systems based on Cre, FLP, Dre and Vika recombinases. In this study, we present an effective method for ex vivo allele conversion using Cre, FLP (flippase), Dre, and Vika recombinases, employing adeno-associated viruses (AAV) as delivery vectors. AAVs enable efficient allele conversion with minimal toxicity in a reporter mouse line. Moreover, AAVs facilitate sequential allele conversion, essential for fully converting alleles with multiple recombination sites, typically found in conditional knockout mouse models. While simple allele conversions show a 100% efficiency rate, complex multiple conversions consistently achieve an 80% conversion rate. Overall, this strategy markedly reduces the need for animals and significantly speeds up the process of allele conversion, representing a significant improvement in genome engineering techniques.

Czech Centre for Phenogenomics Institute of Molecular Genetics of the Czech Academy of Sciences Prumyslova 595 252 50 Vestec Czech Republic

Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova 595 252 50 Vestec Czech Republic

Laboratory of Transgenic Models of Diseases Institute of Molecular Genetics of the Czech Academy of Sciences Videnska 1083 142 20 Prague Czech Republic

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