AAV1-CMV-FLPo

Site-specific recombinases (SSRs) are critical for achieving precise spatiotemporal control of engineered alleles. However, multiple recombination in complex alleles can be laborious. Here, researchers present an efficient method for in vitro allelic conversion using Cre, FLP (flippase), Dre, and Vika recombinases using adeno-associated virus (AAV) as a delivery vector. AAV enables efficient allelic switching in reporter mouse lines with minimal toxicity. Furthermore, AAV facilitates sequential allelic switching, which is critical for complete switching of alleles with multiple recombination sites, often found in conditional knockout mouse models. While simple allelic conversions showed 100% efficiency rates, complex multiple conversions consistently achieved 80% conversion rates. Overall, this strategy significantly reduces the need for animals and significantly speeds up the process of allelic conversion, representing a major improvement in genome engineering technology.

Utilizing the identified optimal titers for AAV recombinases, researchers achieved significant transformation efficiencies of 100% using AAV EF1a-Flpo, AAV CMV-Cre, -Dre, and -Vika vectors. Notably, in the case of the AAV CMV-Flpo vector, researchers observed a transformation rate of 92.6% of born animals, of which 2 (7.4%) showed chimerism and 2 (7.4%) showed no recombination. To increase Flpo conversion efficiency, researchers used an EF1a-Flpo vector with a titer of 1E + 11 GC/mL to convert MuX embryos. Using the EFlpo vector, 100% conversion was achieved. These findings indicate that AAV-Flpo treatment does not interfere with embryo viability at later stages of development and that treated zygotes have the potential to produce fully transformed animals. One fully-converted male animal from each AAV treatment group was then selected and analyzed for EGFP signal in the liver, lungs, skin (ear), heart, intestine, and testicles. All converted animals were EGFP positive in the organs analyzed (Figure 1).

Figure 1. Imaging of EGFP signal in fully-converted animals by AAV-SSR vectors (Cre, Dre, Vika and CMV-Flpo, EF1a-Flpo). (Nickl P, et al., 2024)