Application of complex transgenic alleles, exemplified by EUCOMM/KOMP alleles, often requires multiple recombination events to understand specific biological processes fully. To achieve this level of genetic engineering sophistication, an efficient combinatorial method based on site-specific recombinases (SSRs) is needed. In this context, we introduce a new method that uses AAV vectors, which allows for such an approach.
As shown with Cre protein11, we attempted to produce and purify the recombinant Dre, Flpo, and Vika proteins in multiple cellular heterologous systems, including BL21-DE3-RIPL bacterial (BL21-DE3-RIPL), Hi5 insect cells, and HEK293T mammalian cells (HEK293). Nevertheless, Flpo and Vika proteins were not sufficient for experiments in large scale workflows (unpublished data). To overcome this limitation, SSR coding sequences were packaged in AAV serotype 1 vectors, and their conversion efficiency was compared to established SSR delivery techniques in form of protein electroporation and/or mRNA microinjection.
Titers of the vectors varied. Cre and Dre containing vectors could be prepared in high-titers, while Vika and CMV-Flpo vectors achieved relatively low-titer (Fig.S20). The reason for this discrepancy is unclear. The constructs fall below the AAV packaging limit of 4.7kb (Cre: 3167bp, Dre: 3156bp, Vika: 3159bp, CMV-Flpo: 3369bp), thus, the size of the vector should not influence packaging. Despite employing identical procedure for the production and purification protocols, our data suggest that the expression of Vika and Flpo may influence the AAV replication process in a mammalian system. However, it is important to highlight that our data did not indicate a significant presence of empty capsids in the Vika and CMV-Flpo vectors. If such capsids were prevalent, a disproportionately higher amount of protein relative to the GC (genome copy) count would be expected (Fig. S20). Therefore, it is more plausible that the efficiency of AAV-SSRs production is negatively affected by the overexpression of Vika and Flpo in mammalian producer cells.
Different AAV SSR titers were applied on MuX heterozygote (het) zygotes to determine the most effective and least toxic viral concentration (Fig. 1b-c). Once the optimal titer was identified, the efficiency and scale of conversion was evaluated in adult mice. Performance of AAV vectors was compared with standard delivery methods, such as electroporation of protein (Cre and Dre), or microinjection of mRNA (Vika and Flpo).
Despite the relatively low-yield in production of CMV-Flpo and Vika vectors (reaching a maximum titer of 7E + 10 GC/mL and 3.3E + 11 GC/mL, respectively), the titration screen has identified the minimum effective titers for CMV-Flpo and Vika as 1E + 10 GC/mL and 1E + 9 GC/mL, respectively (Fig. 1b-d). Reduced embryo viability at 1E + 11 GC/mL was observed in the case of Cre and Dre vectors (Fig. 1b and c). The optimal titers were determined based on viability and conversion efficiency ratio, based on quantification in Table 1. However, it is important to note that Cre and Dre vectors can be used at concentrations one order of magnitude lower while still ensuring efficient conversion and low toxicity. In contrast, CMV-Flpo and Vika titration experiments indicated that concentrations below 1E + 10 GC/mL significantly reduce conversion efficiency. A similar trend was observed with the commercial EF1a-Flpo vector. However, unlike Cre and Dre, the highest titer of this vector did not adversely affect embryo viability (Fig. 1b and c).
While AAV vectors are generally considered non-integrative vectors, recent evidence has introduced some uncertainty regarding their potential integration following double-stranded breaks (DSB)20. SSRs, unlike site-specific endonucleases, do not induce open DSB. SSR-mediated recombination represents a more isolated and tightly regulated process compared to the machinery involved in DSB repair21. Hypothetically, this process considerably reduces likelihood of AAV genome integration during SSR-mediated recombination. The presence of expression-competent fragments of the AAV vector within the host genome was analyzed, since AAVs are associated with low integrative potential22. Our data suggest that all used recombinases in AAV form can integrate into the host genome. Based on integration assay in MuX mice, the frequency of integration is on average 9.8%, therefore relatively low (Fig.S2-S6). Due to this integration potential, newborn animals should be screened for Flpo and Cre AAV genomes after AAV-mediated conversions. Based on this selection, there is no need for further back-cross breeding to eliminate the active Flpo gene from the genome, as it is often case with conventional Flpo driver lines.
One of the practical applications of the AAV-based SSRs delivery is in vitro fertilization. A single treatment with AAV Flpo or Cre facilitates the direct conversion of the previously mentioned EUCOMM/KOMP alleles without an elevated risk of recombinase vector integration. This approach offers a simple and effective method for Cre or Flpo-mediated conversion. The AAV system not only enables the conversion from tm1a to tm1c, but also provides the opportunity to perform tm1a to tm1d conversion in whole animal, as shown in Fig. 3b-n. Furthermore, we verified that AAV-mediated conversion is heritably transmitted to the next generation and that AAV vectors exhibit minimal integrative tendencies (Fig. S16-S19).
We have effectively demonstrated the practical application of AAV SSRs in the context of EUCOMM/KOMP allele conversion, specifically transitioning from the tm1a state to tm1c, and further to tm1d in a single animal (Fig. 3b-h and Fig.S15). A distinctive feature of the AAV-based approach is the ability to convert tm1a alleles to tm1d through sequential treatment with two distinct vectors. Current practice for achieving tm1d allele is conversion of tm1a allele to tm1c using either a Flpo driver or mRNA microinjection23,24. Subsequent progeny of tm1c mice were then converted either through a Cre driver or mRNA/protein microinjection/electroporation5,11,23–25. As a result, the conventional approach requires more time and animals until the final model is established, which can be at odds with the principles outlined in the 3Rs guidelines.
The main objective of this study is to underscore the versatility of AAV SSRs and improve conversion methods employing these enzymes. We focused on Flpo, whose protein synthesis is complicated, and the mRNA microinjection is invasive and less efficient. The utilization of AAV Flpo treatment offers an efficient and straightforward tool for effective tm1c conversion without inducing significant adverse effects. Additionally, it enables the potential for combinatorial treatment with AAV Cre vectors to achieve the tm1d allele, resulting in rapid conversion in comparison to conventional methods. Furthermore, combinatorial treatment enables reduction of required animals.
Moreover, the use of recombinant AAVs (rAAVs) can be conducted within a biosafety level 1 (BSL-1) facility, provided that the AAV vector does not carry an oncogenic or toxic payload and is produced in a helper virus-free manner. rAAVs loaded with SSRs adhere to the BSL-1 criteria, making the vector readily applicable in laboratories lacking BSL-2 facilities26. Furthermore, rAAV vectors can be purchased in a high-purity grade and ready-to-use form, making AAV-mediated conversion broadly available method.