Abstract
The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates. Therefore, this study elucidated the effects of culture methods and reagent combinations on the CRISPR/Cas9 gene editing systems by using three lipofection reagents: Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (CM), Lipofectamine™ 2000 Transfection Reagent (LP), and jetCRISPR™ RNP Transfection Reagent (Jet). Porcine zona pellucida–free zygotes were incubated for 5 h with Cas9, a guide RNA targeting CD163, and the above lipofection reagents. When examining the effect of culture methods using 4-well (multiple embryo culture) and 25-well plates (single embryo culture) on the efficiency of CM-mediated zygote transfection, the culture of embryos in 25-well plates significantly increased the blastocyst formation rate; however, there was no difference in mutation rates between the 4-well and 25-well plates. When assessing the effects of individual or combined reagents on the efficiency of zygote transfection, the mutation rate was significantly lower for individual LP compared to individual CM- and Jet-mediated transfections. Moreover, combinations of lipofection transfection reagents did not significantly increase the mutation rate or mutation efficiency.
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Acknowledgements
We thank Nippon Food Packer Shikoku K.K. (Tokushima, Japan) for supplying us with the pig ovaries necessary for our experiments.
Funding
This study was supported in part by KAKENHI Grant Numbers JP22H02499 (to TO and MH) and JP22K19896 (to TO, MH, and FT) of the Japan Society for the Promotion of Science (JSPS). We acknowledge the Uzushio Program of Tokushima University for their financial support (to QL).
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Lin, Q., Takebayashi, K., Torigoe, N. et al. Evaluation of culture methods and chemical reagent combinations on CRISPR/Cas9 gene editing systems by lipofection in pig zygotes. In Vitro Cell.Dev.Biol.-Animal (2024). https://doi.org/10.1007/s11626-024-00908-0
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DOI: https://doi.org/10.1007/s11626-024-00908-0