Evaluation of culture methods and chemical reagent combinations on CRISPR/Cas9 gene editing systems by lipofection in pig zygotes.

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.