156 Genome editing of porcine zygotes through the lipofection of a CRISPR/Cas9 system with two guide RNAs

As a method through which the CRISPR/Cas9 system can be introduced into zygotes without the use of specialised equipment, we successfully performed gene editing through lipofection in in vitro-fertilised pig zygotes and embryos using lipofectamine. Owing to its flexibility and versatility, the lipofection method represents a promising approach for delivering Cas9-gRNA ribonucleoprotein complexes (RNPs) and can substantially improve the value of pig resources as experimental animals, particularly in ill-equipped laboratories. We aimed to investigate whether lipofection-mediated transfection can be used for CRISPR/Cas9-based multiplex genome editing targeting GHR and GGTA1 in pig zygotes. Zona pellucida–free zygotes collected 10 h after IVF were incubated with RNPs and Lipofectamine 2000 reagent (LP2000) for 5 h. In Experiment 1, we evaluated the editing efficiency of gRNAs targeting GHR and GGTA1 alone or in combination in LP2000-mediated zygotes using 4-well plates. In Experiment 2, we examined the effects of culture methods on the development and editing efficiency of GHR and GGTA1 double-edited zygotes using 4- and 25-well plates. In Experiment 3, we assessed the effect of an additional exposure period (5 h) to GHR-mediated lipofection before and after simultaneous GHR and GGTA1 double-mediated lipofection on the editing efficiency of double-edited embryos. For each experiment, four replicates were carried out and the blastocyst formation and mutation rates were examined. Data for blastocyst formation were evaluated using analysis of variance (ANOVA), followed by Fisher’s protected least significant difference test using STATVIEW. Mutation rates were determined by TIDE analysis and analysed using chi-square tests with the Yates correction. No significant differences in mutation rates were observed between single- and combined-targeting gRNAs (11.8% vs 5.3% for GHR-mediated mutation and 25.0% vs 36.8% for GGTA1-mediated mutation). In addition, compared with the 4-well plate, culture in the 25-well plate significantly increased blastocyst formation rate (16.5% vs 32.6%, P < 0.05); however, a significant increase only in the mutation of GGTA1 target gene was observed when zygotes were transfected with two gRNAs (11.1% vs 45.0%, P < 0.05). In Experiment 3, mutation rates did not significantly differ across the GHR-mediated exposure periods (0.0%, 10.5%, and 0.0% for the control, pre-exposure, and post-exposure groups). However, multiple mutations were detected only in blastocysts from zygotes treated with GHR-mediated lipofection before simultaneous double-mediated lipofection (10.5% and 57.9% in GHR and GGTA1-mediated mutation, respectively). We demonstrated the feasibility of lipofection for transferring multiple gRNAs and Cas9 into pig zygotes; however, this newly established chemistry-based technique is still in its infancy and requires further improvement, especially regarding editing efficiency.