Effective generation of maternal genome point mutated porcine embryos by injection of cytosine base editor into germinal vesicle oocytes.

Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T (C>T) or A→G (A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F0 are mosaic, and breeding of F1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing (GVBE) to produce point mutant F0 porcine embryos by editing the maternal alleles during the GV to MII transition. Injection of cytosine base editor 3 (BE3) mRNA and X-linked Dmd-specific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MII oocytes developed into blastocysts after parthenogenetic activation (PA) or in vitro fertilization (IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4% ±2.1%. There 40%-78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five (5/19) F0 male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine (9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F0 embryos with maternal point mutated alleles in a single step.