Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos

Abstract

Liposome-mediated gene transfer has become an alternative method for establishing a gene targeting framework, and the production of mutant animals may be feasible even in laboratories without specialized equipment. However, how this system functions in mammalian oocytes and embryos remains unclear. The present study was conducted to clarify whether blastocyst genome editing can be performed by treatment with lipofection reagent, guide RNA, and Cas9 for 5 h without using electroporation or microinjection. A mosaic mutation was observed in blastocysts derived from zona pellucida (ZP)-free oocytes following lipofection treatment, regardless of the target genes. When lipofection treatment was performed after in vitro fertilization (IVF), no significant differences in the mutation rates or mutation efficiency were found between blastocysts derived from embryos treated at 24 and 29 h from the start of IVF. Only blastocysts from embryos exposed to lipofection treatment at 29 h after IVF contained biallelic mutant. Furthermore, there were no significant differences in the mutation rates or mutation efficiency between blastocysts derived from embryos at the 2- and 4-cell stages. This suggests that lipofection-mediated gene editing can be performed in ZP-free oocytes and ZP-free embryos; however, other factors affecting the system efficiency should be further investigated.

Generation of mutant pigs by lipofection-mediated genome editing in embryos

“…We have previously optimized the timing of lipofection treatment for the introduction of the CRISPR/Cas9 system into ZP-free embryos, demonstrating that the treatment of 1- to 8-cell stage embryos at 29 h from the start of in vitro fertilization (IVF) for 5 h yielded a high gene editing efficiency 17 . In this study, jetCRISPR (Polyplus-transfection, Illkirch-Graffenstaden, France) was used as RNP transfection reagent for CRISPR/Cas9-mediated gene editing in mammalian cells 19 , 20 .…”

“…Without the use of specialized equipment, Cas9 protein and gRNA can be co-delivered into various mammalian cells using the lipofection mechanism 14 – 16 . Recently, we successfully demonstrated lipofection-mediated gene editing in in vitro fertilized porcine zygotes and embryos without zona pellucida (ZP) 17 . Although the efficiency was insufficient, lipofection-mediated gene editing during embryogenesis can substantially improve the value of pig resources as experimental animals, particularly in unequipped laboratories.…”

Timing and duration of lipofection-mediated CRISPR/Cas9 delivery into porcine zygotes affect gene-editing events

“…Techniques to generate genetically modified porcine embryos, e.g., microinjection, electroporation, and somatic cell nuclear transfer, generally require specialized equipment. We have previously demonstrated that lipofection-mediated gene editing by the CRISPR/Cas9 system, without specialized equipment, can be performed in ZP-free oocytes and embryos [ 7 ]. In the present study, to improve the gene-editing efficiency, we evaluated two parameters: the timing of the introduction of the CRISPR/Cas9 system into ZP-free zygotes and the duration of lipofection treatment.…”

“…There were no significant differences in blastocyst formation rates when zygotes were incubated with or without lipofection reagent. We previously found that lipofection does not affect the developmental competence of porcine oocytes during IVF but affects that of embryos collected at 24–29 h from the start of IVF [ 7 ], suggesting that developmental competence after lipofection is affected by the embryonic stage. Although the blastocysts derived from zygotes treated with Lipofectamine 2000 at 10 and 15 h from the start of IVF had mutations, blastocysts derived from zygotes treated at 5 h lacked mutations.…”

“…Lipofectamine 2000 is a cationic liposome-based reagent with sufficient capacity for transfection in vitro [ 6 ]. We have previously demonstrated that a lipofection-mediated gene editing system using Lipofectamine 2000 and CRISPR/Cas9 components can be applied to zona pellucida (ZP)-free oocytes and embryos in pigs [ 7 ]. However, the optimal conditions for lipofection-mediated nucleic acid transfection depend on the cell line [ 3 , 8 ], and an efficient protocol has not been established for pig zygotes.…”