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

Abstract

The specificity and efficiency of CRISPR/Cas9 gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. Given the limited time window for delivery, faster and more reliable methods to introduce Cas9-gRNA ribonucleoprotein complexes (RNPs) into target embryos are needed. In pigs, somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes/embryos by microinjection or electroporation have been used to generate gene-edited embryos; however, these strategies require expensive equipment and sophisticated techniques. In this study, we developed a novel lipofection-mediated RNP transfection technique that does not require specialized equipment for the generation of gene-edited pigs and produced no detectable off-target events. In particular, we determined the concentration of lipofection reagent for efficient RNP delivery into embryos and successfully generated MSTN gene-edited pigs (with mutations in 7 of 9 piglets) after blastocyst transfer to a recipient gilt. This newly established lipofection-based technique is still in its early stages and requires improvements, particularly in terms of editing efficiency. Nonetheless, this practical method for rapid and large-scale lipofection-mediated gene editing in pigs has important agricultural and biomedical applications.

Production of Genetically Modified Porcine Embryos via Lipofection of Zona-Pellucida-Intact Oocytes Using the CRISPR/Cas9 System

“…To the best of our knowledge, only one research group has reported the use of the lipofection method in porcine ZP-free oocytes and ZP-free embryos with some success [26][27][28][29]. As the ZP is a significant physical barrier and appears to reduce the effectiveness of lipofectamine, these researchers treated ZP-free oocytes and ZP-free embryos and achieved a moderate mutation rate (ranging from 8% to 57%) with a high level of mosaicism (ranging from 87.5% to 100% of mutant embryos) [27].…”

“…As the ZP is a significant physical barrier and appears to reduce the effectiveness of lipofectamine, these researchers treated ZP-free oocytes and ZP-free embryos and achieved a moderate mutation rate (ranging from 8% to 57%) with a high level of mosaicism (ranging from 87.5% to 100% of mutant embryos) [27]. Later, with further optimization of the system using ZP-free embryos, they generated mutant embryos for different genes and produced genetically modified piglets with a monoallelic mutation for the MSTN gene [26].…”

“…ZP-free oocytes are considered to be more difficult to manipulate, and their viability is lower than that of ZP-intact oocytes [26,41]. For this reason, the optimization of a lipofection method for ZP-intact oocytes is a desired objective.…”

“…The CRISPR/Cas9 system can also be introduced into cells using the lipofection method, and it can be used to produce genetically modified animals. Currently, there are only a few reports from the same research group in Japan on the use of lipofection in zona pellucida (ZP)-free porcine oocytes and embryos [26][27][28].…”

Effects of the timing of electroporation during in vitro maturation on triple gene editing in porcine embryos using CRISPR/Cas9 system

“…S1). The gRNAs targeting KDR and PDX1 were evaluated in our previous study ( Hirata et al., 2021 ; Tanihara et al., 2020 ). The oocytes incubated for each time point were mechanically freed from cumulus cells in Dulbecco's PBS (DPBS; Invitrogen Co.) supplemented with 1 mg/ml hyaluronidase (Sigma-Aldrich).…”