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.
Highlights
The specificity and efficiency of Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9), which is composed of a guide RNA and a Cas[9] nuclease, are widely used for efficient and versatile gene editing in various organisms by specifying a 20-nucleotide targeting sequence within a gRNA8–10
We have previously optimized the timing of lipofection treatment for the introduction of the CRISPR/Cas[9] system into zona pellucida (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 e fficiency[17]
Summary
The specificity and efficiency of CRISPR/Cas[9] gene-editing systems are determined by several factors, including the mode of delivery, when applied to mammalian embryos. 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, in terms of editing efficiency. Genetically modified pigs are typically established by somatic cell nuclear transfer using gene-modified somatic cells and the direct introduction of gene editors into the cytoplasm of zygotes and embryos via microinjection or e lectroporation[7,11] These conventional methods require expensive equipment and sophisticated techniques. For the first time, we report the generation of genetically modified pigs by the CRISPR/ Cas[9] system via the lipofection-mediated introduction of Cas9-gRNA ribonucleoprotein complexes (RNPs) into porcine embryos
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