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.

Figure 1

Optimization of the jetCRISPR concentration for gene editing of MSTN. (A) Blastocyst formation rates of ZP-intact and ZP-free embryos without RNP transfection. (B) Frequency of gene editing in the target regions of blastocysts derived from the embryos treated with jetCRISPR, Cas9 protein, and gRNA. Gene editing of blastocysts was determined by Sanger sequencing and TIDE. The percentage of blastocysts with gene editing was defined as the ratio of the number of gene edited blastocysts to the total number of blastocysts examined. (C) Mutation efficiency in gene-edited blastocysts. Editing efficiency was defined as the proportion of indel mutation events in blastocysts carrying mutations. Heterozygous without WT: blastocysts carrying multiple types of editing but no WT sequences, Heterogeneous with WT: blastocysts carrying mosaic mutation or heterozygous mutation carrying more than one type of mutation and the WT sequence, and monoallelic mutation. (D) Blastocyst formation rates of embryos treated with various concentrations of jetCRISPR. Each bar represents the mean ± SEM. Four replicate trials were carried out and the numbers in parentheses indicate the total number of oocytes (A,D) and examined blastocysts (B,C). Percentages of blastocysts carrying mutations in target genes were analyzed using chi-squared tests (B). *p < 0.05.

Figure 2

Confirmation of gene editing with jetCRISPR targeting five different genes. (A) The blastocyst formation rate of jetCRISPR treated embryos. Each bar represents mean ± SEM. (B) Frequency of gene editing in the target regions of blastocysts derived from the embryos treated with jetCRISPR, Cas9 protein, and gRNAs. Gene editing of the blastocysts was determined by Sanger sequencing and a TIDE analysis. The percentage of blastocysts with gene editing was defined as the ratio of the number of gene edited blastocysts to the total number of blastocysts examined. (C) Mutation efficiency in gene-edited blastocysts. Editing efficiency was defined as the proportion of indel mutation events in blastocysts carrying mutations. Heterogeneous with WT: blastocysts carrying mosaic mutation or heterozygous mutation carrying more than one type of mutation and the WT sequence, and monoallelic mutation. Four to five replicate trials were carried out and the numbers in parentheses indicate the total number of oocytes (A) and examined blastocysts (B,C).

Figure 3

Photographs of lipofection-treated ZP-free embryos (day 4) (A), ZP-free blastocyst (Day 7) (B), and delivered piglets (C). The scale bar in each panel represents 100 μm.

Figure 4

Deep sequencing analysis of the MSTN target region in delivered piglets. *Nucleotides in blue and red represent the target sequences and PAM sequences of gRNA, respectively. Nucleotides in green represent inserted nucleotids. **The read frequency was defined as the ratio of the number of reads to the total number of aligned read. ***The total mutation rate was defined as the ratio of the total number of modified reads to the total number of aligned reads. WT wild-type; ♂, male.

Figure 5

Sanger sequencing analysis of muscle and ear tissues derived from MSTN-mutant and wild-type pigs, and their total mutation frequency of indel mutations. Total efficiency was defined as the frequency of indel mutations decomposed from Sanger sequence data by TIDE analysis.

Figure 6

Immunohistochemical assessment and quantification of MSTN protein concentration of wild-type (WT) and MSTN heterogeneous mutant piglets. (A) The longissimus dorsi muscles biopsies derived from WT (#4) and mutant piglets (#3 and #5) were immunohistochemically stained for slow (red) and fast (green) skeletal muscle myosin. The scale bar in each panel represents 100 μm. (B) Proportion of slow myofibers in longissimus dorsi muscle tissues. The slow myofiber areas were calculated as percentages from seven images after immunofluorescence staining for slow and fast type muscle fiber markers in longissimus dorsi muscle tissues obtained from 40-day-old piglets. WT, wild-type. Each bar represents a mean ± SEM. ^a–cp < 0.05. (C) Comparison of MSTN protein concentrations. Equal concentrations (1.0 mg mL⁻¹) of total protein extracts obtained from the longissimus dorsi muscle of the wild type (WT; #4) and MSTN-mutant pigs (#5, #7 and #8) were used for ELISA. Each sample was assessed in quadruplet (n = 4), and the data are expressed as the mean ± SEM. ^a–cp < 0.05.