Animals
Animal husbandry and procedures of anesthesia/euthanasia were performed as described previously27. One sexually mature Landrace gilt was obtained from the Tokushima Prefectural Livestock Research Institute (Tokushima, Japan), housed in a temperature-controlled room (25 ± 3 °C) under a 12-h light/12-h dark cycle with free access to water, and provided with commercial feed (JA Nishinihon Kumiai Shiryou, Hyogo, Japan). The health condition of each pig was observed daily by the animal husbandry staff under the supervision of an attending veterinarian. To minimize animal suffering, all surgical procedures were performed under anesthesia by intramuscular injection of 10 mg/kg ketamine (Ketalar, ketamine hydrochloride, Daiichi Sankyo Pharmaceutical, Tokyo, Japan) and continuous inhalation of 2–3% isoflurane (Mylan, Osaka, Japan) in the operating room. Euthanasia was performed by intravenous injection of a potassium chloride solution (3 mmol/kg) under deep anesthesia by isoflurane according to the American Veterinary Medical Association Guidelines for the Euthanasia of Animals.
Design of gRNA sequence
Alt-R CRISPR crRNAs and tracrRNA system purchased from IDT was used as gRNA. The gRNAs were designed using the CRISPR direct web tool (https://crispr.dbcls.jp/)52. To minimize off-target effects, the 14 nucleotides at the 3′ end of the designed gRNAs only matched the target regions of each genes and had no other sequence matches in the pig genome, as determined using the COSMID web tool (https://crispr.bme.gatech.edu/)53.
Oocyte collection, in vitro maturation, and fertilization
Oocyte collection, IVM, and IVF were performed as described previously54. Briefly, pig ovaries were obtained from prepubertal crossed gilts (Landrace × Large White × Duroc breeds) at a local slaughterhouse. Cumulus-oocyte complexes (COCs) were collected from ovaries and cultured in maturation medium at 39 °C in a humidified incubator containing 5% CO2. The matured oocytes were subjected to IVF. frozen-thawed ejaculated spermatozoa were transferred into 5 mL of fertilization medium (PFM; Research Institute for the Functional Peptides Co.) and washed by centrifugation at 500×g for 5 min. The pelleted spermatozoa were resuspended in fertilization medium and adjusted to a density of 1 × 106 cells/mL. Approximately 50 oocytes were transferred to 500 µL of sperm-containing fertilization medium, covered with mineral oil in 4-well dishes, and co-incubated for 5 h at 39 °C in a humidified incubator containing 5% CO2, 5% O2, and 90% N2. After co-incubation, the attached spermatozoa were gently removed from the oocytes by mechanical pipetting. The putative zygotes were transferred to PZM-5 and cultured for 24 h until RNP transfection.
ZP removal and RNP transfection
Embryos at 1- to 8-cell stages collected at 29 h from the start of IVF were exposed to 0.5% (w/v) actinase-E in Dulbecco’s phosphate-buffered saline (Nissui Pharmaceutical, Tokyo, Japan) for 20–30 s, transferred to PZM-5 without actinase-E, and freed completely from their ZP by gentle pipetting. The ZP-free embryos were incubated at 39 °C in a humidified incubator containing 5% CO2, 5% O2, and 90% N2 for 1 h before the reagent-mediated introduction of the CRISPR/Cas9 system using jetCRISPR.
RNP transfection solution was prepared by adding 0.5, 1, or 2 μL of jetCRISPR to the nucleic acid-free duplex buffer (IDT) containing RNP complex prepared by mixing gRNA (Supplementary Table S1) and Cas9 protein at a final concentration of 167 ng/μL and 500 ng/μL, respectively, to make a final volume of 30 μL. After 15 min of incubation at 25 °C, the RNP transfection solution was added to 470 μL of PZM-5 containing ZP-free embryos and then co-incubated for 5 h in a humidified incubator containing 5% CO2, 5% O2, and 90% N2. After 5 h of incubation, ZP-free embryos were washed and cultured in PZM-5 for 2 days. Subsequently, the embryos were cultured in porcine blastocyst medium (PBM; Research Institute for the Functional Peptides Co.) for 4 days to evaluate their ability to develop to the blastocyst stage and the genotype of resulting blastocysts. As a control for the analysis of embryonic development, some ZP-free embryos without RNP transfection were cultured in the same manner. ZP-free embryos were cultured together, because single culture of ZP-free embryos showed decreased blastocyst formation rates in our culture conditions.
Electroporation
Electroporation was performed as described previously11. Briefly, an electrode (LF501PT1-20; BEX, Tokyo, Japan) was connected to a CUY21EDIT II electroporator (BEX) and was set under a stereoscopic microscope. The inseminated ZP-intact 50 zygotes collected at 10 h from the start of IVF were washed with Opti-MEM I solution (Gibco/Invitrogen, Carlsbad, CA, USA) and were placed in a line in the electrode gap in a chamber slide filled with 10 μL of Nuclease-Free Duplex Buffer (IDT) containing 100 ng/μL gRNA targeting KDR and 100 ng/μL Cas9 protein (Takara Bio). After electroporation (five 1-ms square pulses at 25 V), the zygotes were washed with PZM-5 and were cultured until embryo transfer (for 12 h) or for 3 days. The embryos that were cultured for 3 days were subsequently incubated in PBM for 4 days, and resulting blastocysts were used for genotyping analysis. Zygotes and embryos were incubated at 39 °C in a humidified incubator containing 5% CO2, 5% O2, and 90% N2.
Analysis of the targeted gene in embryos
Analysis of the targeted gene in embryos was performed as described previously27. Genomic DNA was isolated from blastocysts by boiling in a 50 mM NaOH solution. After neutralization, the DNA samples were subjected to polymerase chain reaction (PCR) using KOD One PCR Master Mix (Toyobo, Osaka, Japan) according to the manufacturer’s instructions using specific primers (Supplementary Table S1). The PCR products were extracted by agarose gel electrophoresis using a Fast Gene Gel/PCR Extraction Kit (Nippon Genetics, Tokyo, Japan). The PCR products were directly sequenced by Sanger sequencing using the BigDye Terminator Cycle Sequencing Kit (version 3.1; Thermo Fisher Scientific K.K., Tokyo, Japan) and an ABI 3500 genetic analyzer (Applied Biosystems, Foster City, CA, USA). The TIDE bioinformatics package was used to determine the genotype of each blastocyst25. Genotypes of blastocysts were classified as homozygous editing (including only single types of editing), heterozygous editing without WT (including multiple types of editing but carrying no WT sequences), heterogeneous editing with WT (including mosaic or heterozygous mutation carrying more than one type of mutation and the WT sequence, and monoallelic mutation), or WT (carrying only the WT sequence). The editing rate was defined as the ratio of the number of gene-edited blastocysts to the total number of sequenced blastocysts. Editing efficiency was defined as the proportion of indel mutation events in mutant blastocysts.
Embryo transfer
Recipient gilt, after synchronization of estrous cycles, was prepared for embryo transfer as described previously55. In brief, 0.2 mg of cloprostenol (Planate; MSD Animal Health, Tokyo, Japan) was administered by intramuscular injection to pregnant gilt 4–7 weeks after mating. Subsequently, a second intramuscular injection of 0.2 mg of cloprostenol and 1000 IU of eCG (PMSG, ZENOAQ, Fukushima, Japan) was administered to the gilt 24 h after the first injection of cloprostenol. At 72 h after the intramuscular injection of eCG, 1500 IU of hCG (Gestron 1500, Kyoritsu Seiyaku, Tokyo, Japan) was administered to the gilt. Approximately 125 h after the hCG intramuscular injection, early blastocysts derived from embryos treated with the RNP transfection reagent were transferred into the uterus of a recipient gilt under anesthesia.
Mutation analysis in piglets by deep sequencing and Sanger sequencing
Genomic DNA was isolated from ear biopsies by boiling in a 50 mM NaOH solution. After neutralization, the genomic regions flanking the gRNA target sequences were amplified by two-step PCR using specific primers and the index PCR primers following the manufacturer’s instructions (Illumina, Hayward, CA, USA) (Supplementary Table S2). After gel purification, the amplicons were subjected to MiSeq sequencing using the MiSeq Reagent Kit v. 2 (250 cycles) (Illumina, San Diego, CA, USA). CRISPResso256 was used for data analysis. The genotypes of piglets were classified according to the definition of genotypes in embryos described above.
Genomic DNA was isolated from ear, muscle, lung, heart, liver, and kidney by boiling in 50 mM NaOH. After neutralization, the DNA samples were subjected to PCR using specific primers targeting MSTN (Supplementary Table S1). The PCR products were extracted by agarose gel electrophoresis and subjected to Sanger sequencing as described above.
Off-target effects determined by deep sequencing
An off-target analysis was performed as described previously11. The COSMID webtool was used to predict off-target candidates53. The genomic regions flanking potential off-target sites were amplified by two-step PCR using specific primers (Supplementary Table S3) and analyzed by a MiSeq sequencing analysis, as described above. Indels or substitutions were measured within a 5-bp window around the predicted Cas9 cleavage site in each off-target site. A small number of amplicons carrying different sequences that were also detected in the WT sample were considered as sequencing errors.
Immunofluorescence staining
Longissimus dorsi muscle biopsy samples obtained from the 40-day-old piglets were fixed in a 4% paraformaldehyde neutral-buffered solution (Wako, Osaka, Japan) and manually embedded in paraffin. To analyze the distribution of skeletal muscle fiber types, paraffin-embedded sections were deparaffinized and antigen retrieval was performed by autoclaving the slides in citrate buffer (pH 6.0) for 15 min. Slow and fast myofibers were detected using mouse anti-slow skeletal muscle myosin (ab11083, 1/500; Abcam, Cambridge, UK) and rabbit anti-fast skeletal muscle myosin (ab91506, 1/500; Abcam), respectively. The sections were subsequently incubated for 2 h at 25 °C with Alexa Fluor 594 goat anti-mouse IgG (ab150116, 1/500; Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (ab150077, 1/500; Abcam). After staining, seven images were obtained per sample using a BZ-X710 microscope (KEYENCE, Osaka, Japan), and the slow and fast muscle fiber areas were calculated using BZ-X Analyzer (KEYENCE). The percentage of slow myofibers was defined as the percentage of slow myofibers to the sum of the slow and fast myofiber areas.
Quantifying muscle MSTN protein concentration
Longissimus dorsi muscle biopsy samples were obtained from 6-month-old pigs under anesthesia. MSTN protein concentration was determined using an ELISA kit (R&D Systems, Minneapolis, MN, USA). Total protein extraction from muscle samples and the quantification of MSTN protein concentration were performed according to the manufacturer’s instructions. The concentration of all protein extracts were quantified using the BCA protein assay kit (Takara Bio). The samples were diluted to 1.0 mg mL−1 concentration before starting the assay.
Statistical analyses
Data for blastocyst formation and mutation efficiencies were evaluated using analysis of variance (ANOVA) followed by protected Fisher’s least significant difference tests using StatView (Abacus Concepts, Berkeley, CA, USA). All percentage data were subjected to arcsine transformation before ANOVA. The percentage of mutated blastocysts was analyzed using chi-squared tests with Yates’ correction. Differences with a p value of ≤ 0.05 were considered statistically significant.
Study approval
The animal experiments were approved by the Institutional Animal Care and Use Committee of Tokushima University (approval number: T2019-11). All animal care and experimental procedures were performed in accordance with the Guidelines for Animal Experiments of Tokushima University and in compliance with the ARRIVE guidelines.
