The experimental procedures and methods used in this study were approved by the Animal Welfare and Ethics Office (2019012A-CNU-174), Chungnam National University, Daejeon, and performed according to “The Guide for the Care and Use of Laboratory Animals” published by IACUC of Chungnam National University. Female mixed dogs from 2 to 6 years of age were used in this study as oocyte donors and embryo transfer recipients. The dogs were housed indoors and fed once daily with water ad libitum. All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments in the Methods section.
Construction of prime editor vector and production of lentiviral particles
The vector for PE was purchased from Addgene (Watertown, MA, USA: #135955) and modified to correct HD-related SNPs. Briefly, the CMV promoter was obtained by PCR using the primer sets 5′-gaattcttgacattgattattgactag-3′ and 5′-tctagaaatttcgataagccagtaagc-3′, and inserted into the vector by EcoRI and XbaI (NEB Inc., MA, USA: #R0101M and #R0145M) enzyme cuts. The pegRNA targeting the HD locus was newly synthesized and then added to the vector using PacI (NEB Inc., MA, USA: #R0547S) and EcoRI. Finally, the vector was confirmed through sequencing. The lentiviral particles of PE vector were produced by commercial vendor (Lugen SCI, Inc., Bucheon, South Korea).
Collection and establishment of canine fibroblast cell lines, transduction, and transgene analysis
Fibroblasts were collected from the ears of an 18-month-old Labrador retriever diagnosed with HD (donor patient). The primary fibroblasts were cultured in vitro using culture medium composed of DMEM-GlutaMAX, 15% fetal bovine serum, and 1% penicillin/streptomycin solution (GIBCO, Inc.). For transduction, 100 multiplicity of infection (MOI) of the PE lentiviral particles, containing 1 μg/mL of polybrene, was transduced into 1 × 105 fibroblasts per a well of 12-well plate. Transgene expression was confirmed by EGFP and integration of the vector was confirmed by sequence analysis.
Collection of in vivo matured canine oocytes
We collected mature oocytes from dogs as described previously27. The concentration of progesterone in the blood was measured to optimize the hormone concentration for harvesting mature oocytes. After confirming the time of estrus, blood was collected, and progesterone was measured using VET Chroma (ANIVET Inc., Chuncheon, South Korea). When the analyzed progesterone level was in the range of 4–7 ng/mL we considered that day as ovulation. Three days after ovulation, mature oocytes were surgically collected. During the procedure, all dogs were treated with ketamine and xylazine at a concentration of 6 mg/Kg, and anesthesia was maintained with 2% isoflurane. After exposing the ovary and uterus, a 24G intravenous catheter was inserted into the oviductal lumen near the uterotubal junction, and the culture medium was flowed to collect mature oocytes. The culture medium was prepared by adding 2 mM NaHCO31% penicillin/streptomycin, 0.5% bovine serum albumin, and 10% FBS to medium 199 containing 25 mM HEPES.
SCNT and embryo transfer
For generating gene corrected dogs, SCNT followed by embryo transfer was performed following the method described elsewhere27. Briefly, in vivo matured oocytes with the first polar body were used for micromanipulation. Metaphase chromosomes were removed by aspiration from the oocytes. A single cell (C>T cell) was transferred into the perivitelline space of an enucleated oocyte, and each donor cell-cytoplast couplets were fused by two pulses of direct current (24–26 V for 15 μsec) using an Electro-Cell fusion apparatus. The fused SCNT embryos were chemically activated by incubating with 10 μM calcium ionophore (Sigma) and then 1.9 mM 6-dimethylaminopurine (6-DMAP). The activated SCNT embryos were surgically transferred into the oviducts of estrus-synchronized surrogates. Pregnancy was confirmed by ultrasonography at 30 days after embryo transfer.
PCR validation and sequencing analysis
Transgene integration into the genome of transduced fibroblasts and gene-corrected dogs was confirmed by PCR. The PCR primers used to validate the Cas9 sequence in the vector were 5′-catcgctattaccatggtgat-3′ and 5′-ctcttgcagatagcagatcc-3′. These primer sets detected the linkage between the CMV promoter and dCas9 of the vector used in this study. Sequencing of the target locus was performed to validate the PE-mediated gene correction. The sequencing primers used were 5′-gacgccaagggagcagatatt-3′ and 5′-cctctcttatgagaacagcat-3′ (Bioneer Inc., Daejeon, South Korea). In addition, TA cloning was performed for accurate sequencing analysis using the products generated through PCR (Supplement Fig. 1). PCR products and T vector (Promega Inc., WI, USA: #A1360) were mixed at a ratio of 1:3, and DNA was isolated and purified from the bacterium colony generated by ligation (NEB Inc., MA, USA: # M0202) to extract DNA. After confirming the extracted DNA with EcoRI restriction enzyme, sequencing analysis was performed.
Analysis of off-target mutations in gene-corrected dogs
Potential off-target loci were determined in silico using Cas-OFFinder (http://www.rgenome.net/cas-offinder/). We selected two potential off-target loci with two mismatches and another three loci with three mismatches compared to the genomic target sequences of pegRNA used in the study. The potential off-target loci were PCR amplified with genomic DNA from the C>T dog #1 and C>T dog #2 and sequencing analysis was performed (Supplementary Table 2).
Ethics approval and consent to participate
In conducting this study, we collected cells from a retriever with hip dysplasia, and this was done after explaining the study to the retriever owner and consent was obtained. The experimental procedures and methods used in this study were approved by the Animal Welfare and Ethics Office (CNU-01090) of Chungnam National University, Daejeon, and performed according to the Guide for the Care and Use of Laboratory Animals published by the IACUC of Chungnam National University. All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org) for the reporting of animal experiments in the Methods section.
Consent for publication