ArticleThe Journal of reproduction and development2019
Effects of concentration of CRISPR/Cas9 components on genetic mosaicism in cytoplasmic microinjected porcine embryos.
Article in The Journal of reproduction and development, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 25 papers.
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Who cites it
25 citing papers in PubMed, 51 citations in OpenAlex.
- Harnessing CRISPR-Cas technology for insect pest control: current advances and future perspectives.Molecular biology reports · 2026Review
- JAK3-deficient mini-pigs exhibit impaired lymphoid organogenesis, intestinal structure, and leukocyte/cytokine production.Journal of advanced research · 2026Article
- CRISPR-Cas9 in the Tailoring of Genetically Engineered Animals.Current issues in molecular biology · 2025Review
- Genome editing of porcine zygotes via lipofection of two guide RNAs using a CRISPR/Cas9 system.The Journal of reproduction and development · 2024Article
- Efficient gene editing of pig embryos by combining electroporation and lipofection.Veterinary world · 2024Article
- Perspectives in Genome-Editing Techniques for Livestock.Animals : an open access journal from MDPI · 2023Review
- Oviduct epithelial cell‑derived extracellular vesicles promote the developmental competence of IVF porcine embryos.Molecular medicine reports · 2023Article
- CRISPR/Cas9-induced Mutation of Sex Peptide Receptor Gene Bdspr Affects Ovary, Egg Laying, and Female Fecundity in Bactrocera dorsalis (Hendel) (Diptera: Tephritidae).Journal of insect science (Online) · 2023Article
- Multiple gene editing in porcine embryos using a combination of microinjection, electroporation, and transfection methods.Veterinary world · 2022Article
- Triple gene editing in porcine embryos using electroporation alone or in combination with microinjection.Veterinary world · 2022Article
- Cell Cycle Stage and DNA Repair Pathway Influence CRISPR/Cas9 Gene Editing Efficiency in Porcine Embryos.Life (Basel, Switzerland) · 2022Article
- Establishment, characterization, and validation of novel porcine embryonic fibroblasts as a potential source for genetic modification.Frontiers in cell and developmental biology · 2022Article
- CRISPR/Cas9-editing ofFrontiers in genetics · 2022Article
- Timing and duration of lipofection-mediated CRISPR/Cas9 delivery into porcine zygotes affect gene-editing events.BMC research notes · 2021Article
- Generation and first characterization of TRDC-knockout pigs lacking γδ T cells.Scientific reports · 2021Article
- Current status of the application of gene editing in pigs.The Journal of reproduction and development · 2021Review
- Lipofection-Mediated Introduction of CRISPR/Cas9 System into Porcine Oocytes and Embryos.Animals : an open access journal from MDPI · 2021Article
- MicroRNA-210 Regulates Endoplasmic Reticulum Stress and Apoptosis in Porcine Embryos.Animals : an open access journal from MDPI · 2021Article
- Assessment of Mosaicism and Detection of Cryptic Alleles in CRISPR/Cas9-Engineered Neurofibromatosis Type 1 andFrontiers in genetics · 2021Article
- Electroporation-Mediated Genome Editing of Livestock Zygotes.Frontiers in genetics · 2021Review
Corrections and comments
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/Cas9 components for introduction into oocytes/zygotes by CI, to reduce mosaicism in the resulting blastocysts. First, we introduced 20 ng/μl of Cas9 protein and guide RNA (gRNA), targeting the α-1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes/zygotes before and after IVF, twice. CI treatment had no detrimental effects on blastocyst formation rates. The highest value of the rate of mutant blastocysts was observed in zygotes injected after IVF. Next, we injected Cas9 protein and gRNA into zygotes after IVF at a concentration of 20 ng/μl each (20 ng/μl group) or 100 ng/μl each (100 ng/μl group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/μl group was significantly higher (P < 0.05) than that in the 20 ng/μl group. Although no blastocysts from the 20 ng/μl group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/μl group carried a biallelic mutation. In conclusion, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI, and the concentration of CRISPR/Cas9 components, is needed.
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