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Published on: April 30, 2018
Efficient biallelic mutation in porcine parthenotes using a CRISPR-Cas9 system.
Li Tao1, Mingyao Yang1, Xiaodong Wang1
1National Engineering Laboratory for Animal Breeding, Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture, College of Animal Science and Technology, China Agricultural University, No. 2 Yuan Ming Yuan Xi Lu, Haidian District, Beijing 100193, PR China.
Optimizing CRISPR/Cas9 delivery in porcine parthenotes significantly enhanced gene editing efficiency. This study achieved a high biallelic mutation rate with reduced mosaicism, improving genetically modified cell line generation.
Area of Science:
- * Reproductive biology and developmental genetics.
- * Gene editing technologies and applications in livestock.
Background:
- * Porcine parthenotes are valuable models for studying embryonic development and serve as a source for pluripotent cell lines.
- * CRISPR/Cas9 gene editing in parthenotes is crucial for creating genetically modified pigs, but faces challenges with low biallelelic mutation rates and high mosaicism.
- * Parthenogenetically activated (PA) embryos offer a rapid assay for assessing gene editing efficiency.
Purpose of the Study:
- * To optimize CRISPR/Cas9 delivery parameters (concentration and injection timing) in porcine parthenotes.
- * To enhance the biallelelic mutation rate while reducing mosaicism in genetically modified porcine parthenotes.
- * To establish a more efficient and homogeneous method for generating modified porcine cell lines.
Main Methods:
- * Cytoplasmic injection of Cas9 mRNA/sgRNA mixture into porcine parthenotes at varying concentrations and time points post-activation.
- * Optimization of the Cas9/sgRNA concentration and injection timing after parthenogenetical activation.
- * Analysis of mutation rates (biallelic and monoallelic) and mosaicism frequency in edited porcine parthenotes.
Main Results:
- * An optimized cytoplasmic injection of Cas9 mRNA/sgRNA at 125/12.5 ng/μl, 8 hours post-parthenogenetic activation, yielded efficient biallelic mutation.
- * Achieved a high biallelelic mutation rate of 93% with significantly reduced mosaicism at 33%.
- * Demonstrated improved population homogeneity in genetically modified porcine parthenotes.
Conclusions:
- * The optimized CRISPR/Cas9 delivery strategy effectively increases biallelelic mutation rates and reduces mosaicism in porcine parthenotes.
- * This improved method enhances the utility of porcine parthenotes for studying early embryonic development and assessing gene editing efficiency.
- * The findings provide a robust approach for generating homogeneous genetically modified porcine cell lines using CRISPR/Cas9 technology.
