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Updated: Nov 2, 2025
Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes
Published on: December 16, 2022
CRISPR/Cas13d-mediated efficient KDM5B mRNA knockdown in porcine somatic cells and parthenogenetic embryos
Dengfeng Bi1,2,3, Jing Yao2,3,4,5, Yu Wang2,3,5
1School of Life Sciences, University of Science and Technology of China, Hefei, China.
Abstract:
An efficient mRNA knockdown strategy is needed to explore gene function in cells and embryos, especially to understand the process of maternal mRNA decay during early embryo development. Cas13, a novel RNA-targeting CRISPR effector protein, could bind and cleave complementary single-strand RNA, which has been employed for mRNA knockdown in mouse and human cells and RNA-virus interference in plants. Cas13 has not yet been reported to be used in pigs. In the current study, we explored the feasibility of CRISPR/Cas13d-mediated endogenous RNA knockdown in pigs. KDM5B, a histone demethylase of H3K4me3, was downregulated at the transcriptional level by 50% with CRISPR/Cas13d in porcine fibroblast cells. Knockdown of KDM5B-induced H3K4me3 expression and decreased the abundance of H3K27me3, H3K9me3, H3K4ac, H4K8ac, and H4K12ac. These changes affected cell proliferation and cell cycle. Furthermore, stable integration of the CRISPR/Cas13d system into the porcine genome resulted in the continuous expression of Cas13d and persistent knockdown of KDM5B. Finally, the RNA-targeting potential of Cas13d was further validated in porcine parthenogenetic embryos. By microinjection of Cas13d mRNA and gRNA targeting KDM5B into porcine oocytes, the expression of KDM5B was downregulated, the abundance of H3K4me3 increased as expected, and the expression of embryonic development-related genes was changed accordingly. These results indicate that CRISPR/Cas13d provides an easily programmable platform for spatiotemporal transcriptional manipulation in pigs.
Insights
Researchers explored CRISPR/Cas13d for mRNA knockdown in pigs, successfully downregulating KDM5B in cells and embryos. This RNA-targeting system offers a new tool for gene function studies in pigs.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Efficient mRNA knockdown is crucial for understanding gene function and maternal mRNA decay in early embryonic development.
- CRISPR-Cas13 systems offer RNA-targeting capabilities, previously demonstrated in various cell types and plants, but not yet in pigs.
Purpose of the Study:
- To investigate the feasibility of using CRISPR/Cas13d for endogenous RNA knockdown in porcine cells and embryos.
- To assess the impact of KDM5B knockdown on epigenetic modifications and cellular processes in pigs.
Main Methods:
- CRISPR/Cas13d system was employed to target and downregulate KDM5B mRNA in porcine fibroblast cells.
- Stable integration of the CRISPR/Cas13d system was achieved for persistent gene knockdown.
- Cas13d's RNA-targeting potential was validated in porcine parthenogenetic embryos via microinjection.
Main Results:
- CRISPR/Cas13d successfully downregulated KDM5B mRNA by 50% in porcine cells, altering histone modifications (H3K4me3, H3K27me3, H3K9me3) and affecting cell proliferation.
- Stable integration led to continuous Cas13d expression and sustained KDM5B knockdown.
- In porcine embryos, KDM5B knockdown increased H3K4me3 and altered embryonic development-related gene expression.
Conclusions:
- CRISPR/Cas13d is a feasible and effective tool for endogenous RNA knockdown in pigs.
- This system enables spatiotemporal transcriptional manipulation for studying gene function and developmental processes in porcine models.
