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Dengfeng Bi School of Life Sciences, University of Science and Technology of China, Hefei, China
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China

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Jing Yao State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
University of Chinese Academy of Sciences, Beijing, China

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Yu Wang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
University of Chinese Academy of Sciences, Beijing, China

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Guosong Qin State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China

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Yunting Zhang State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

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Yanfang Wang Laboratory of Animal (Poultry) Genetics Breeding and Reproduction, Ministry of Agriculture, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China

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Jianguo Zhao School of Life Sciences, University of Science and Technology of China, Hefei, China
State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing, China
Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China
University of Chinese Academy of Sciences, Beijing, China

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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.

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Yanfang Wu College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, P.R. China
Key Laboratory of Animal Biotechnology, Ministry of Agriculture and Rural Affairs, Northwest A&F University, Yangling, P.R. China

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Zhenzi Zuo State Key Laboratory of Agrobiotechnology, College of Biological Science, China Agricultural University, Beijing, P.R. China

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Zheng Wang College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, P.R. China
Key Laboratory of Animal Biotechnology, Ministry of Agriculture and Rural Affairs, Northwest A&F University, Yangling, P.R. China

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Hanghang Liu College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, P.R. China
Key Laboratory of Animal Biotechnology, Ministry of Agriculture and Rural Affairs, Northwest A&F University, Yangling, P.R. China

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Qi Zhou College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, P.R. China
Key Laboratory of Animal Biotechnology, Ministry of Agriculture and Rural Affairs, Northwest A&F University, Yangling, P.R. China

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Subi Ren College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, P.R. China
Key Laboratory of Animal Biotechnology, Ministry of Agriculture and Rural Affairs, Northwest A&F University, Yangling, P.R. China

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Xinrui Lan College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, P.R. China
Key Laboratory of Animal Biotechnology, Ministry of Agriculture and Rural Affairs, Northwest A&F University, Yangling, P.R. China

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Yong Zhang College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, P.R. China
Key Laboratory of Animal Biotechnology, Ministry of Agriculture and Rural Affairs, Northwest A&F University, Yangling, P.R. China

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Yongsheng Wang College of Veterinary Medicine, Northwest A & F University, Yangling, Shaanxi, P.R. China
Key Laboratory of Animal Biotechnology, Ministry of Agriculture and Rural Affairs, Northwest A&F University, Yangling, P.R. China

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In brief

Almost total lack of sperm-borne RNAs is regarded as one of the key factors that leads to the abnormal development of somatic cell nuclear transfer embryo. This paper reveals a need for us to further explore the roles of the paternal regulatory factors on embryonic development in early embryos.

Abstract

Mature sperm contain both coding and non-coding RNAs, which can be delivered into an oocyte with the sperm at fertilization. Accumulating evidences show that these sperm-borne RNAs play crucial roles in epigenetic reprogramming, cytoskeleton remodeling, embryonic development, and offspring phenotype. Almost total lack of sperm-borne RNAs is regarded as one of the key factors that leads to the abnormal development of somatic cell nuclear transfer (SCNT) embryo. bta-miR-183 was found to be highly expressed in bovine sperm and can be delivered into oocytes during fertilization in our previous study, and in this study, EZR was confirmed as a target gene of bta-miR-183 in early embryos by bioinformatics, luciferase, and gain-of-function and loss-of-function experiments. Scanning electron microscopy showed that the density of microvilli on the surface of SCNT embryos was significantly higher than that onin vitro fertilized embryos and was significantly reduced by injection of bta-miR-183 mimic. EZR-siRNA injected into SCNT embryos had a similar effect. This indicated that the lack of bta-miR-183 might lead to abnormal changes in microvilli by downregulating ezrin protein. In addition, gain-of-function studies showed that bta-miR-183 significantly improved developmental competence of SCNT embryo in terms of cleavage (76.63% vs 64.32%, P < 0.05), blastocyst formation (43.75% vs 28.26%, P < 0.05), apoptotic index (5.21% vs 12.64%, P < 0.05), and the trophoblast ratio (32.65% vs 25.58%, P < 0.05) in day 7 blastocysts. Thus, the present study indicated that bta-miR-183 might play crucial roles in the formation of microvilli and embryo development by regulating expression of EZR mRNA.

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