CRISPR/Cas13d-mediated efficient KDM5B mRNA knockdown in porcine somatic cells and parthenogenetic embryos.

Dengfeng Bi,Jing Yao,Yu Wang,Yunting Zhang,Guosong Qin,Jianguo Zhao,Yanfang Wang

doi:10.1530/rep-21-0053

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.

Highlights

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated genes (Cas) system has been rapidly used for various genomic engineering in a wide range of organisms, from yeast to mammals (Cong et al 2013, Hsu et al 2014)
Ten-eleven translocation (TET) gene family members had been reported to regulate interactions between DNA methylation and histone modification in mouse embryonic stem cells (ESCs), and our results showed that TET2 and TET3 were increased and TET1 was decreased by knockdown of KDM5B, which might be a reason for the decreased developmental competence (Fig. 4O)
The h ttps://rep.bioscientifica.com activity of CRISPR/Cas13d is severely decreased or eliminated if 1–2 mismatches occur in the seed region of gRNA, which greatly reduces the risk of off-target effects

Summary

Introduction

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated genes (Cas) system has been rapidly used for various genomic engineering in a wide range of organisms, from yeast to mammals (Cong et al 2013, Hsu et al 2014). A novel RNA-targeting CRISPR effector protein, called Cas (Koonin et al 2017, Shmakov et al 2017), has been shown to bind and cleave RNA rather than DNA substrates, suggesting a potential for RNA editing and programming. This HEPN domain can be converted to active conformation and cleaves target RNA molecules (Fig. 1A) (cis-cleavage), further cleaving random other RNA it encounters (trans- or collateral cleavage). CRISPR-Cas13d has been developed for unique applications, such as interference against RNA viruses in plants (Mahas et al 2019), knockdown of DNA ligase IV

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Conclusion