CRISPR/Cas13d-mediated Endogenous Gene Transcriptional Knockdown In Pigs

The discovery and modified variants of the CRISPR/Cas system has brought revolutionary changes to gene editing,which makes gene modifications,such as gene deletion and insertion in large animals more efficiently and precisely.CRISPR/Cas9,CRISPR/Cas12a(Cpfl)and CRISPR/Cas 13 systems,have been widely used in plants and animals.Both CRISPR/Cas9 and CRISPR/Cas12a are capable of modifying genome DNA,which regulate target gene expression continuously.By contrast,the CRISPR/Cas13 system could target RNA to achieve accurate transcriptive regulation of target genes which is safe.Since 2018,as a new member of the Cas13 family,Cas13d has been widely used in studying expression regulation of genes in plant,mouse and human cells,but has not been reported in large animals.This study aims to explore the CRSIPR/Cas13d system mediated gene regulation at the transcription level in porcine somatic cells and preimplantation embryos.The main findings are as follows:1.sgRNA expression vectors were constructed to target KDM5B(histone demethylase),SUV39H1 and SUV39H2(histone methyltransferases)genes which related to histone modification.The efficient sgRNA was selected by transient transfection of porcine embryonic fibroblast cells(PEFs).Transfection ratio and detection time of Cas13d and sgRNA were also optimized and results showed that the knockdown efficiency of CRISPR/Cas 13d could reach 70%at 24 h after transfection when the molar ratio of Cas13d:sgRNA was 1:2.2.After knocking down KDM5B in PEF cells,the changes of histone methylation and acetylation levels and effects on cell proliferation and cell cycle were detected.The results showed that the level of H3K4me3 was up-regulated,and that of H3K27me3 and H3K9me3 was down-regulated after knocking down KDM5B via CRISPR/Cas 13d.At the same time,histone acetylation such as H3K4ac,H4K8ac,and H4K12ac was down-regulated.The proliferation capacity of KDM5B-knockdown PEF cells was reduced and most of that were blocked in the G0/G1 phase of cell cycle.3.Stable site-specific integration of Cas13d-KDM5B-sgRNA into PEFs was transfected and screened.In single cell colonies,Cas 13d was expressed stably at different time points and knockdown effect of the KDM5B gene was also continuously observed.4.Down-regulation of KDM5B can be detected in 2-cell embryos after injecting in vitro transcribed Cas 13d mRNA and sgRNA into porcine embryos.The level of H3K4me3 in KDM5B-knockdown blastocysts was significantly up-regulated,causing the significant decrease of embryo quality and in vitro development potential.In this study,the CRISPR/Cas13d-mediated knockdown system to target porcine endogenous gene in transcription levels was established in porcine somatic cell and embryo for the first time,providing an important platform for the study of gene function.CRISPR/Cas13d interfere with embryonic development-related gene expression,which will provide a new research system for the study of molecular mechanisms at early embryo development.