基于CRISPR系统技术鉴定抑制PED病毒感染的宿主（猪）抗性基因及其作用机理研究

Porcine Epidemic Diarrhea (PED) is one of worldwide serious viral infectious diseases that results in huge harm and has difficulties in prevention and control. Breeding the disease-resistant pig breed is an effective way to control disease, but there is no pig resistant gene that can be applied in breeding. Recently CRISPR system technology was developed for high-through-put and rapidly screening and identification of important genes. Therefore we will establish the Genome-Wide/CRISPRa Screening（GW-CRISPRa）technology by using the activating sgRNA library including the pig genome-wide genes that we have previously constructed; screen the candidate resistant genes of suppressing PED virus infection from the pig whole genome by using this GW-CRISPRa technology; analyze the antiviral effects of the candidate resistant genes on suppressing PED virus infection by using the previously established two techniques of knockout CAS9 and activating CRISPRa pig single gene, and then identify the pig important resistant genes from the candidate resistant genes; and finally investigate the mechanisms of the resistant genes on suppressing PED virus infection through studying gene interactions, gene signaling pathways and regulatory networks. ..The results obtained from this project will provide target genes with independent intellectual property rights for breeding the PED-resistant pig breeds, enhance to better understanding of the porcine antiviral mechanism and PEDV-host interaction mechanism. What is more, the CRISPR system technology suitable for the researches of pig genes established and perfected in this project can also be applied in many fields of pig genome research, including the pig other diseases and economic traits. Therefore, the project will have important theoretical and practical value.

猪流行性腹泻（PED）是危害大防治难的重大病毒性传染病之一，培育抗病品种是控制疾病的有效途径，而目前尚无可用于育种的抗PED的抗性基因。鉴于CRISPR系统技术是高通量快速筛选鉴定基因的新型工具，本项目拟基于本课题组已构建的全基因组的猪源sgRNA激活文库建立猪全基因组基因GW-CRISPRa激活筛选技术，利用该技术从猪全基因组中筛选抑制PED病毒感染的抗性候选基因；并利用已建立的单个猪基因CAS9敲除和CRISPRa激活两个技术分析猪抗性候选基因的抗病毒作用，从猪抗性候选基因中鉴定出猪重要抗性基因；最后从基因互作和信号通路及调控网络角度研究猪抗性基因的抗病毒作用机理，旨为培育抗PED新品种提供具有自主知识产权的靶基因，为解析猪抗病、PEDV-宿主互作机理奠定基础，建立的适于猪基因研究的CRISPR系统技术亦可用于猪其它疾病及经济性状等许多研究领域，故具有重要理论与应用价值。

猪流行性腹泻（PED）是PEDV病毒感染导致的，是养猪业中危害最严重的病毒性传染病，而疫苗免疫不能安全有效地防控PED流行。猪抗病育种成为控制猪疾病的重要手段，但还缺乏可用于PED抗病育种的重要基因。病毒感染同时受宿主易感和抗性基因的调控。为了全面研究PEDV与宿主互作、宿主抗病毒机理，需要在猪全基因组范围内挖掘PEDV感染的重要易感和抗性基因。全基因组基因激活和敲除CRISPR-based及其配套的快速高通量遗传筛选鉴定技术，是挖掘病毒感染的易感和抗性基因的有效新工具。然而，目前还没有构建猪全基因组基因激活的猪源突变细胞库及建立配套的抗性基因筛选技术。. 本项目开展了（1）建立GW-CRISPRa激活筛选技术，（2）构建全基因组基因激活的猪源激活细胞，（3）从全基因组中筛选出抗PEDV候选基因、重要基因，（4）猪抗性基因的作用与机理等研究。. 本项目取得了：（1）在猪全基因组范围内挖掘出了抗PEDV 感染的猪重要基因5个、PEDV易感基因14个；（2）解析了猪抗性基因SPHKAP抑制PEDV的调控机理：SPHKAP基因可能通过调节鞘氨醇信号通路影响抑制PEDV感染诱导的TNF-α表达；（3）构建和完善了Genome-Wide CRISPR-dCas9 所需要的猪源全基因组靶基因 gRNA 文库（PGW-gRNA）和稳定表达dCas9 的猪源细胞以及CRISPR-dCas9 所需要的猪单基因激活gRNA 表达载体和稳定表达SAM-dCas9 的猪源细胞；（4）以通讯作者发表了第一标注基金委资助项目编号的文章3篇，在投文章1篇；（5）授权发明专利 2 项；（6）申请发明专利5项；（7）培养了3名博士研究生、1名硕士研究生。. 本项目研究成果为研究抗PEDV感染机理、病毒与宿主互作机理打下基础；为防治病毒感染的药物和疫苗设计提供新靶分子，为培育抗病品种提供基因资源。