利用CRISPR-Cas9技术研究PRRSV感染相关基因及其在病毒增殖感染中的作用机理

Porcine Reproductive and Respiratory Syndrome (PRRS) that is caused by PRRS virus (PRRSV) is worldwide serious infectious diseases, and results in huge economic loss and difficulties of prevention and control, is therefore the industry leading killer. Due to the complex immunological characteristics of the PRRSV constantly variation and suppression of pig immune response, these increase the difficulties to investigate the PRRSV pathogenic mechanism and the porcine immune antiviral mechanism, so that it is still lack of effective security vaccines and drugs and techniques. ..This project will (1) establish the pig Genome-Wide gRNA library and use the Genome-Wide CRISPR-CAS9 technique to knockout genome-wide genes, to screen and select the candidate genes related with PRRSV-infection based on pig whole genome; (2) establish the pig targeted gene gRNA vector and apply the CRISPR-CAS9 tool that knockouts a single specific gene, to study the roles of candidate genes on PRRSV proliferation and infection, and then identify the pig important genes related PRRSV proliferation and infection; and (3) investigate the mechanisms of pig important genes on PRRSV proliferation and infection using CRISPR-CAS9. ..The results obtained from this project will contribute to the development of the new types of vaccines and drugs to prevent and control PRRS, and to breeding disease-resistance pigs through the important genetic strategy that knockout or modify the pig important genes related with PRRSV proliferation and infection, and also enhance to better understand the PRRSV pathogenic mechanism and porcine immune antiviral mechanism, so this project will have important applied and theoretical value.

猪繁殖呼吸综合症(PRRS)是由PRRS病毒（PRRSV）感染引起的危害大和防治难的世界性传染病，目前仍缺乏安全有效的疫苗药物和技术。本项目将（1）构建猪源全基因组靶基因gRNA文库和利用全基因组基因敲除编辑Genome-Wide CRISPR-Cas9技术，在猪全基因组中筛选PRRSV感染相关候选基因；（2）构建猪单个特定基因gRNA表达载体和利用单个特定基因敲除编辑CRISPR-Cas9技术，鉴定感染候选基因在PRRSV增殖感染中作用，挖掘PRRSV感染相关猪重要基因；（3）利用CRISPR-Cas9等技术研究该重要基因对PRRSV增殖感染的作用机理，为研究PRRSV致病、猪免疫抗病等机理奠定基础，为防治PRRS新型疫苗药物的研制、通过敲除或修饰感染相关猪重要基因的策略培育抗病新品种等提供参考，将具有重要的理论和应用价值。

猪繁殖呼吸综合症(PRRS)是由PRRS病毒（PRRSV）感染引起的危害大和防治难的世界性传染病，目前仍缺乏安全有效的疫苗药物和技术。. 本项目已经（1）构建了猪源全基因组基因敲除gRNA文库，利用全基因组基因敲除编辑Genome-Wide CRISPR-Cas9技术在猪全基因组中筛选出了PRRSV感染相关候选基因；（2）构建了猪单基因敲除gRNA表达载体，利用单基因敲除CRISPR-Cas9技术研究了感染候选基因在PRRSV感染中作用，挖掘出了PRRSV感染相关的猪重要基因9个；（3）利用CRISPR-Cas9等技术研究了4个重要基因对PRRSV增殖感染的作用机理，将为研究抗PRRSV感染、猪免疫抗病等机理奠定基础，为防治PRRS新型疫苗药物的研制、通过敲除或修饰感染相关猪重要基因的策略培育抗病新品种等提供参考，具有重要的理论和应用价值。. 此外，项目主持人张淑君（4）以通讯作者发表了标注基金委资助项目编号的文章10篇（8篇SCI收录文章、2篇中文核心期刊论文）；（5）申请了专利 2 项；（6）被邀请参与撰写了英文书籍《The CRISPR/Cas System: Emerging Technology and Application》；（7）被邀请在2018年基因编辑学术研讨会上作了大会报告《全基因组CRISPR/Cas9文库筛选与猪流感病毒感染相关的宿主（猪）基因》；（8）培养了2名博士研究生、4名硕士研究生；（9）选送了4名博士生去英国合作研究和联合培养；1名博士生于2016年申请和获得了英国牛顿青年人才基金项目（Newton International Young Scientist Fellowship）；（10）主办了《第五届中英动物育种与疾病防治研讨会》。