家蚕CRISPR/Cpf1基因编辑系统建立  

Development of a CRISPR/Cpf1 gene editing system in silkworm Bombyx mori

作　　者：董战旗 秦琪 张新铃 李柯洁 陈鹏[1] 潘敏慧[1,2] Zhanqi Dong;Qi Qin;Xinling Zhang;Kejie Li;Peng Chen;Minhui Pan(State Key Laboratory of Silkworm Genome Biology,Southwest University,Chongqing 400716,China;Key Laboratory for Sericulture Functional Genomics and Biotechnology of Agricultural and Rural Affairs Ministry,Southwest University,Chongqing 400716,China)

机构地区：[1]西南大学家蚕基因组生物学国家重点实验室,重庆400716 [2]西南大学农业农村部蚕桑生物学与遗传育种重点实验室,重庆400716

出　　处：《生物工程学报》2021年第12期4342-4350,共9页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(Nos.31902214,31872427);中央高校基本科研业务费(Nos.SWU120008,XDJK2020C010);财政部和农业农村部:国家现代农业产业技术体系资助;重庆市自然科学基金项目(Nos.cstc2019jcyjmsxmX0096,cstc2020jscxmsxmX0045)资助。

摘　　要：自CRISPR/Cas9基因编辑系统成功应用于模式生物以来,因其快速、高效、便捷等特点,广泛应用于基因功能研究、基因治疗和基因工程等研究领域。与此同时,CRISPR/Cas系统不断在微生物界的发现也加速了新的基因编辑工具的不断涌现。CRISPR/Cpf1是第二类(Ⅴ型)能够编辑哺乳动物基因组的CRISPR系统,相比于CRISPR/Cas9基因编辑系统,能够利用5′T-PAM富集区增加基因组覆盖率,具有其切割位点为粘性末端和更不易同源重组修复等诸多优势。基于此,本研究构建了能够在家蚕细胞表达的3个不同来源的CRISPR/Cpf1 (AsCpf1、FnCpf1和LbCpf1)表达载体,选择高度保守的家蚕热休克蛋白基因BmHSP60和家蚕ATP酶家族BmATAD3A基因分别设计靶标gRNA,构建gHSP60-266R和gATAD3A-346R基因编辑载体。通过T7E1酶切分析和T克隆测序,鉴定3个Cpf1基因编辑系统AsCpf1、FnCpf1和LbCpf1对靶标基因BmHSP60和BmATAD3A的编辑效率。同时,利用Western blotting分析不同基因编辑系统敲除靶基因后对其BmATAD3A和BmHSP60蛋白翻译的影响。本研究成功构建了家蚕CRISPR/Cpf1基因编辑系统,能够在家蚕细胞中有效编辑家蚕基因组,为家蚕基因功能研究、基因工程和遗传育种开发了新技术与新方法。The CRISPR/Cas9 gene editing system has been widely used in basic research, gene therapy and genetic engineering due to its high efficiency, fast speed and convenience. Meanwhile, the discovery of novel CRISPR/Cas systems in the microbial community also accelerated the emergence of novel gene editing tools. CRISPR/Cpf1 is the second type(V type)CRISPR system that can edit mammalian genome. Compared with the CRISPR/Cas9, CRISPR/Cpf1 can use 5′T-PAM rich region to increase the genome coverage, and has many advantages, such as sticky end of cleavage site and less homologous recombination repair. Here we constructed three CRISPR/Cpf1(AsCpf1, FnCpf1 and LbCpf1) expression vectors in silkworm cells. We selected a highly conserved BmHSP60 gene and an ATPase family BmATAD3 A gene to design the target gRNA, and constructed gHSP60-266 and gATAD3 A-346 knockout vectors. The efficiency for editing the target genes BmATAD3 A and BmHSP60 by AsCpf1, FnCpf1 and LbCpf1 were analyzed by T7 E1 analysis and T-clone sequencing. Moreover, the effects of target gene knockout by different gene editing systems on the protein translation of BmHSP60 and BmATAD3 A were analyzed by Western blotting. We demonstrate the CRISPR/Cpf1 gene editing system developed in this study could effectively edit the silkworm genome, thus providing a novel method for silkworm gene function research, genetic engineering and genetic breeding.

关 键 词：家蚕 基因编辑 CRISPR/Cpf1 

分 类 号：Q78[生物学—分子生物学] S881[农业科学—特种经济动物饲养]