Efficient genome editing in medaka(Oryzias latipes)using a codon-optimized SaCas9 system  

作　　者：Yuewen JIANG Qihua PAN Zhi WANG Ke LU Bilin XIA Tiansheng CHEN 

机构地区：[1]Key Laboratory of Freshwater Animal Breeding,Ministry of Agriculture and Rural Affairs,College of Fisheries,Huazhong Agricultural University,Wuhan 430070,China [2]State Key Laboratory of Mariculture Breeding,Engineering Research Center of the Modern Technology for Eel Industry,Ministry of Education,Key Laboratory of Healthy Mariculture for the East China Sea,Ministry of Agriculture and Rural Affairs,Fisheries College of Jimei University,Xiamen 361021,China

出　　处：《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》2024年第12期1083-1096,共14页浙江大学学报(英文版)B辑(生物医学与生物技术)

基　　金：supported by the National Natural Science Foundation of China(Nos.32273127,31771648,and 31672653);the Scientific Research Foundation of Jimei University(No.ZQ2020003),China。

摘　　要：The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system,belonging to the typeⅡCRISPR/Cas system,is an effective gene-editing tool widely used in different organisms,but the size of Streptococcus pyogenes Cas9(SpCas9)is quite large(4.3 kb),which is not convenient for vector delivery.In this study,we used a codon-optimized Staphylococcus aureus Cas9(SaCas9)system to edit the tyrosinase(tyr),oculocutaneous albinismⅡ(oca2),and paired box 6.1(pax6.1)genes in the fish model medaka(Oryzias latipes),in which the size of SaCas9(3.3 kb)is much smaller and the necessary protospacer-adjacent motif(PAM)sequence is 5'-NNGRRT-3'.We also used a transfer RNA(tRNA)-single-guide RNA(sgRNA)system to express the functional sgRNA by transcription either in vivo or in vitro,and the combination of SaCas9 and tRNA-sgRNA was used to edit the tyr gene in the medaka genome.The SaCas9/sgRNA and SaCas9/tRNA-sgRNA systems were shown to edit the medaka genome effectively,while the PAM sequence is an essential part for the efficiency of editing.Besides,tRNA can improve the flexibility of the system by enabling the sgRNA to be controlled by a common promoter such as cytomegalovirus.Moreover,the all-in-one cassette cytomegalovirus(CMV)-SaCas9-tRNA-sgRNA-tRNA is functional in medaka gene editing.Taken together,the codon-optimized SaCas9 system provides an alternative and smaller tool to edit the medaka genome and potentially other fish genomes.

关 键 词：Staphylococcus aureus Cas9(SaCas9) MEDAKA Transfer RNA(tRNA) Gene editing Tyrosinase(tyr) Oculocutaneous albinismⅡ(oca2) Paired box 6.1(pax6.1) 

分 类 号：Q78[生物学—分子生物学]