Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted primordial germ cells  

作　　者：Fenghua Zhang Xianmei Li Mudan He Ding Ye Feng Xiong Golpour Amin Zuoyan Zhu Yonghua Sun 

机构地区：[1]State Key Laboratory of Freshwater Ecology and Biotechnology,Innovation Academy for Seed Design,Institute of Hydrobiology,Chinese Academy of Sciences,Wuhan,430072,China [2]College o f Advanced Agricultural Sciences,University of Chinese Academy of Sciences,Beijing,100049,China

出　　处：《Journal of Genetics and Genomics》2020年第1期37-47,共11页遗传学报（英文版）

基　　金：supported by the National Key R&D Project of China (2018YFA0801000 and 2018YFD0901205);National Natural Science Foundation of China (Nos. 31721005, 31671501 and 31222052);the Youth Innovation Association of CAS;the State Key Laboratory of Freshwater Ecology and Biotechnology (No. 2019FBZ05).

摘　　要：The clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rate during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cell(PGC) transplantation(PGCT) to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. Firstly, we optimized the procedure for CRISPR/Cas9 targeted PGCT by increasing the efficiencies of genome mutation in PGCs and induction of PGC fates in donor embryos for PGCT. Secondly, the optimized CRISPR/Cas9 targeted PGCT was utilized for generation of maternal-zygotic(MZ) mutants of tcf7l1a(gene essential for head development), pou5f3(gene essential for zygotic genome activation) and chd(gene essential for dorsal development) at F1 generation with relatively high efficiency. Finally, we revealed some novel phenotypes in MZ mutants of tcf7l1 a and chd, as MZtcf7l1 a showed elevated neural crest development while MZchd had much severer ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish. It is also feasible to speed up the genome editing in commercial fishes by utilizing a similar approach by surrogate production of CRISPR/Cas9 targeted germ cells.The clustered regularly interspaced short palindromic repeats（CRISPR）/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rate during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cell（PGC） transplantation（PGCT） to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. Firstly, we optimized the procedure for CRISPR/Cas9 targeted PGCT by increasing the efficiencies of genome mutation in PGCs and induction of PGC fates in donor embryos for PGCT. Secondly, the optimized CRISPR/Cas9 targeted PGCT was utilized for generation of maternal-zygotic（MZ） mutants of tcf7l1a（gene essential for head development）, pou5f3（gene essential for zygotic genome activation） and chd（gene essential for dorsal development） at F1 generation with relatively high efficiency. Finally, we revealed some novel phenotypes in MZ mutants of tcf7l1 a and chd, as MZtcf7l1 a showed elevated neural crest development while MZchd had much severer ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish. It is also feasible to speed up the genome editing in commercial fishes by utilizing a similar approach by surrogate production of CRISPR/Cas9 targeted germ cells.

关 键 词：ZEBRAFISH CRISPR/Cas9 Primordial GERM cells TRANSPLANTATION MATERNAL zygotic MUTANT 

分 类 号：Q78[生物学—分子生物学]