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

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

doi:10.1016/j.jgg.2019.12.004

Volume 47 Issue 1

Jan. 2020

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

doi: 10.1016/j.jgg.2019.12.004

Fenghua Zhang^{a, b},
Xianmei Li^{a, b},
Mudan He^{a, b},
Ding Ye^{a, b},
Feng Xiong^{a, b},
Golpour Amin^{a, b},
Zuoyan Zhu^{a, b},
Yonghua Sun^{a, b}

a
State Key Laboratory of Freshwater Ecology and Biotechnology, Innovation Academy for Seed Design, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China
b
College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, 100049, China

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Corresponding author: E-mail address: yhsun@ihb.ac.cn (Yonghua Sun)
Publish Date: 2020-01-25

Abstract

Abstract

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 oftcf7l1a (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 tcf7l1a and chd, as MZtcf7l1a 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.
- Zebrafish,
- CRISPR/Cas9,
- Primordial germ cells,
- Transplantation,
- Maternal zygotic mutant

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References(43)

References

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