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 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.

成簇规则间隔短回文重复序列 (CRISPR)/Cas9 技术已广泛用于敲除斑马鱼中参与各种生物过程的基因。尽管这项技术可以有效地产生不同的突变，但主要缺点之一是在胚胎发生过程中敲除一些胚胎致死基因时存活率较低。为了克服这个问题，我们开发了一种新策略，将 CRISPR/Cas9 介导的基因敲除与原始生殖细胞 (PGC) 移植 (PGCT) 相结合，以促进和加速斑马鱼突变体的产生过程，特别是胚胎致死基因。首先，我们通过提高PGC基因组突变的效率以及在PGCT供体胚胎中诱导PGC命运来优化CRISPR/Cas9靶向PGCT的程序。其次，优化的 CRISPR/Cas9 靶向 PGCT 用于生成tcf7l1a （头部发育必需基因）、 pou5f3 （合子基因组激活必需基因）和chd （背部发育必需基因）的母本合子 (MZ) 突变体。 F ₁代效率相对较高。最后，我们揭示了tcf7l1a和chd的 MZ 突变体中的一些新表型，因为 MZ tcf7l1a显示出神经嵴发育升高，而 MZ chd比其合子对应物具有更严重的腹侧化。因此，本研究提出了一种有效且强大的方法来生成斑马鱼胚胎致死基因的 MZ 突变体。通过利用类似的方法替代生产 CRISPR/Cas9 靶向生殖细胞，加速商业鱼类的基因组编辑也是可行的。