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

ABSTRACT The 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 rates 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 cells (PGCs) transplantation to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. First, we optimized the procedure for gRNA targeted PGCs transplantation (PGCT), by increasing the efficiencies of genome mutation in PGCs and induction of PGCs fates in donor embryos for PGCT. Second, the combined CRISPR/Cas9 with PGCT was utilized for generation of maternal zygotic (MZ) mutants of tcf7l1a (essential gene for head development), pou5f3 (essential gene for zygotic genome activation) and chd (essential gene for dorsal development) at F1 generation with high efficiency. Finally, we revealed some novel phenotypes in the maternal zygotic mutant of tcf7l1a and chd , while MZ tcf7l1a showed elevated neural crest development, and MZ chd have stronger ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish.