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

Publication date: Available online 29 January 2020Source: Journal of Genetics and GenomicsAuthor(s): Fenghua Zhang, Xianmei Li, Mudan He, Ding Ye, Feng Xiong, Golpour Amin, Zuoyan Zhu, Yonghua SunAbstractThe 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. Firstly, we optimized the procedure for CRISPR/Cas9 targeted PGCs transplantation (PGCT), by increasing the efficiencies of genome mutation in PGCs and induction of PGCs fates in donor embryos for PGCT. Secondly, the optimized CRISPR/Cas9 targeted PGCs transplantation 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 ventraliz...

Source: Journal of Genetics and Genomics - January 30, 2020 Category: Genetics & Stem Cells Source Type: research