Rapid generation of maternal mutants via oocyte transgenic expression of CRISPR-Cas9 and sgRNAs in zebrafish

Abstract

Maternal products are exclusive factors to drive oogenesis and early embryonic development. As disrupting maternal gene functions is either time-consuming or technically challenging, early developmental programs regulated by maternal factors remain mostly elusive. We provide a transgenic approach to inactivate maternal genes in zebrafish primary oocytes. By introducing three tandem single guide RNA (sgRNA) expression cassettes and a green fluorescent protein (GFP) reporter into Tg(zpc:zcas9) embryos, we efficiently obtained maternal nanog and ctnnb2 mutants among GFP-positive F₁ offspring. Notably, most of these maternal mutants displayed either sgRNA site-spanning genomic deletions or unintended large deletions extending distantly from the sgRNA targets, suggesting a prominent deletion-prone tendency of genome editing in the oocyte. Thus, our method allows maternal gene knockout in the absence of viable and fertile homozygous mutant adults. This approach is particularly time-saving and can be applied for functional screening of maternal factors and generating genomic deletions in zebrafish.