Generation of Zebrafish Maternal Mutants via Oocyte-Specific Knockout System

Maternal mRNAs and proteins are produced during oogenesis by more than 60% of zebrafish genes. They are indispensable for fertilization and early embryogenesis. Generation and analysis of the maternal mutant is the most direct way to characterize the maternal function of the specific gene. However, due to the lethality of zygotic mutants, the maternal function of most genes in zebrafish remains elusive. Several methods have been developed to circumvent this obstacle, including mRNA rescue, germ-line replacement, oocyte microinjection in situ, mosaic mutation, and bacterial artificial chromosome (BAC)-mediated conditional rescue. Here, we provide an alternative approach to generate zebrafish maternal mutants rapidly and efficiently by introducing four tandem sgRNA expression cassettes into Tg(zpc:zcas9) embryos. This method is more technically feasible and cost- and time-effective than other established methods. Key features • This protocol can circumvent the lethality or infertility of the zygotic mutants to obtain maternal mutants of the target gene. • This protocol is time-saving (one fish generation). • Using this protocol, double-gene maternal mutants can be obtained in a single generation. • Stable lines can be established to continuously produce maternal mutant embryos for the gene of interest.