A Time-Saving Strategy To Generate Double Maternal Mutants By An Oocyte-Specific Conditional Knockout System In Zebrafish

Simple Summary Maternally supplied mRNAs and proteins, termed maternal factors, are produced by over 14,000 coding genes in zebrafish. They play exclusive roles in controlling the formation of oocytes and the development of early embryos. These maternal factors can also compensate for the loss of function of its corresponding zygotic gene products. Thus, eliminating both maternal and zygotic gene products is essential to elucidate the functions of more than half of zebrafish genes. However, it is always challenging to inactivate maternal factors, because traditional genetic methods are either technically demanding or time-consuming. Our recent work established a rapid conditional knockout method to generate maternal or maternal and zygotic mutants in one fish generation. Here, we further test the feasibility of this approach to knock out two maternal genes with functional redundancy simultaneously. As a proof of principle, we successfully generated double maternal mutant embryos for dvl2 and dvl3a genes in three months for the first time. The cell movement defects in mutant embryos obtained by this approach mimic the genuine mutant embryos generated after fifteen months of time-consuming screening following the previously reported mosaic strategy. Therefore, this method has the potential to speed up the functional study of paralogous maternal genes. Maternal products are those mRNAs and proteins deposited during oogenesis, which play critical roles in controlling oocyte formation, fertilization, and early embryonic development. However, loss-of-function studies for these maternal factors are still lacking, mainly because of the prolonged period of transgenerational screening and technical barriers that prevent the generation of maternal (M) and maternal and zygotic (MZ) mutant embryos. By the transgenic expression of multiple sgRNAs targeting a single gene of interest in the background of a transgenic line Tg(zpc:zcas9) with oocyte-specific cas9 expression, we have successfully obtained maternal or maternal-zygotic mutant for single genes in F1 embryos. In this work, we tandemly connected a maternal GFP marker and eight sgRNA expression units to target dvl2 and dvl3a simultaneously and introduced this construct to the genome of Tg(zpc:zcas9) by meganuclease I-Sce I. As expected, we confirmed the existence of Mdvl2;Mdvl3a embryos with strong defective convergence and extension movement during gastrulation among outcrossed GFP positive F1 offspring. The MZdvl2;MZdvl3a embryos were also obtained by crossing the mutant carrying mosaic F0 female with dvl2(+/-);dvl3a(-/-) male fish. This proof-of-principle thus highlights the potential of this conditional knockout strategy to circumvent the current difficulty in the study of genes with multiple functionally redundant paralogs.