Background

Maternal factors are mRNAs and proteins deposited in the oocyte. They play vital roles in oocyte maturation, fertilization, and blastocyst development. Approximately 66% of zebrafish genes are expressed maternally [1]. As zygotic genome activation (ZGA) starts at around the 1k-cell stage, maternal factors are dominant to function in the developmental events before this time point. In addition, even after zygotic genome activation (ZGA), maternal factors still function in later embryogenesis, including axis formation, germ layers differentiation, morphogenesis, etc. [2–4].

Generation of the corresponding maternal mutant is the most straightforward way to address the maternal function of a gene in zebrafish. The genotype of primary oocyte is the same as that of somatic cells. Hence, viable and fertile female zygotic mutants are indispensable for giving birth to maternal mutants or maternal and zygotic mutants. However, zygotic mutants usually cannot survive to adulthood or spawn because of the crucial and uncompensable functions of the mutated genes [5–7]. This obstacle represents the major bottleneck to studying maternal factors. Several methods have been developed to bypass this technical hurdle. The first is mRNA rescue. Through microinjection of in vitro–synthesized wild-type mRNA, the zygotic mutant can be rescued to live through a critical period, in which the zygotic products contribute to normal development [4]. Considering that the ectopic expression may disrupt normal embryogenesis and transient expression of mRNA cannot support long-term gene function, this method is not applicable to most genes. In 2002, Ciruna et al. developed the germ-line replacement technology. They injected dead end1 morpholino into wild-type embryos to block primordial germ cell development and then transplanted germ cells from zygotic mutants into these primordial germ cell (PGC)-free hosts. This ensured that the germ lines of the host were entirely replaced by donor PGCs. Upon maturation, all the embryos laid by the female chimera were maternal mutants [8]. Transplantation of PGCs is technically demanding and the chimeric embryos tend to develop into males because of the limited number of transplanted PGCs. Wu et al. established a surgery-based method [9]. Following the opening of the female fish abdomen, the fluorescent lineage tracer and the morpholino targeting the specific gene were co-injected into the stage- oocyte. After recovery, the laid fluorescent embryos were the desired maternal morphants [9]. However, the intricate operative skills required for the application of this method are significantly challenging. In 2018, Xing et al. generated mosaic mutants containing homozygous mutant cells through secondary genome editing in heterozygotes [2]. This approach was designed to circumvent the lethality associated with double zygotic mutants of dvl2 and dvl3a. Some of these homozygous mutant cells were incorporated into the germline, eventually resulting in maternal mutants after fertilization. However, this method is inefficient and time-consuming, requiring at least three generations. In addition to the methods mentioned above, the bacterial artificial chromosome-rescue-based knockout (BACK) is another method to bypass the zygotic lethality and obtain maternal mutants [10]. To obtain such mutants, first, the authors generated mutants of the target gene through nuclease-mediated genome editing. Second, they rescued the zygotic mutants by introducing a bacterial artificial chromosome (BAC) containing the target gene expression cassette flanked by loxP sites via Tol2-mediated transgenesis. Third, they crossed these fish with a germline-specific Cre line. This approach ultimately allowed them to obtain maternal mutants of the target gene. However, this strategy is time-consuming and labor-intensive, as it involves gene knock-out, transgenesis, and crossing with the Cre line.

Recently, we have developed a new strategy to generate single or double-gene maternal mutants through transgenic expression of Cas9 and sgRNA in oocytes [11,12]. Through I-Sce I-mediated transgenesis, we introduced an eGFP reporter and multiple tandem sgRNA expression cassettes into Tg(zpc:zcas9) transgenic fish, where Cas9 is specifically expressed in oocytes. This approach enables genome editing during the early stages of oogenesis. As a result, some of the GFP-positive embryos will have their maternal products completely ablated, thereby becoming maternal mutants. Consequently, we can analyze their phenotypes after identifying them through genotyping. Compared to other methods, it has several advantages. First, it is technically feasible. The main techniques are plasmid construction and transgenesis, which are conventional in most zebrafish labs. Second, it is time saving, taking one generation (2–3 months) for either single or double-genes maternal knockout. Third, the efficiency is generally stable despite individual variation. Through generating maternal mutants of nanog, ctnnb2, rbm24a, dvl2, and dvl3a, we find that the average ratio of the maternal mutant for a single gene is approximately 25% and the maximum efficiency can reach 63.3%. For every single founder fish, the ratio of maternal mutants remains stable in each spawning, so that we can obtain maternal mutants repeatedly once we get a founder. Finally, this approach can be utilized to generate double-gene maternal mutants, which takes nearly the same time.