Abstract
Simple SummaryMaternal gene products accumulated during oogenesis as mRNAs and proteins, which are referred to as maternal contributions, play essential roles in proper development before and after the transcription of the zygotic genome. Zebrafish represent an attractive model for the systematic characterization of genetic mutants using the CRISPR/Cas9 genome editing technology. The traditional approach for generating maternal mutants to study the functions of maternal gene products consists of creating founder fish with germ-line transmission of mutated genes, followed by successive crossing to obtain zygotic homozygous mutant females, which can produce offspring lacking the corresponding maternal gene products. This is time-consuming and normally requires three generations. Nevertheless, zygotic mutations of many genes often lead to embryonic lethality or adult sterility, making it impossible to study their maternal functions. Different approaches were used to overcome this difficulty and allowed for the characterization of several important maternal-effect genes. However, they are often technically challenging or lack versatile applications. Recently, an oocyte-specific knockout strategy has been developed, which inactivates genes of interest in the developing oocytes. It is particularly accessible, generating maternal mutants in one fish generation. By further improving its efficiency, this method can be used for the large-scale analysis of maternal gene functions.Maternal gene products accumulated during oogenesis are essential for supporting early developmental processes in both invertebrates and vertebrates. Therefore, understanding their regulatory functions should provide insights into the maternal control of embryogenesis. The CRISPR/Cas9 genome editing technology has provided a powerful tool for creating genetic mutations to study gene functions and developing disease models to identify new therapeutics. However, many maternal genes are also essential after zygotic genome activation; as a result, loss of their zygotic functions often leads to lethality or sterility, thus preventing the generation of maternal mutants by classical crossing between zygotic homozygous mutant adult animals. Although several approaches, such as the rescue of mutant phenotypes through an injection of the wild-type mRNA, germ-line replacement, and the generation of genetically mosaic females, have been developed to overcome this difficulty, they are often technically challenging and time-consuming or inappropriate for many genes that are essential for late developmental events or for germ-line formation. Recently, a method based on the oocyte transgenic expression of CRISPR/Cas9 and guide RNAs has been designed to eliminate maternal gene products in zebrafish. This approach introduces several tandem guide RNA expression cassettes and a GFP reporter into transgenic embryos expressing Cas9 to create biallelic mutations and inactivate genes of interest specifically in the developing oocytes. It is particularly accessible and allows for the elimination of maternal gene products in one fish generation. By further improving its efficiency, this method can be used for the systematic characterization of maternal-effect genes.
Highlights
Maternal gene products accumulated during oogenesis as mRNAs and proteins make an important contribution to early development, such as egg polarity and activation, cytokinesis, germ layer and embryonic axis specification, morphogenetic cell movements, and organogenesis [1,2,3,4,5]
Several methods were used to circumvent this difficulty and allowed for the generation of maternal mutants for zygotic lethal genes. They mainly include the rescue of zygotic defective phenotypes by injecting the wild-type mRNA into fertilized eggs [15], germ-line replacement [16,17], oocyte microinjection in situ (OMIS) combined with gene knockout or knockdown [18], the generation of genetic mosaic females with biallelic mutations in the oocytes [19], conditional gene inactivation by the artificial chromosome-rescue-based knockout (BACK) approach [20], or the production of maternal crispants [21]
The elegant procedure of germ-line replacement transfers zygotic lethal mutations through the germ-line [16], but its success is conditioned by the number of transplanted primordial germ cells (PGCs); it is influenced by the sex bias
Summary
Maternal gene products accumulated during oogenesis as mRNAs and proteins make an important contribution to early development, such as egg polarity and activation, cytokinesis, germ layer and embryonic axis specification, morphogenetic cell movements, and organogenesis [1,2,3,4,5]. Several methods were used to circumvent this difficulty and allowed for the generation of maternal mutants for zygotic lethal genes They mainly include the rescue of zygotic defective phenotypes by injecting the wild-type mRNA into fertilized eggs [15], germ-line replacement [16,17], oocyte microinjection in situ (OMIS) combined with gene knockout or knockdown [18], the generation of genetic mosaic females with biallelic mutations in the oocytes [19], conditional gene inactivation by the artificial chromosome-rescue-based knockout (BACK) approach [20], or the production of maternal crispants [21]. By further improving the efficiency, this oocyte-specific conditional knockout strategy should open the door for the systematic identification and characterization of maternal gene functions during early development
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