generation-of-maternal-mutants-using-zpccas9-knock-in-zebrafish

Generation of Maternal Mutants Using <em>zpc:cas9</em> Knock-in Zebrafish

Oogenesis and early embryonic development are critically dependent on maternally derived mRNAs and proteins. Eliminating these maternal factors ...

Shandong University

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(:) with oocyte-specific  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  and  simultaneously and introduced this construct to the genome of Tg(:) by meganuclease I- I. As expected, we confirmed the existence of M;M embryos with strong defective convergence and extension movement during gastrulation among outcrossed GFP positive F1 offspring. The MZ;MZ embryos were also obtained by crossing the mutant carrying mosaic F0 female with ; 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.

A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish.

Lens transparency is established by abundant accumulation of crystallin proteins and loss of organelles in the fiber cells. It requires an efficient translation of lens messenger RNAs (mRNAs) to overcome the progressively reduced transcriptional activity that results from denucleation. Inappropriate regulation of this process impairs lens differentiation and causes cataract formation. However, the regulatory mechanism promoting protein synthesis from lens-expressed mRNAs remains unclear. Here we show that in zebrafish, the RNA-binding protein Rbm24 is critically required for the accumulation of crystallin proteins and terminal differentiation of lens fiber cells. In the developing lens, Rbm24 binds to a wide spectrum of lens-specific mRNAs through the RNA recognition motif and interacts with cytoplasmic polyadenylation element-binding protein (Cpeb1b) and cytoplasmic poly(A)-binding protein (Pabpc1l) through the C-terminal region. Loss of Rbm24 reduces the stability of a subset of lens mRNAs encoding heat shock proteins and shortens the poly(A) tail length of  mRNAs encoding lens structural components, thereby preventing their translation into functional proteins. This severely impairs lens transparency and results in blindness. Consistent with its highly conserved expression in differentiating lens fiber cells, the findings suggest that vertebrate Rbm24 represents a key regulator of cytoplasmic polyadenylation and plays an essential role in the posttranscriptional control of lens development.

Rbm24 controls poly(A) tail length and translation efficiency of  mRNAs in the lens via cytoplasmic polyadenylation.

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(:) embryos, we efficiently obtained maternal  and  mutants among GFP-positive F 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.

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

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() 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.

Generation of Zebrafish Maternal Mutants via Oocyte-Specific Knockout System.

Double-stranded RNAs (dsRNAs), known as conserved pathogen-associated molecular patterns, activate the integrated stress response via interferon-induced protein kinase R (PKR), leading to global translation inhibition. However, the interferon system is inactive in pluripotent cells, leaving the mechanisms of dsRNA sensing and translational control unclear. In this study, we utilized early zebrafish embryos as a model of pluripotent cells and discovered a PKR-independent blockage of translation initiation by dsRNA stimulation. Prkra dimer was identified as the genuine dsRNA sensor. Upon dsRNA binding, the dimerized dsRNA-binding domain 3 of Prkra becomes activated to sequester the eIF2 complexes from the translation machinery, inhibiting global protein synthesis. This distinctive embryonic stress response restricts RNA virus replication in zebrafish embryos, is conserved in mouse embryonic stem cells, and compensates PKR function in differentiated cells. Therefore, the Prkra-mediated dsRNA sensing and translation control may serve as a common strategy for cells to adapt to environmental stresses.

Prkra dimer senses double-stranded RNAs to dictate global translation efficiency.

The germ granules are ribonucleoprotein (RNP) biomolecular condensates that determine the fate of primordial germ cells (PGCs) and serve as a model for studying RNP granule assembly. Here, we show that the maternal RNA-binding protein Rbm24a is a key factor governing the specific sorting of mRNAs into germ granules. Mechanistically, Rbm24a interacts with the germ plasm component Buc to dictate the specific recruitment of germ plasm mRNAs into phase-separated condensates. Germ plasm particles lacking Rbm24a and mRNAs fail to undergo kinesin-dependent transport toward cleavage furrows where small granules fuse into large aggregates. Therefore, the loss of maternal Rbm24a causes a complete degradation of the germ plasm and the disappearance of PGCs. These findings demonstrate that the Rbm24a/Buc complex functions as a nucleating organizer of germ granules, highlighting an emerging mechanism for RNA-binding proteins in reading and recruiting RNA components into a phase-separated protein scaffold.

Article	Year
A Time-Saving Strategy to Generate Double Maternal Mutants by an Oocyte-Specific Conditional Knockout System in Zebrafish. Biology\| PubMed ID: 34440009	2021
Rbm24 controls poly(A) tail length and translation efficiency of mRNAs in the lens via cytoplasmic polyadenylation. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 32170011	2020
Rapid generation of maternal mutants via oocyte transgenic expression of CRISPR-Cas9 and sgRNAs in zebrafish. Science advances\| PubMed ID: 34362733	2021
Generation of Zebrafish Maternal Mutants via Oocyte-Specific Knockout System. Bio-protocol\| PubMed ID: 39525965	2024
Prkra dimer senses double-stranded RNAs to dictate global translation efficiency. Molecular cell\| PubMed ID: 40280134	2025
Rbm24a dictates mRNA recruitment for germ granule assembly in zebrafish. The EMBO journal\| PubMed ID: 40281355	2025

Yizhuang Zhang

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