| The mRNAs and proteins deposited in the oocytes are termed as maternal factors.Maternal factors are indispensable for oocyte maturation and early embryo development.Over half of the zebrafish genes are maternally expressed.In zebrafish,zygotic genome activation starts from lk-cell stage(3 hpf),before which all of the developmental processes are regulated by maternal factors.Even after zygotic genome activation,maternal fators still play a crucial role in multiple developmental processes,including epiboly,convergence and extension movements and axis formation.Hence eliminating both the maternal and zygotic products is essential for elucidating their functions.Because maternal factors are cell-autonomously produced in primary oocytes,maternal mutants can only be produced from homozygous mutant females.Thus,viable and fertile homozygous mutant female animals are necessary for obtaining maternal mutants.The traditional method for obtaining maternal mutants is through repeated crossing and screening.which is time-consuming and usually needs three generations of time(about nine months).For many genes,the homozygous mutants are lethal or sterile,which prevents the acquisition of maternal mutants.Many new methods were developed to circumvent these obstacles and listed as follows:1,Rescue by exogenous wild-type mRNA of the target gene.2,Germline replacement.3,Oocyte microinjection in situ.4,Making mosaic animals by genome editing in heterozygotes.5,BACK approach.All of these new methods contributed to the study of the maternal function of zebrafish genes.However,all of them remain technically challenging,time-consuming,or less efficient.Therefore,we set to develop a simpler,more rapid,and straightforward method to obtain maternal mutants.CRISPR-Cas9 system has become a powerful tool for genome editing.It facilitated the investigation in many biological fields.CRISPR-Cas9-based conditional knockout has been tested in several species.The strategy to achieve spatially or temporally controlled gene inactivation relies on the transgenesis of the spatiotemporal expression of Cas9 driven by different promoters.However,the application of Cas9-mediated conditional knockout is still limited,probably due to the low reproducibility of genome-editing efficieny,which often leads to incomplete removal of tissue-specific gene products.It will finally result in functional compensation from wild-type or heterozygously mutated cells,thus generating animals without detectable mutant phenotypes.Distinct from tissues in which component cells function as a whole,every oocyte with biallelic maternal gene disruption in the ovary can lead to the generation of a homogeneous maternal mutant animal.Therefore,we reasoned that CRISPR-Cas9-mediated oocyte-specific conditional knockout should still be highly applicable to create maternal mutants,despite the moderate efficiency and mosaic feature of the CRISPR-Cas9 system in transgenesis.Based on this idea,we developed a new approach to obtain maternal mutants through oocyte-specific conditional knockout.In this strategy,transgenic expression of Cas9 and sgRNA in the oocyte was necessary.Firstly,we constructed a transgenic line Tg(zpc:zcas9),in which sufficient Cas9 was expressed and accumulated in the oocyte.Then we constructed the transgenic vector containing the U6 promoter to drive the expression of sgRNA and a ubiquitous GFP expression cassette as a selection marker.After that,we inserted the sgRNA expression plasmid into the genome of Tg(zpc:zcas9)via I-Sce I-mediated transgenesis.The embryos that displayed high and widespread expression of GFP were selected and raised.After sexual maturation,some of the oocytes could express both Cas9 and sgRNA.Thus the gene of interest could be possibly knocked out in the oocytes.If this strategy could work,it would overcome the obstacles of zygotic lethality and save much time.As a proof of principle,we constructed nanog maternal mutants using one sgRNA expression cassette.We obtained 2 F0 females that could give birth to nanog maternal mutants.The average ratio of nanog maternal mutants in GFP-positive F1 embryos was 16.5%.Next,we conducted the same procedures but using three distinct sgRNA expression cassettes,and the ratio was increased to 38.8%.To verify the nanog maternal mutants,we examined the maternal nanog mRNA,Nanog protein and downstream target genes expression.We found that the maternal nanog mRNA and protein were absent,and the expression of downstream target genes was decreased or disrupted.In addition,-injection of nanog wild-type mRNA could significantly rescue the phenotypes of nanog maternal mutants.All of the above results confirmed the presence of nanog maternal mutants.To precisely identify and study the maternal mutants,we developed a new method that could combine the phenotype analysis and the identification of maternal mutant mRNA.Through this method,we linked the mRNA mutation to the nanog maternal mutants.Furthermore,we found that multiple Cas9 RNPs intended to generate large deletions in oocytes.Some of the large deletions spanned the sgRNA sites and others extended beyond them.Few articles have reported this phenomenon in zebrafish,but it is reminiscent of the "On Target Effect(OnTE)" of genome editing in early mammal embryos or embryonic stem cells.To further confirm the feasibility of oocyte-specific conditional knockout for generating maternal mutants and the tendency of creating large deletions by this approach,we tried to construct ctnnb2 maternal mutants using three sgRNA cassettes.We successfully obtained ctnnb2 maternal mutants and verified them by examing the expression of downstream target genes and rescue through injecting wild-type ctnnb2 mRNA.After identifying the mutations of ctnnb2 maternal mRNA,we found that most were large-deletion forms.So we asked the question of whether the deletion bias of genome editing was oocyte-specific.For comparison,we conducted conventional gene knockout using the same sets of sgRNAs targeting nanog or ctnnb2 and a high dose Cas9 protein.After examination,we found that the ratio of large deletions in conventional gene knockout was significantly lower than that in oocyte-specific conditional knockout.Besides,the large deletion beyond the sgRNA sites was absent in conventional gene knockout.Hence,generating large deletions is much more efficient in oocytes comparing to that in somatic cells.After that,we also proved that these large-deletion mutations were hereditable.Rbm24a is a conserved RNA binding protein among different vertebrates.Its expression is restricted in the lens,heart,somites,and otic vesicles.The research on zebrafish rbm24a and mouse Rbm24 mutants demonstrated that deficiency of this gene led to impaired development of the tissues where it is expressed.So Rbm24 is crucial for different developmental processes.From the RNA-Seq data,we found that rbm24a showed maternal expression.Thus we want to examine its maternal function.Through the oocyte-specific conditional knockout technology,we successfully obtained and verified the rbm24a maternal mutants.However,the maternal mutants appear like wild-type embryos,and there was no visible difference between maternal zygotic mutants and zygotic mutants.These results suggest that rbm24a maternal products may be dispensible for zebrafish early morphogenesis.However,we could not exclude the possibility that maternal rbm24a products play a role in other developmental processes that we have not examined during the study.In zebrafish,many paralogous genes can function redundantly.Hence,to study the role of one specific gene,we need to knock out all the paralogous genes simultaneously.If there are paralogous genes,the construction of double maternal mutants was necessary for investigation of their maternal function.This will need at least five generations(15 months).What is worse,the double zygotic mutants are more likely to be lethal than single zygotic mutants.So it is intriguing to test whether our method could be used to construct double maternal mutants.Our previous work proved that the maternal products of dvl2 and dvl3a could be redundantly involved in anteroposterior axis formation as well as convergence and extension movements in zebrafish embryos.So we chose these two genes to test the oocyte-specific conditional knockout.We successfully got the dvl2/dvl3a double maternal mutants in three months and found similar large deletions.It showed the great potential of the oocyte-specific conditional knockout technology in studying the maternal function of paralogous genes.In summary,we developed a new genetic approach for the rapid generation of zebrafish maternal mutants via oocyte transgenic expression of Cas9 and sgRNAs.This approach overcomes the lethality and infertility of homozygous mutants and allows single or double maternal gene knockout.This method tends to generate large deletions in the genome and is feasible and time-saving.Therefore,we can apply it to the functional screening of maternal factors,investigating the functions of paralogous genes,and generating genomic deletions in zebrafish. |
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