Name: Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish (Video) | JoVE Journal
Uploaded: 2025-07-22T14:30:00.000+00:00
Duration: 9 min 17 s
Description: Learn an improved oocyte-specific conditional knockout method in zebrafish using zpc:cas9 knock-in lines and Tol2-mediated sgRNA delivery. This protocol enables rapid, efficient generation of maternal mutants for studying developmental genetics.

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5 minutes

We thank Jianlin Shen, Yiteng Xu, and Qingqing Wei from the Core Facility and Service Platform, School of Lifesciences. This work was supported by the National Natural Science Foundation of China (grants 32170816, 32370860, 32450630, and 31871451), Program of Outstanding Middle-aged and Young Scholars of Shandong University, the Taishan Scholars of Shandong Province.

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<li>Ciruna, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+maternal-zygotic+mutant+zebrafish+by+germ-line+replacement%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Production of maternal-zygotic mutant zebrafish by germ-line replacement.</a> Proc Natl Acad Sci U S A. 99 (23), 14919-14924 (2002).</li>
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<li>Wu, X., Shen, W., Zhang, B., Meng, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+genetic+program+of+oocytes+can+be+modified+in+vivo+in+the+zebrafish+ovary%5BTitle%5D)+AND+%22J+Mol+Cell+Biol%22%5BJournal%5D)">The genetic program of oocytes can be modified in vivo in the zebrafish ovary.</a> J Mol Cell Biol. 10 (6), 479-493 (2018).</li>
<li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+zebrafish+maternal+mutants+via+oocyte-specific+knockout+system%5BTitle%5D)+AND+%22Bio+Protoc%22%5BJournal%5D)">Generation of zebrafish maternal mutants via oocyte-specific knockout system.</a> Bio Protoc. 14 (21), e5092(2024).</li>
<li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+generation+of+maternal+mutants+via+oocyte+transgenic+expression+of+CRISPR-Cas9+and+sgRNAs+in+zebrafish%5BTitle%5D)+AND+%22Sci+Adv%22%5BJournal%5D)">Rapid generation of maternal mutants via oocyte transgenic expression of CRISPR-Cas9 and sgRNAs in zebrafish.</a> Sci Adv. 7 (32), abg4243(2021).</li>
<li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intron+targeting-mediated+and+endogenous+gene+integrity-maintaining+knockin+in+zebrafish+using+the+CRISPR%2FCas9+system%5BTitle%5D)+AND+%22Cell+Res%22%5BJournal%5D)">Intron targeting-mediated and endogenous gene integrity-maintaining knockin in zebrafish using the CRISPR/Cas9 system.</a> Cell Res. 25 (5), 634-637 (2015).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rbm24a+dictates+mRNA+recruitment+for+germ+granule+assembly+in+zebrafish%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Rbm24a dictates mRNA recruitment for germ granule assembly in zebrafish.</a> EMBO J. 44 (11), 3121-3149 (2025).</li>
<li>Xia, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T5+exonuclease-dependent+assembly+offers+a+low-cost+method+for+efficient+cloning+and+site-directed+mutagenesis%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">T5 exonuclease-dependent assembly offers a low-cost method for efficient cloning and site-directed mutagenesis.</a> Nucleic Acids Res. 47 (3), e15(2019).</li>
<li>Yin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multiplex+conditional+mutagenesis+using+transgenic+expression+of+Cas9+and+sgRNAs%5BTitle%5D)+AND+%22Genetics%22%5BJournal%5D)">Multiplex conditional mutagenesis using transgenic expression of Cas9 and sgRNAs.</a> Genetics. 200 (2), 431-441 (2015).</li>
<li>Shao, M., Cheng, X. N., Liu, Y. Y., Li, J. T., Shi, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transplantation+of+zebrafish+cells+by+conventional+pneumatic+microinjector%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">Transplantation of zebrafish cells by conventional pneumatic microinjector.</a> Zebrafish. 15 (1), 73-76 (2018).</li>
<li>Siegfried, K. R., Nüsslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Germ+line+control+of+female+sex+determination+in+zebrafish%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Germ line control of female sex determination in zebrafish.</a> Dev Biol. 324 (2), 277-287 (2008).</li>
<li>Aharon, D., Marlow, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sexual+determination+in+zebrafish%5BTitle%5D)+AND+%22Cell+Mol+Life+Sci%22%5BJournal%5D)">Sexual determination in zebrafish.</a> Cell Mol Life Sci. 79 (1), 8(2021).</li>
<li>Ye, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Abundance+of+early+embryonic+primordial+germ+cells+promotes+zebrafish+female+differentiation+as+revealed+by+lifetime+labeling+of+germline%5BTitle%5D)+AND+%22Mar+Biotechnol+%28NY%29%22%5BJournal%5D)">Abundance of early embryonic primordial germ cells promotes zebrafish female differentiation as revealed by lifetime labeling of germline.</a> Mar Biotechnol (NY). 21 (2), 217-228 (2019).</li>
<li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+formation+of+primordial+germ+cells+from+zebrafish+blastomeres+by+germplasm+factors%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Induced formation of primordial germ cells from zebrafish blastomeres by germplasm factors.</a> Nat Commun. 14 (1), 7918(2023).</li>
<li>Thermes, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((I-SceI+meganuclease+mediates+highly+efficient+transgenesis+in+fish%5BTitle%5D)+AND+%22Mech+Dev%22%5BJournal%5D)">I-SceI meganuclease mediates highly efficient transgenesis in fish.</a> Mech Dev. 118 (1-2), 91-98 (2002).</li>
<li>Bertho, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+dazl+regulates+cystogenesis+and+germline+stem+cell+specification+during+the+primordial+germ+cell+to+germline+stem+cell+transition%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Zebrafish dazl regulates cystogenesis and germline stem cell specification during the primordial germ cell to germline stem cell transition.</a> Development. 148 (7), dev.187773(2021).</li>
<li>Houwing, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+role+for+Piwi+and+piRNAs+in+germ+cell+maintenance+and+transposon+silencing+in+Zebrafish%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish.</a> Cell. 129 (1), 69-82 (2007).</li>
<li>Shao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Vegetally+localised+Vrtn+functions+as+a+novel+repressor+to+modulate+bmp2b+transcription+during+dorsoventral+patterning+in+zebrafish%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Vegetally localised Vrtn functions as a novel repressor to modulate bmp2b transcription during dorsoventral patterning in zebrafish.</a> Development. 144 (18), 3361-3374 (2017).</li>
</ol>

This protocol has been granted as a patent by the China National Intellectual Property Administration, which will relieve the restricted usage of the method after gaining the permission of the authors. The authors declare that they have no other competing or financial interests.

Lethal zygotic mutations hinder analysis of a gene&#39;s maternal contribution. Alternative strategies, most notably germline replacement and Oocyte Microinjection In Situ (OMIS), are technically demanding6,8. In cell transplantation process of germline replacement, the recipient embryo receives very few PGCs, often preventing normal female development16,17,18. Using ind...

The early embryonic development of vertebrates relies heavily on the RNA and proteins stored within the egg, collectively referred to as maternal factors. These maternal products are predominantly synthesized during the diplotene stage of meiosis I1,2,3. To investigate their functional roles, it is essential to generate homozygous mutant female individuals, as only homozygous mutation in the oocytes can fully deplete these maternal factors. However, obtaining such homozygous mutant females becomes challenging when zygotic homozygous mutations result in lethality or sterility4,5. This poses a significant barrier to elucidating the functions of maternal products encoded by zygotic lethal or sterile genes. To overcome this technical limitation, several innovative approaches have been developed6,7,8. Nevertheless, these techniques often suffer from prolonged experimental timelines or substantial technical challenges.
In our previous work, we also developed a conditional knockout strategy that allows for the generation of maternal mutants within a single generation9,10. The system comprises two key components: Tg(zpczcas9) transgenic zebrafish and an I-SceI transgenic plasmid system harboring 3-4 sgRNA expression cassettes. Leveraging this system, we successfully achieved conditional knockout of maternal factors in oocytes9,10, but there is still room for improvement. First, the genome editing efficiency of the previously reported Tg(zpczcas9) transgenic line was not stable in maintaining the transgenic expression of Cas9. Furthermore, since this transgenic line was generated using the Tol2 transposon system, Tol2 transposition cannot be further used for the integration of the sgRNA expression cassette; instead, an I-SceI-mediated transgenesis is required. However, this approach is less efficient than Tol2 and is also more susceptible to transgenerational silencing.
To overcome these limitations, we sought to establish a knock-in line enabling robust and stable oocyte-specific Cas9 expression. This line allows the use of the Tol2 transposon system for the highly efficient delivery of sgRNA expression plasmids in zebrafish. Utilizing an intron-based genome editing strategy11, we successfully generated a zpccas9 knock-in line with the integrated site in the last intron of the rbm24a gene (Figure 1A,B). We chose this locus as a candidate genomic safe harbor for maternal transgene expression on the basis of two independent observations. 1) Harmless integration: in our recent study12, homozygous knock-in fish carrying exogenous DNA at this site remained fully viable and fertile. 2) Robust expression: when a zpccas9 cassette was inserted at the same locus, Cas9 was expressed at high levels, yielding consistently strong genome-editing efficiencies across generations (Figure 1C). As a core germ plasm component, the RNA-binding protein Rbm24a partners with Buc to incorporate germ plasm RNAs into germ granules. Consequently, maternal Rbm24a deficiency leads to a complete absence of primordial germ cells (PGCs)12. Here, by targeting rbm24a as an example in our recent work, we provide an improved video protocol for generating and characterizing maternal mutants for zygotic lethal genes.

<table><tbody><tr><td>2x M5 HiPer Taq PCR mix</td><td>Mei5bio</td><td>Cat#MF001-BD-100</td><td></td></tr><tr><td>Agarose</td><td>MDbio</td><td>Cat#A006L</td><td></td></tr><tr><td>AxyPrep Plasmid Miniprep Kit</td><td>Axygen</td><td>Cat#AP-MN-P-4</td><td></td></tr><tr><td>Capillary electrophoresis instrument (Qsep400)</td><td>BiOptic</td><td>N/A</td><td></td></tr><tr><td>DIG-labeling mix</td><td>Roche</td><td>Cat#11277073910</td><td></td></tr><tr><td>DNase I</td><td>Roche</td><td>Cat#4716728001</td><td></td></tr><tr><td>DTT</td><td>Thermo Fisher Scientific</td><td>Cat#R0861</td><td></td></tr><tr><td>First-strand cDNA synthesis kit</td><td>TransGen Biotech</td><td>Cat#AT301</td><td></td></tr><tr><td>GenCrispr NLS-Cas9-NLS Nuclease</td><td>GenScript</td><td>Cat#Z03389</td><td></td></tr><tr><td>Glass capillaries (1.0 x 100 mm)</td><td>Rantai Educational Equipment Factory</td><td>Cat#OS-2B</td><td></td></tr><tr><td>Glycogen</td><td>BBI Life Science</td><td>Cat#28985</td><td></td></tr><tr><td>GraphPad Prism 9</td><td>GraphPad</td><td>https://www.graphpad.com/</td><td></td></tr><tr><td>ImageJ</td><td>Open source</td><td>https://imagej.net/software/fiji/</td><td></td></tr><tr><td>Low-melting agarose</td><td>Biotech</td><td>Cat#CA1351</td><td></td></tr><tr><td>Microforge (MF2)</td><td>NARISHIGE</td><td>N/A</td><td></td></tr><tr><td>Micro-spectrophotometer (Nano-300)</td><td>ALLSHENG</td><td>N/A</td><td></td></tr><tr><td>OlyVIA</td><td>OLYMPUS</td><td>N/A</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Gibco</td><td>Cat#15140122</td><td></td></tr><tr><td>Pico-Liter Injector (PLI-100A)&amp;nbsp;</td><td>Harvard Apparatus</td><td>N/A</td><td></td></tr><tr><td>Pointed tweezers</td><td>WPI</td><td>Cat#500341</td><td></td></tr><tr><td>Pointed tweezers</td><td>&amp;nbsp;WPI&amp;nbsp;</td><td>500341</td><td></td></tr><tr><td>Puller (PC-100)</td><td>NARISHIGE</td><td>N/A</td><td></td></tr><tr><td>RiboLock RNase Inhibitor</td><td>Thermo Fisher Scientific</td><td>Cat#EO0381</td><td></td></tr><tr><td>rNTP</td><td>Thermo Fisher Scientific</td><td>Cat#R0481</td><td></td></tr><tr><td>Silicon hydroxyl magnetic beads</td><td>Sangon Biotech</td><td>Cat#B518720-0001</td><td></td></tr><tr><td>T7 RNA polymerase</td><td>NEB</td><td>Cat#M0251L</td><td></td></tr><tr><td>Zold</td><td>WPI</td><td>Cat# z-molds</td><td></td></tr></tbody></table>

generation of maternal mutants using zpc:cas9 knock-in zebrafish

Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish

Oogenesis and early embryonic development are critically dependent on maternally derived mRNAs and proteins. Eliminating these maternal factors necessitates homozygous mutations in female zebrafish, often resulting in lethal or infertile phenotypes, which prevent the acquisition of maternal mutant embryos. Our previous work introduced a rapid approach to bypass zygotic lethality through oocyte-specific genome editing. However, the previously reported cas9 transgene exhibits instability and undergoes gradual silencing over successive generations. Furthermore, the presence of Tol2 transposable elements flanking the zpc:cas9 cassette in this line hinders the potential for further sgRNA transgenesis using Tol2 system, which is currently the most efficient transgenic system in zebrafish. Consequently, there is a critical need for a Tol2-free zebrafish line that ensures stable and robust oocyte-specific Cas9 expression. Here, we present a line with zpccas9 knock-in at the rbm24a locus that addresses this requirement. Using this enhanced tool, we provide a pipeline for the rapid generation of maternal mutants of genes with zygotically lethal mutant phenotypes within the zebrafish model.

Rapid generation of rbm24a maternal mutants 
We constructed a transgenic vector enabling expression of a maternal BFP marker and four highly efficient sgRNAs targeting the rbm24a coding sequence. Following the introduction of this vector into homozygous rbm24a-RFP KIzpc:cas9 embryos via Tol2 transposition, Mrbm24a was easily and rapidly identified among BFP-positive F1 embryos due to the absence of Rbm24a-RFP pro...

Here, we describe a protocol for generating maternal mutant that couples a stable zpc:cas9 knock-in line with Tol2-mediated delivery of sgRNA expression cassettes.

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

In this video, we present an improved oocyte-specific condition knockout method in zebrafish, offering a versatile platform for studying maternal factors with zygotic mutations resulting in lethality or sterility. Current maternal knockout method are either technically challenging or time-consuming. We had previously reported zpc:cas9 transgene is transcriptionally silenced over generations.Using zpc:cas9 knock-in line, we have established a robust platform for generating conditional knockout during oogenesis, enabling the rapid and highly-efficient production of maternal mutants. To begin, collect embryos that have been spawned by homozygous rbm24a RFPKI zpc:cas9 fish. Combine one microliter of PGG dust EB rbm24a 4sGRNA with one microliter of Tol2 transposase messenger RNA to prepare the injection mixture.Dilute both the plasmid and the Tol2 messenger RNA directly in pure water. Now place a glass coverslip in a 90-millimeter dish. Align the embryos along the edge of the coverslip, and gently use a pipette to remove any excess water.Then inject two nanoliters of the prepared mixture into the blastodisc of each one-cell stage embryo. Gently rinse the injected embryos with blue water, and transfer them to another dish. Place the dish in an incubator set to 28 degrees Celsius for cultivation.At 24 hours post-fertilization, select embryos that show robust and ubiquitous blue fluorescent protein expression. At four days post-fertilization, raise only the embryos that maintain strong transgenic fluorescent signals. Collect embryos produced from mating mosaic transgenic founder females with wild-type males.Using a fluorescent stereomicroscope, pick up the blue fluorescent protein-positive embryos at the one-cell stage. Then fix the embryos at appropriate stages in 4%paraformaldehyde at four degrees Celsius overnight. To prepare a 1.5%OGER solution, weigh and combine OGER powder in 1/3-strength Ringer's buffer and boil until fully dissolved.Pour the melted OGER solution into a 90-millimeter Petri dish and insert a Z-mold into the molten agarose. Once the agarose has solidified, gently remove the mold to create a ready-to-use plate. Next, adjust the settings on the needle puller to use two light weights, and select the step one procedure.Load the glass capillaries into the puller to make melt-sealed needles. Using pointed tweezers, trim the capillary tips to a 30 to 40-micrometer diameter. Then use a microforge to form a spike at the tip, which will aid in embryo penetration and reduce tissue damage.Mate the putative female founders with wild-type zebra fish and collect embryos. At the one-cell stage, isolate embryos that are positive for blue fluorescent protein using a fluorescent stereomicroscope. At three hours post-fertilization, incubate the embryos in pronase, dissolved in one third strength Ringer's buffer for 10 minutes with gentle pipetting to dechorionate them.Carefully transfer the dechorionated embryos onto the prepared argarose plate flooded with 1/3 Ringer's buffer, supplemented with penicillin streptomycin. Reorient the embryos so that the blastomere is facing toward the capillary tip for cell aspiration. Now use a microinjector to aspirate 20 to 40 cells from each embryo by gently reducing the equilibrium pressure to counter the capillary aspiration force.Increase the equilibrium pressure to release the aspirated cells into two microliters of deionized water at the edge of 1.5-milliliter Eppendorf tubes. Then add 200 microliters of RNA extraction reagent to the tube to wash down the aspirated cells, and place the tube on ice for temporary storage. Keep the embryos after cell aspiration on the agarose plate until aberrant phenotypes are visualized.Next, add 40 microliters of chloroform to the cells from selected embryos in the extraction reagent and mix gently. Then centrifuge the mixture at 12, 000 G for one minute at room temperature. After collecting the supernatant, add one microliter of glycogen solution.Then add an equal volume of isopropanol based on the supernatant volume and mix thoroughly. Incubate the tube at minus 20 degrees Celsius for 30 minutes to allow RNA precipitation. Now centrifuge the sample at 12, 000 G for 10 minutes at four degrees Celsius and discard the supernatant.Then to wash the RNA pellet twice, add 500 microliters of 70%ethanol and centrifuge at 12, 000 G for one minute at four degrees Celsius. After discarding the supernatant, open the tube lid to let the pellet dry at room temperature for five minutes. Add seven microliters of water to dissolve the RNA pellet.Perform reverse transcription using a First Strand cDNA synthesis kit, then conduct PCR and Sanger sequencing to analyze mutations in maternal mutant embryos. Maternal rbm24a mutants were identified by the absence of rbm24a RFP, while other maternal mutants were detected by specific phenotypes or cell aspiration-based genotyping. Among BFP-positive embryos, those lacking RFP signal were identified as maternal rbm24a mutants.In situ hybridization using NANOS3 probe revealed that Mrbm24a embryos failed to recruit germ plasm mRNAs to germ granules at the four-cell stage, and lacked primordial germ cells at 24 hours post-fertilization. All adult Mrbm24a males failed to fertilize eggs spawned by wild-type females, showing anatomical abnormalities with fatty deposits replacing normal testes. Histological analysis confirmed the complete absence of germ cells and spermatozoa in Mrbm24a testes.Western blot analysis confirmed nearly undetectable levels of rbm24a protein in BFP-positive RFP-negative embryos. RT-qPCR results showed significantly lower rbm24a transcript levels in RFP-negative BFP-positive embryos relative to controls. RT-PCR and Sanger sequencing revealed large deletions and indels in both RFP-negative BFP-positive and RFP-positive BFP-positive embryos with wild-type transcripts detectable only in RFP-positive embryos.A maternal GFP marker and nanog sgRNAs were introduced to generate maternal nanog mutants, and GFP-positive embryos showed a range of dorsalized phenotypes. Germline transmission rates ranged from 12%to 32%among GFP-positive transgenic lines. A dorsal phenotype consistent with maternal nanog mutants was observed in 22%to 60%of GFP-positive embryos.

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Learn an improved oocyte-specific conditional knockout method in zebrafish using zpc:cas9 knock-in lines and Tol2-mediated sgRNA delivery. This protocol enables rapid, efficient generation of maternal mutants for studying developmental genetics.

Generation of Maternal Mutants Using zpc:cas9 Knock-in Zebrafish

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