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

Summary

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.

Abstract

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.

Introduction

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 I^1,2,3. 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 sterility^4,5. 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 developed^6,7,8. 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 generation^9,10. 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 oocytes^9,10, 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.

Protocol

The experiments were carried out in accordance with ARRIVE guidelines and were ethically approved by Shandong University's Institutional Animal Care Committee (Approval No. SYDWLL-2021-15). The overview of this protocol is shown in Figure 2.

1. Generation of the rbm24a-RFP ^zpc:cas9 knock-in line

1. Screen of sgRNA for knock-in
   1. Select and synthesize knock-in sgRNA candidates
      1. Submit the sequence of last intron of rbm24a to CRISPRscan (http://www.crisprscan.org) to identify optimal sgRNA candidates exhibiting both high CRISPRscan scores (indicative of on-target efficiency) and low CFD scores (reflecting off-target effects). Synthesize the long oligo for each sgRNA (sgRNA oligo).
         NOTE: CRISPRscan generates sgRNA oligos containing essential functional elements, including the T7 promoter sequence, the 20-nucleotide sgRNA target sequence, and a 16-nucleotide sgRNA scaffold sequence. Here, we selected an sgRNA with the following sequence: 5′-taatacgactcactataGGGCTGCTGTCATGTTGG
         GTgttttagagctagaa-3′ (T7 promoter lowercase, 20-nt guide uppercase, partial scaffold lowercase).
      2. To obtain the DNA template for sgRNA synthesis, prepare 50 µL of Fill-in PCR reaction: 2x Taq Master mix 25 µL; sgRNA primer (10 µM) 2 µL; Universal primer (10 µM) 2 µL; Deionized H₂O 21 µL.
         NOTE: The sequences of all primers are shown in the Table of Materials.
      3. Mix thoroughly and transfer the reaction mixture to a preprogrammed thermal cycler and initiate the amplification protocol using the cycling parameters as follows: 1 cycle at 94 °C for 3 min, 35 cycles (94 °C for 15 s, 50 °C for 15 s, 72 °C for 30 s) and 1 cycle at 72 °C for 5 min.
      4. Prepare the reaction mixture for sgRNA synthesis in a 1.5 mL microcentrifuge tube; combine all components at room temperature in the following order: 11 µL of distilled water, 2 µL of ATP/GTP/CTP/UTP (10 mM each), 10 µL of PCR product, 4 µL of 10x RNA polymerase reaction buffer, 2 µL of T7 RNA polymerase, and 1 µL of RNase inhibitor. The total reaction volume is 40 µL. Mix thoroughly by flicking and perform brief centrifugation.
      5. Incubate at 37 °C for 1.5 h. Add 1 µL of DNase I, mix by flicking, and centrifuge briefly. Incubate at 37 °C for 15 min to ensure complete DNA template removal.
      6. Add 5 µL of silica magnetic beads, followed by 120 µL (4x volume) of RNA binding buffer (Gu-HCl 1 M 9.55 g, Tween-20 0.05%, ethanol 100 mL). Mix thoroughly by pipetting. Incubate on a thermomixer at 1,500 rpm for 10 min at room temperature.
      7. Place the tube on a magnetic stand and carefully remove the supernatant using either a pipette or vacuum system.
      8. Add 400 µL of 90% ethanol and vortex to resuspend the beads. Place the tube on the magnetic stand and discard the supernatant.
      9. Repeat the ethanol wash steps to complete a total of two wash cycles.
      10. Remove residual supernatant and air-dry the open tube on a 50 °C heating block for 10 min.
      11. Add 15 µL of nuclease-free water and incubate on a thermomixer at 1,500 rpm for 5 min at room temperature.
      12. Place the tube on the magnetic stand and transfer the eluate to a fresh microcentrifuge tube.
      13. Confirm the 98 bp sgRNA band by agarose electrophoresis. Quantify RNA concentration by micro-spectrophotometer and agarose electrophoresis, label clearly, and store at -20 °C.
   2. Test sgRNA efficiency
      1. To determine the actual efficiency of sgRNAs, prepare the ribonucleoprotein (RNP) complex for microinjection. Mix 1 µL of each sgRNA (50 ng/µL final concentration) with 1 µL of Cas9 protein (100 ng/µL final concentration) on ice by pipetting. Deliver 2 nL of the RNP complex into each embryo at one-cell stage by microinjection.
      2. At 24 h post fertilization (hpf), collect 10 embryos for each injected Cas9 RNP into a 1.5 mL microcentrifuge tube, and carefully remove excess medium. Add 100 µL of 50 mM NaOH (10 µL per embryo) and incubate at 95 °C for 15 min using a heating block or thermocycler. Neutralize the lysate by adding 10 µL of 1 M Tris-HCl (pH 7.0).
      3. Use the neutralized embryo lysate directly as template for PCR amplification. Design primers to amplify a fragment containing the target site with ≥100 bp flanking sequences on both sides. Verify PCR products by agarose gel electrophoresis and purify using gel extraction when necessary. Perform Sanger sequencing of the amplified products.
      4. CRISPR editing efficiency quantification: analyze sequencing chromatograms using the ICE (Inference of CRISPR Edits) analysis tool (https://ice.synthego.com/#/). Normalize editing efficiency against uninjected control samples following protocols recommended by the web site.
2. Construction of a donor plasmid for knock-in
   1. Use the established TEDA (T5 exonuclease DNA assembly) methodology to perform the ligation reaction¹³.
   2. Employ a three-fragment Gibson assembly strategy for the construction of the rbm24a-RFP KI^zpc:cas9 knock-in donor plasmid. The first fragment includes a 1,704 bp 5' homology arm encompassing the genomic region immediately upstream of the rbm24a stop codon. The second is the GFP coding sequence. The third contains a 2,274 bp 3' homology arm downstream of the rbm24a stop codon, followed by the zpccas9 expression cassette positioned in the 3' direction distal to the rbm24a 3'-UTR.
   3. Assemble the three fragments into the ApaI site of pBluescript SK- vector using Gibson assembly.
   4. Verify the final plasmid through restriction digestion and Sanger sequencing.
3. Microinjection and selection of embryos with early integration
   1. Prepare the injection mixture containing 12 pg of rbm24a-RFP KI^zpc:cas9 knock-in donor plasmid, 200 pg of sgRNA, and 600 ng of recombinant Cas9 protein in nuclease-free water. Deliver 1-2 nL of the mixture into the blastodisc of one-cell stage embryos using standard microinjection techniques. At 30 hpf, screen embryos for RFP expression patterns that mimic the endogenous expression of rbm24a, which indicates integration of the donor plasmid as early as the cleavage stage. Select these embryos with early integration for breeding, which showed RFP signal in the lens, heart, skeletal muscle, and inner ear hair cells (Figure 3A,B).
      NOTE: Purify the plasmid by phenol-chloroform extraction before injection to eliminate any residual RNases introduced during plasmid preparation.
   2. Analyze the expression patterns of maternal Rbm24a-RFP in F1 generation embryos derived from rbm24a-RFP KI^zpc:cas9 incrosses (Figure 4A,B).
4. Assessment of Cas9 protein expression and editing efficiency
   1. To assess Cas9 editing efficiency, microinject 200 pg bmp2b sgRNA into 1-cell rbm24a-RFP KI^zpc:cas9 embryos (Figure 1C,D).
   2. To evaluate the stability of Cas9 editing, quantify the number of embryos exhibiting dorsalization phenotypes and perform Sanger sequencing analysis to assess editing efficiency (Figure 5A-D).
      NOTE: Although we have not detected any reduction in Cas9 expression over three generations, we still recommend verifying the editing efficiency in every generation of this knock-in line.

2. sgRNA transgenesis in rbm24a-RFP KI ^{zpc cas9} line

1. Select and synthesize sgRNAs for rbm24a knockout.
   1. Select and synthesize sgRNA candidates as described in step 1.1.1.
      NOTE: It is crucial to select sgRNAs that target the anterior half of the coding region to ensure a highly efficient loss of function. Additionally, if critical protein domains are identified in the C-terminal region, sgRNAs targeting the 3' end of the coding sequence are also acceptable. CRISPRscan generates sgRNA oligos containing essential functional elements, including the T7 promoter sequence, the 20-nucleotide sgRNA target sequence, and a 16-nucleotide sgRNA scaffold sequence. To achieve an effective maternal gene knockout, it is advisable to design 6-8 candidates to reliably identify 3-4 highly efficient sgRNAs. A detailed protocol can be found in a previous paper⁹.
   2. Test the sgRNA efficiency in early zebrafish embryo as described in step 1.1.2.
      NOTE: sgRNAs with an editing efficiency exceeding 50% are preferred.
2. Construction of sgRNA-expressing transgenic plasmid
   NOTE: To maximize editing efficiency, we recommend using four distinct, non-overlapping sgRNAs, each separated by at least 36 bp. These sgRNAs are individually ligated into pU6x:sgRNA vectors (pU6x:sgRNA #1, #2, #3, #4), each harboring a unique U6 promoters along with the 3' scaffold¹⁴. Subsequently, these four U6 vectors, each containing an sgRNA expression cassette, are assembled into the pGGDestISceIEB-4sgRNA destination vector via Golden Gate ligation.
   The pU6x:sgRNA vectors should be selected sequentially. For example, if cloning three sgRNAs, use vectors #1 to #3; if cloning four sgRNAs, use vectors #1 to #4.
   1. Subcloning sgRNA target sequence into pU6x:sgRNA vectors
      1. According to the sequence of selected sgRNA, synthesize the annealing primers: Forward primer: TTCGN(19); Reverse primer: AAAC[N(19)]*. Where GN(19) is the 20 nt target sequence of sgRNA. [N(19)]* represents the reverse complement sequence of the 19 nt of the sgRNA target in the 3' direction.
      2. To anneal the primers, prepare the mixture as follows: 1 µL of forward primer (100 µM), 1 µL of reverse primer (100 µM), 2 µL of 10x buffer, and 16 µL of deionized H₂O to reach a total volume of 20 µL. Mix thoroughly.
         NOTE: Sequences of all primers were shown in the Table of Materials.
      3. Place the mixture in a thermocycler and run the program as follows: 1 cycle at 95 °C for 15 min, then gradually cool to 50 °C at a controlled ramp rate of 0.1 °C/s, and maintain this temperature for 10 min; hold at 4 °C for 10 min.
      4. Prepare the reaction mixture for cloning the annealed sgRNA target sequence into pU6x-sgRNA vectors as outlined: 1 µL of 10x restriction enzyme buffer, 1 µL of 10x T4 ligase buffer, 0.3 µL of T4 ligase, 0.3 µL of BsmBI, 0.2 µL of PstI, 0.2 µL of SalI, 1 µL of annealed sgRNA primer, 5 µL of pU6x:sgRNA#x (20 ng/µL), and 1 µL of deionized H₂O, adjusting the final reaction volume to 10 µL.
      5. Thoroughly mix the reaction components and run the following thermocycler program: 6 cycles (37 °C for 20 min, 16 °C for 15 min), 1 cycle at 37 °C for 10 min, 1 cycle at 55 °C for 15 min, 1 cycle at 80 °C for 15 min.
      6. For transformation, thaw DH5α competent cells on ice, gently mix 50 µL of the cells with the ligation mixture by flicking the tube, and incubate the mixture on ice for 30 min. Perform a heat shock at 42 °C for 60 s, then immediately transfer the tube back to ice for 2 min. Spread the transformed cells onto an LB plate containing 100 µg/mL spectinomycin and incubate the plate upside down at 37 °C for at least 16 h to allow colony formation.
      7. For colony PCR, pick each bacterial colony with a pipette tip and transfer into 10 µL of deionized H₂O. Resuspend the colony thoroughly by pipetting up and down to ensure complete dispersion for downstream PCR analysis. Prepare the PCR reaction mixture by combining 5 µL of 2x Taq Mix, 2 µL of colony liquid, 0.4 µL each of M13 forward primer (10 µM) and reverse primer (10 µM), and 2.2 µL of deionized H₂O, yielding a total reaction volume of 10 µL.
      8. Run the program as shown below: 1 cycle at 94 °C for 3 min, 23 cycles (94 °C for 15 s, 55 °C for 15 s, 72 °C for 30 s) , and 1 cycle at 72 °C for 5 min.
      9. Subject the PCR products to electrophoresis. Select the positive colony with the expected size of 482 bp (Figure 6).
      10. Inoculate the remaining colony liquid into 3 mL of LB medium supplemented with 100 µg/mL spectinomycin. Incubate at 37 °C with shaking at 200 rpm for 14 h.
      11. Extract plasmids from the bacterial culture using the Plasmid Miniprep Kit according to the manufacturer's instructions. Measure the plasmid concentration using a microspectrophotometer.
      12. Subject the extracted plasmids to Sanger sequencing using the M13 forward primer. Align the sequencing results with the expected U6 vector map to verify the correct insertion of the sgRNA target.
   2. Assembly of pGGDestEB-4sgRNA vector
      NOTE: As illustrated in Figure 7, four U6-driven sgRNA expression cassettes can be efficiently ligated into the pGGDestEB-4sgRNA backbone vector using Golden Gate assembly. The number of sgRNAs to be ligated determines the use of vectors. For example, when assembling four sgRNA cassettes, use pU6a:sgRNA#1, pU6a:sgRNA#2, pU6b:sgRNA#3, pU6c:sgRNA#4, and pGGDestEB-4sgRNA.
      1. Prepare the 20 µL of Golden Gate reaction mixture containing: 2 µL of 10× restriction enzyme buffer, 2 µL of 10x T4 DNA ligase buffer, 100 ng of each pU6x:sgRNA#x construct, 50 ng of pGGDestIEB-4sgRNA destination vector, 1 µL of BsaI, 1 µL of T4 DNA ligase, and deionized water to adjust the final volume to 20 µL.
      2. Run the Golden Gate assembly reaction through 3 cycles of 20 min at 37 °C followed by 15 min at 16 °C, then complete with a final 15 min incubation at 80 °C for enzyme inactivation.
      3. To transform, transfer 5 µL of the reaction mixture into competent cells and plate onto ampicillin-supplemented LB medium.
      4. Perform colony PCR by screening colonies using Dest forward and Reverse primers (see Table of Materials) to confirm the presence of insert (Figure 8).
         NOTE: The main band of 2,415 bp and a laddering pattern should be present for a positive clone.
      5. Extract plasmids from positive colonies and sequence using Dest forward and Reverse primers for each sgRNA target.
      6. Align Sanger sequencing results with the predicted vector sequence for verification.
3. Microinjection and prescreening of transgenic founders
   1. Collect embryos spawned by homozygous rbm24a-RFP KI^zpc:cas9 fish.
   2. Prepare the injection mixture by combining 1 µL of pGGDestEB-rbm24a-4sgRNA (12 ng/µL) with 1 µL of Tol2 transposase mRNA (50 ng/µL).
      NOTE: Purify the plasmid using phenol-chloroform extraction before injection to remove any residual RNase that may have been introduced during the plasmid extraction process. The plasmid and Tol2 mRNA were diluted directly in pure water.
   3. Place a glass coverslip into a 15 cm dish. Align the embryos along the edge of the coverslip and carefully remove excess water using a pipette. Inject 2 nL of the mixture into the blastodisc of each 1-cell stage embryo. Following the injection, gently rinse the embryos with blue water and transfer them to a fresh dish. Finally, place the dish in a 28 °C incubator for cultivation.
      NOTE: Prepare methylene blue working solution (blue water) as follows: Dissolve 180 g of sea salt in 2.7 L of distilled water and sterilize to make 100x seawater stock. Dilute this stock 200-fold with sterile distilled water to obtain 1/100x seawater. Add 10 µL of 5% (m/v) methylene blue stock solution to 1 L of this 1/100x seawater to prepare the final working solution.
   4. At 24 hpf, select embryos exhibiting robust and ubiquitous BFP expression. At 4 dpf, raise only those maintaining strong transgenic fluorescent signals (Figure 9).

3. Characterization of double transgenic embryos

1. Phenotyping double transgenic embryos
   1. Collect embryos from mosaic transgenic founder females mating with wild-type males.
   2. Pick up the BFP-positive (BFP⁺) embryos at the one-cell stage under a fluorescence steromicroscope.
   3. Identify developmental defects emerging in BFP⁺ embryos but not in BFP^- embryos.
   4. Fix embryos in 4% paraformaldehyde (PFA) at 4 °C overnight. Perform whole-mount in situ hybridization, immunofluorescence staining, or other histological analyses when necessary.
      NOTE: Only defective phenotypes that arise exclusively in GFP- or BFP-positive sgRNA-expressing embryos are reliable. Conclusions should be drawn with caution, and maternal mutant phenotypes must be substantiated by more than 10 cases.
2. Genotyping maternal mutant embryos
   NOTE: For identification of Mrbm24a embryos, they can be easily picked up by the absence of Rbm24a-RFP under a fluorescence microscope. In contrast, identifying maternal mutants of other genes relies mainly on a cell aspiration strategy prior to zygotic genome activation, followed by the RT-PCR analysis of maternal mRNAs in the collected cells (Figure 10).
   1. Cell aspiration
      1. Prepare a 1.5% agarose solution by dissolving it in 1/3x Ringer's buffer and boiling until melted. Pour the solution into a 90 mm Petri dish and insert a mold (WPI, z-molds) into the molten agarose. Once solidified, gently remove the mold, resulting in a ready-to-use plate.
      2. Utilize glass capillaries and a puller to create melt-sealed needles. For optimal cell aspiration needles, set two light weights and the heating power at Lv1: 60 and Lv2: 90, and select the Step2 procedure. Trim the capillary tips with pointed tweezers (see Table of Materials) to achieve an opening diameter of 30-40 µm. Subsequently, create a spike at the needle tip using a microforge, which aids in penetration and minimizes damage to the embryo.
      3. Mate putative female founders with wild-type zebrafish and collect the embryos. Isolate BFP-positive embryos at the one-cell stage.
      4. At 3 hpf, dechorionate the embryos by incubation in 1 mg/mL Pronase in 1/3x Ringer's buffer for 10 min and gentle pipetting, and carefully place the embryos onto the agarose plate flooded with 1/3x Ringer's buffer supplemented with 1x penicillin-streptomycin. Reorient the embryos so that the blastomere is directed toward the capillary tip.
         NOTE: It is crucial to transfer the embryos with care, because they are extremely fragile without the protection of the chorion. Do not let them touch the surface of the medium!
      5. Employ a microinjector to aspirate 20-40 cells from each embryo by carefully reducing the equilibrium pressure that compensates for the aspiration force from the capillary action¹⁵. Release these cells into 2 µL of deionized H₂O at the opening edge of PCR tubes by increasing the equilibrium pressure.
         NOTE: To avoid cross-contamination, the glass needle should be cleaned by suctioning and ejecting deionized H₂O 3x before each aspiration.
      6. Add 200 µL of RNA extraction reagent to wash down the cells, and temporarily store the tubes on ice. Subsequently, analyze the genotypes of cells from embryos with or without developmental defects.
         NOTE: Cells preserved in the extraction reagent can be stored at -20 °C until the corresponding embryos exhibit aberrant phenotypes.
      7. Leave the embryos after cell aspiration in the agarose plate or place them individually into 24-well plates flooded with 1/3x Ringer's solution containing 1x penicillin-streptomycin.
         NOTE: Care should be taken for transferring the dechorionated embryos! Typically, we examine 20-40 embryos in one experiment, which takes approximately 1 h.
   2. Analysis of maternal mRNA of interest in aspired cells
      1. Add 40 µL of chloroform to the cells in the extraction reagent and mix gently. Centrifuge the mixture at 12,000 × g for 1 min at 4 °C.
      2. Carefully collect the supernatant. Add 1 µL of glycogen solution (20 mg/mL) and an equal volume of isopropanol (If the volume of the supernatant is 100 µL, then add 100 µL of isopropanol) and mix well. Incubate the mixture at -20 °C for 30 min to facilitate precipitation.
         NOTE: Due to the small quantity of total RNA in the aspirated cells, glycogen must be added to enhance precipitation efficiency and facilitate the visualization of the precipitate.
      3. Centrifuge the sample at 12,000 × g for 10 min at 4 °C and discard the supernatant.
      4. Wash the pellet 2x by the addition of 500 mL of 70% ethanol and centrifugation at 12,000 × g for 1 min.
      5. Discard the liquid and open the lid to allow the pellet dry at room temperature for 5 min.
      6. Add 7 µL of water to dissolve the pellet. Conduct reverse transcription using the First-strand cDNA synthesis kit, following the detailed protocol provided in the instruction manual.
      7. Amplify the entire coding region with appropriate primers (see Table of Materials) and run agarose electrophoresis.
      8. Clone the PCR products and sequence at least 30 clones for each embryo to analyze the mutation emerged in mRNAs.
         ​NOTE: To confirm the presence of a defective phenotype is caused by targeted mutation of the interested maternal factor, all embryos (n > 5) with defective phenotypes should show the absence of wild-type maternal transcripts. Conversely, wild-type maternal mRNAs should be present in all normally developing sgRNA-expressing embryos (n > 5). At the same time, in sgRNA-negative embryos, only wild-type mRNAs are detectable (n > 5).

Results

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 KI^zpc:cas9 embryos via Tol2 transposition, Mrbm24a was easily and rapidly identified among BFP-positive F1 embryos due to the absence of Rbm24a-RFP pro...

Discussion

Lethal zygotic mutations hinder analysis of a gene's maternal contribution. Alternative strategies, most notably germline replacement and Oocyte Microinjection In Situ (OMIS), are technically demanding^6,8. In cell transplantation process of germline replacement, the recipient embryo receives very few PGCs, often preventing normal female development^16,17,18. Using ind...

Materials

List of materials used in this article

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