Improved Genome Editing via Oviductal Nucleic Acids Delivery-based In Vivo Electroporation Technique for Knockout Mice Generation

Summary

The efficiency of the Improved Genome Editing via Oviductal Nucleic Acids Delivery (I-GONAD) method is comparable to traditional microinjection, which requires zygote collection from donor females and transfer to pseudo-pregnant females. This protocol demonstrates its effectiveness by introducing CRISPR/Cas9-induced mutations into the ROSA26 locus on chromosome 6.

Abstract

Methods for creating knockout mice typically involve three main steps: (1) collecting embryos from donor females, (2) microinjecting genetic constructs into the zygotes ex vivo, and (3) surgically transferring them into the oviduct of pseudo-pregnant females. This process requires a significant number of animals, as it involves not only donor females but also vasectomized males and pseudo-pregnant females. Moreover, microinjections into the cytoplasm or pronucleus of mouse zygotes present challenges such as needle clogging, membrane permeability issues due to high elasticity, and potential embryo death. The development of advanced electroporators, such as the Nepa21, provides a unique opportunity to generate mice with targeted gene knockouts in a single step through a method known as Improved Genome Editing via Oviductal Nucleic Acids Delivery (I-GONAD). This technique involves microinjecting CRISPR-Cas components (Cas9 protein and guide RNA) into the oviducts of pregnant females at 0.7 days post-conception, followed by in vivo electroporation to deliver these components directly into the zygotes. Following the I-GONAD procedure, the pregnant mouse carries and gives birth to pups with the targeted gene knockout. This article provides a detailed, step-by-step protocol for implementing the I-GONAD method in mice, offering a more efficient and accessible alternative to traditional knockout mouse generation techniques.

Introduction

Understanding the genetic basis of diseases is critical for identifying the molecular mechanisms underlying their etiology and pathogenesis. A key step in this research is the development of animal models with targeted gene knockouts that simulate human diseases. Mice are particularly valuable for these studies due to their general genomic similarity to humans, small size, short lifespan, and high fertility. Mice models are important for both basic research and preclinical testing¹.

To create a knockout mouse using the CRISPR-Cas9 system, researchers typically inject a complex of guide RNA and Cas9 nuclease into a zygote, causing a double-strand break in the target DNA and thereby activating the cell's repair system to cause a mutation at the site of the break^1,2. Microinjection into zygotes remains the gold standard for this purpose. However, this method requires several steps: isolating embryos from donor females, performing microinjections of genetic constructs into zygotes, and surgically transferring them into the oviducts of pseudo-pregnant females. As a result, maintaining a large number of animals is necessary, including donor females, vasectomized males, and pseudo-pregnant females. Additionally, the isolation of zygotes necessitates the euthanasia of donor females. Therefore, protocols that minimize animal use are preferable, in line with the principles of the 3Rs (Replacement, Reduction, Refinement)³.

Advancements in electroporation technology, such as the use of the Nepa21 system, provide a unique opportunity to generate targeted gene knockout mice in a single step using a technique known as Improved Genome editing via Oviductal Nucleic Acids Delivery (I-GONAD)⁴. The Super Electroporator NEPA21 Type II employs a 4-step multi-pulse electroporation system, ensuring high electroporation efficiency while maintaining high embryo viability. I-GONAD involves microinjecting CRISPR-Cas components (Cas9 protein (500-600 ng/µl) and guide RNA (80-100 ng/µl)) into the oviducts of pregnant mice 0.7 days after conception, followed by in vivo electroporation to deliver these components directly into zygotes⁵. After the I-GONAD procedure, the pregnant mouse gives birth to pups with the targeted gene knockout^6,7,8. The efficacy of this method varies depending on the desired target, but is comparable with standard microinjection-based techniques (about 50%)^9,10. Additionally, I-GONAD could potentially be adapted for genome editing in other species.

This article presents a step-by-step protocol for implementing the I-GONAD method to generate knockout mice. As an example, its effectiveness was demonstrated by targeting the ROSA26 genome locus on mouse chromosome 6. This locus was selected since it undergoes constitutive transcription without subsequent translation. Therefore, introducing a mutation at this locus is unlikely to have a negative effect on the health of the resulting pups.

Protocol

All animal studies were approved by the Animal Research Ethics Committee of Saint Petersburg State University (Approval No. 131-03-5, October 11, 2022). All procedures strictly adhered to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals. Common laboratory outbred strain CD1 mice were obtained from the Collective Use Center of the Institute of Physiologically Active Compounds, Russian Academy of Sciences (IPAC RAS). Female CD1 mice aged 2-3 months (average weight 27-30 g) were used for mating with male CD1 mice aged 2.5-4 months (average weight 35-37 g). The reagents and the equipment used in this study are listed in the Table of Materials.

1. Single-guide RNA (sgRNA) design and its preparation for transfection

1. Use computational analysis tools like CHOPCHOP, SYNTHEGO Knockout Guide Design, or CRISPOR to identify potential sgRNA sequences targeting the selected gene of interest.
   NOTE: The software can identify potential sgRNA sequences throughout the whole gene and suggests multiple variants at the output. These tools will suggest the most optimal sgRNA options and will allow the user to choose among all identified variants if needed. When making a selection, consider the following criteria:
   1. Exon proximity: Choose sequences located in exons that are as close as possible to the desired mutation point. For better results, consider the alternative splice isoforms and select the target sequence that is present in the most isoforms. Consider genome browsers (e.g., Ensembl) for accurate exon selection.
   2. Off-target considerations: Analyze potential off-target effects using the tool's scoring system, selecting sequences that exhibit the lowest predicted off-target activity.
      NOTE: Computational tools like CHOPCHOP or Synthego are highly effective for assessing exon proximity and off-target effects, helping to identify optimal sgRNA sequences for precise genome editing. When designing guide RNAs, it is important to consider the specific sequence of the gene of interest in the mouse strain being used. For some mouse strains (e.g., C57Bl, CBA, C3H), the full genomic sequence is available. If such data are unavailable, it may be advisable to perform preliminary sequencing of the target gene in the planned editing region. Single mismatches in the guide RNA, caused by polymorphisms, may compromise editing efficiency. The ROSA 26 mouse genome locus was targeted with the sgRNA sequence: 5'-ACTCCAGTCTTTCTAGAAGA-3'.
2. Order a set of primers for sgRNA template PCR-amplification as described in Table 1.
   NOTE: gRNA can be ordered elsewhere, either as sgRNA or as crRNA:tracrRNA duplex.
3. Produce a linear dsDNA template.
   1. Prepare the master mix as indicated in Table 1. Add the polymerase at the end and keep the master mix on ice to maintain stability.
   2. Run the PCR under the following conditions: Initial denaturation: 30 s at 95 °C; 30 cycles of: 20 s at 95 °C (denaturation), 20 s at 55 °C (annealing), 15 s at 72 °C (extension); Final elongation: 2 min at 72 °C; Hold at 4 °C.
4. Use the PCR reaction mixture in further In Vitro Transcription (IVT) reaction.
   1. Prepare the IVT master mix as indicated in Table 2 and incubate for 1.5 h at 37 °C. Use a thermocycler for consistent temperature control.
   2. Add 2 µL of DNase I and incubate for another 30 min at 37 °C to remove the DNA template.
   3. Inactivate DNase I by incubating the reaction mixture at 75 °C for 5 min.
   4. Purify RNA using TRIZOL reagent according to the manufacturer's protocol.
      NOTE: Disposal of the TRIZOL reagent and DNAse1, its solutions, or any by-products, should be placed in designated chemical waste containers.
   5. Measure the RNA concentration using a spectrophotometer (the typical yield of one reaction is 3-5 µg). Check the A260/ A280 and A260/A230 ratios. A260/ A280 should be at least 2.0, and the A260/A230 ratio should also be at least 2.0 (meaning there is no contamination by proteins or organic solvents). Store the sgRNAs at -80 °C until use.
5. Prepare the RNP mix for transfection
   1. The transfection mix consists of an RNP complex, which is a mixture of the Cas9 protein and sgRNA. Follow these steps to prepare the RNP mix:
   2. Dilute 1 M Tris-HCl (pH 7.5) buffer to 100 mM using nuclease-free water and filter using a syringe filter with 0.022 µM pore size, aliquot in 10 µL portions, and store at 4 °C until use.
   3. In a nuclease-free tube, prepare the transfection mix of the following components in the final concentrations: 500 ng/µL spCas9 nuclease, 80 ng/µL sgRNA, 10 mM Tris-HCl diluted with DEPC-treated water.
      NOTE: High concentrations of the RNP mix components are required to ensure proper efficiency of the protocol.
   4. Incubate the transfection mix at 37 °C for 10 min to allow the formation of the RNP complex.
   5. Aliquot the mix into small volumes (2 µL) suitable for a single I-GONAD session.
   6. Keep the aliquots at 4 °C during the I-GONAD session. The mix is prepared fresh for each I-GONAD electroporation session to maintain stability.
      NOTE: The RNP mix should be prepared fresh for each I-GONAD session to ensure optimal activity and efficiency.

2. Preparation of female mice for genome editing via oviductal nucleic acid delivery

1. Mating and selection of female mice: Mate CD1 female mice with single-housed CD1 male mice overnight. In the morning, check for the presence of copulation plugs in the female mice. Select females with copulation plugs and transfer them to a new cage for further procedures.
   NOTE: To minimize genetic mosaicism, the optimal time for delivering the genetic construct is 0.7 days post-mating at the single-cell (zygote) stage¹⁰. This approximately corresponds to 16:00 p.m. on the day when the copulation plug was detected. At this stage, dissection of the oviduct reveals that zygotes have fewer cumulus cells, though they remain in the ampulla. Notably, cumulus cells can also take up exogenous nucleic acids or proteins during electroporation. If the procedure is performed earlier, for example, at 10 h post-mating, the cumulus cells still tightly surround the zygotes, which may hinder efficient electrophoretic delivery of nucleic acids into the zygotes. Do not use the hormonally stimulated mice, since they might give an excessive number of oocytes, and this might lead to miscarriage.
2. Preparation of surgical instruments: Autoclave all stainless-steel surgical instruments to ensure sterility. All non-stainless steel items that come in contact with the mouse's internal organs should be provided as sterile or single-use items.
3. Analgesia, anesthesia and preparation of mice: Weigh the selected female mice and apply meloxicam solution (5 mg/kg, s.c.) 15 min prior to anesthesia induction. Anesthetize mice via intraperitoneal injection using a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) (following institutionally approved protocols).
   1. Monitor the mice for loss of the toe pinch reflex to confirm adequate anesthesia. Place the anesthetized mouse on a heating pad to maintain body temperature during the procedure. Protect the mouse's eyes from dryness during anesthesia by applying veterinary ointment.

3. Surgical procedure

1. Remove hair from the surgical area and disinfect the skin with a chlorhexidine or betadine scrub or solution, followed by 70% ethanol in 3 alternating passes.
2. Make a 0.7 cm longitudinal skin incision in the lower back region..
3. Extend the incision to 1.3 cm by pushing laterally with slightly opened scissor tips.
4. Carefully make a small incision (0.7 cm) in the peritoneum to access the abdominal cavity.
5. Extend the incision to 1.1 cm by pushing laterally with slightly opened scissor tips.
6. Locate the fat pad attached to the ovary and gently pull it out using forceps until the ovary, oviduct, and uterus are clearly visible.
7. Secure the fat pad in place using a vessel clamp.
8. Visualize the oviduct under a stereoscopic microscope for precise manipulation (Figure 1).
9. Load 2 µL of the injection mix into a sterile plastic capillary using a Stripper Pipettor.
   NOTE: The Stripper Pipettor with sterile plastic capillaries, originally designed for manipulations with embryos in IVF clinics, can support multiple applications, including embryo transfer and CRISPR/Cas9 construct delivery in I-GONAD procedures. The pipettor functions as a precision liquid dispenser; in combination with sterile disposable flexible capillaries, it allows sterility and prevents cross-contamination. Plastic capillaries are preferable to glass for injecting RNP mix for transfection into the oviduct, as they are less traumatic to delicate tissues. Unlike glass capillaries, disposable plastic ones do not require flame-polishing to smooth sharp edges before insertion, reducing preparation time and risk of damage.
10. Using micro-scissors, make a small incision (0.2 cm) in the oviduct wall a few millimeters upstream of the ampulla.
11. Carefully insert the Stripper Pipettor with plastic capillary into the incision and expel the injection mix into the oviduct.
    NOTE: Ensure that no air enters the oviduct, as this may affect the efficiency of electroporation.
    To prevent this, fill the injection plastic capillary with more injection mix than necessary and avoid complete injection. Plastic capillaries are suitable for both infundibulum transfer and direct oviduct wall dissection between the infundibulum and ampulla. The direct oviduct wall dissection method might be less traumatic than infundibulum transfer since the latter requires an initial incision through the thin bursa membrane covering the oviduct and infundibulum. In CD1 mice, this membrane is highly vascularized, and cutting it may cause minor bleeding, which can obstruct visibility and complicate microinjection of the RNP mix.
12. Gently remove the plastic capillary from the oviduct.

4. Electroporation and suturing

1. Turn on the electroporator by pushing the switch on the front panel.
2. Set electroporation parameters: Poring pulse: Voltage: 50 V, Pulse length: 5 m, Pulse interval: 50 ms, Number of pulses: 3, Decay rate - 10%, Polarity: +/-; Transfer pulse: Voltage: 10 V, Pulse length: 50 ms, Pulse interval: 50 ms, Number of pulses: 3, Decay rate - 40%, Polarity: +/-; Current limit: in vivo.
3. Using the tweezer-type electrodes, carefully grasp the oviduct and check the resistance value by pressing the kΩ button on the electroporator. Ensure that the electrodes are compressed sufficiently so that the resistance remains within the optimal range of 350-400 Ω.
   NOTE: This can be achieved by adjusting the distance between the two electrodes when the intact oviduct is sandwiched between them.
   1. Immediately after measuring the resistance, press the Start button to initiate electroporation. This step is critical to ensure efficient delivery of CRISPR-Cas components into the zygotes⁴. Wait for the Ω button to display an "end" signal. Write down the values shown in the Measurements frame.
      NOTE: The impedance value should be approximately 250-400 Ω/150-250 mA of the voltage value to ensure an optimal balance between viability and transfer efficiency. To maintain this balance, avoid the coverage of the oviduct regions with a piece of wet paper soaked in Dulbecco's modified phosphate-buffered saline (DPBS), recommended in some protocols, as this step can significantly decrease the resistance beyond the optimal range. Decreased resistance can raise the voltage to more than 250 mA, leading to cell death.
4. After completing electroporation on one oviduct, carefully place the reproductive tract back into the abdominal cavity. Suture the incision using 4-0 polyglycolic acid surgical suture.
5. Repeat the operation and electroporation on the second oviduct. Once completed, return the reproductive tract to the abdominal cavity and suture the incision as described above.
6. Post-procedure monitoring: Place the mouse in a warm (37 °C), clean cage for recovery and monitor until it fully recovers from anesthesia.
   NOTE: After the surgery, house the mice in cages containing two mice per cage. If only one mouse underwent surgery, provide her with a companion female mouse. This practice helps to reduce unnecessary stress for the surgical mouse and promotes a more natural social environment. After 12 h, perform a second injection of analgesic (meloxicam, 5 mg/kg, s.c.).

5. Genotyping

1. Primer design
   1. For genotyping, design primers to amplify a PCR product between 300-600 bp in length. Primers should be 18-21 nucleotides long with a melting temperature (Tm) of approximately 60 °C.
   2. Avoid sequences that form secondary structures or primer dimers. Use software tools such as PerlPrimer 1.1.21¹¹, In-Silico PCR V39x1, or similar to ensure primer quality.
   3. Perform a BLAST search against the mouse genome database to confirm primer specificity and avoid non-specific amplification¹¹.
      NOTE: The following primers were designed for genotyping of the target sequence: Forward primer: 5'-CTCTCCCAAAGTCGCTCTGA-3'; Reverse primer: - 5'-TCTGTGGGAAGTCTTGTCCC-3', expected PCR product length: 320 bp, and Tm 62°C. For Sanger sequencing, design a sequencing primer that anneals 150-200 bp away from the Cas9 cut site.
2. Perform DNA extraction. For rapid DNA extraction, the Hotshot isolation protocol¹² is the simplest one.
   1. Using an ear punch, collect a small tissue fragment (approximately 2 mm) from the mouse's ear.
   2. Add 100 µL of Lysis Buffer (25 mM NaOH, 0.2 mM EDTA, pH = 12).
   3. Incubate at 95 °C for 60 min, vortexing the mixture every 20 min.
   4. Add 100 µL of Neutralizing Buffer (40 mM Tris-HCl, pH = 5) and vortex thoroughly.
   5. Use 1-5 µL of the final preparation for PCR.
3. PCR amplification and sequencing. Prepare the PCR master mix as outlined in Table 3. Use a proofreading DNA polymerase to ensure high fidelity. Aliquot the master mix into the required number of tubes and add genomic DNA.
   NOTE: Always include a wild-type DNA sample as a control for sequencing analysis.
4. Run the PCR for ROSA26 genotyping. The following conditions were used: Initial denaturation: 30 s at 95 °C; 28 cycles of: 20 s at 95 °C (denaturation), 20 s at 62 °C (annealing), 20 s at 72 °C (extension); Final elongation: 2 min at 72 °C; Hold at 4 °C.
   NOTE: The provided protocol is accurate for ROSA26 locus fragment (estimated size 320 bp) amplification using primers from step 1.1.2. For more specific and accurate amplification, limit the number of amplification cycles to 28-30.
5. Sequencing and analysis
   1. Perform Sanger sequencing of PCR fragments obtained (step 5.4).
   2. Analysis of the sequencing results
      NOTE: Successful transfection will result in mosaic animals, identifiable by the emergence of additional DNA sequences near the nuclease cut site in the sequencing chromatogram. Use computational tools such as EditCo ICE Analysis or TIDE¹³ to identify and quantify exact indels in the genome.
      1. To use ICE Analysis, simply upload obtained Sanger sequencing files (control .ab1 file for wt mouse PCR product sequence with no editing and experiment .ab1 file for experiment mouse PCR product sequence), enter the guide RNA sequence, and select the nuclease used in the CRISPR experiment.
         NOTE: The ICE Analysis tool will calculate the overall editing efficiency, and determine the profiles and relative abundances of all of the different types of edits present in the sample.
      2. To use TIDE, enter the guide RNA sequence, upload obtained Sanger sequencing files (control .ab1 file for wt mouse PCR product sequence with no editing and experiment .ab1 file for experiment mouse PCR product sequence), and check Parameters (in some cases, Advanced settings can help to find indels larger than 10 bp).

Results

If the electroporation was successful, the experimental mice will: (1) give birth to a handful of pups, and (2) some animals will have different indels in the genome. If the genotyping primers are well-designed, and the PCR program is optimal, the PCR product will be present on the agarose gel as one clear band of the desired size. If the nuclease successfully cuts the target DNA, then the obtained animals will likely be mosaic and have a range of indels in the genome. Computational tools, such as EditCo ICE Analysis or ...

Discussion

The efficiency of generating knockout mice using the I-GONAD method is comparable to that of traditional microinjection techniques. However, traditional methods involve isolating zygotes from donor females, performing microinjections, and transplanting embryos into the oviducts of pseudo-pregnant females. In contrast, the I-GONAD method simplifies this process by delivering genome-editing components directly into the oviducts of pregnant females, eliminating the need for zygote isolation and transplantation. The presente...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5–5 uL pipette	Thermo Scientific	4642020
1.5 mL tube	Aptaca	1003/G
100-1000 uL pipette	Thermo Scientific	4642090
200 uL tube	Axygen	PCR-02-C
20-200 uL pipette	Thermo Scientific	4642080
2-20 uL pipette	Thermo Scientific	4642060
4x Gel Loading Dye	Biolabmix	D-3002
Agarose	AppliChem	A8963,0500
DNase I, RNase-free (1 U/μL)	Thermo Fisher	EN0521
dNTP Set, 100 mM Solution	Thermo Scientific	R0181
DreamTaq Green DNA Polymerase (5 U/μL)	Thermo Scientific	EP0713
Dulbecco’s modified phosphate-buffered saline	Sigma Aldrich	D8537
EditCo ICE Analysis	https://ice.editco.bio/#/		accessed on 04.06.2025
EDTA 0.5M solution, pH = 8	AppliChem	A4892,0500
Ensembl	https://www.ensembl.org/index.html		accessed on 09.06.2025
Ethidium bromide	Biolabmix	EtBr-10
Eye gel drops “Oftagel”	URSAPHARM	204604
Gel-documenting system ChemiDoc XRS+	Bio-Rad	1708265
In-Silico PCR V39x1	https://genome.ucsc.edu/cgi-bin/hgPcr		accessed on 09.06.2025
Integrated DNA Technologies	https://eu.idtdna.com		accessed on 09.06.2025
Microsurgical scissors	Medin URAL	M124
NEPA21 electroporator	Nepa Gene	NEPA21	With CUY652P2.5X4 electrode
NTP Set, 100 mM Solution	Thermo Scientific	R0481
Oligonucleotides	Evrogen		direct order, standard desalting purification
Opti-MEM I Reduced Serum Medium	Thermo Fisher	31985070
PerlPrimer	https://perlprimer.sourceforge.net		Version 1.1.21.
Phusion High-Fidelity DNA Polymerase (2 U/μL)	Thermo Scientific	F530L
pipette 10 uL tips	Ulplast	HT-10-960
pipette 1000 uL tips	Ulplast	HT-1000-576
pipette 200 uL tips	Ulplast	HT-200-960
Power supply “Elf-4”	DNA-technology	PS-400 NEW
RNAse/DNAse free water	Invitrogen	10977049
Saline solution	PanEco	P011n
SE-1 horisontal electrophoresis system	Helicon	SE-1
Sodium Hydroxide	Sigma-Aldrich	S5881-500G
Step100 DNA Ladder	Biolabmix	S-8100
Stereomicroscope SMZ1000	Nikon	5-363376
Stripper micropipetter	CooperSurgical	MXL3-STR
Stripper tips	ORIGIO	MXL3-125
Surgical Needle	KMIZ	4A1- 0,6×20
Suturing forceps	Medin URAL	M302.2
Suturing forceps	Medin URAL	M303
Suturing thread POLYCON N0	Tonzos 95	234
SYNTHEGO Knockout Guide Design	https://design.synthego.com/#/		accessed on 09.06.2025
T100 amplificator	BioRad	10014822
T7 RNA polymerase	Thermo Scientific	EP0111
TBE 10x buffer	Biolabmix	TBE-500
Tide	https://tide.nki.nl/		accessed on 04.06.2025
Tris hydrochloride	Suzhou Yacoo Science	SYS-S0005-0.5
Trizma hydrochloride solution	Sigma Aldrich	T2663
TRIzol Reagent	Invitrogen	15596026
TrueCut Cas9 Protein	Invitrogen	A36499
Universal scissors	Medin URAL	M147
Xylazine	Interchemie Werken de Adelaar	IX2
Zoletil	Virbac Lab	2000443714739.00