GONAD: A new method for germline genome editing in mice and rats

Recent advances in the CRISPR/Cas9 system have demonstrated it to be an efficient gene‐editing technology for various organisms. Laboratory mice and rats are widely used as common models of human diseases; however, the current standard method to create genome‐engineered animals is laborious and involves three major steps: isolation of zygotes from females, ex vivo micromanipulation of zygotes, and implantation into pseudopregnant females. To circumvent this, we recently developed a novel method named Genome‐editing via Oviductal Nucleic Acids Delivery (GONAD). This method does not require the ex vivo handling of embryos; instead, it can execute gene editing with just one step, via the delivery of a genome‐editing mixture into embryos in the oviduct, by electroporation. Here, we present a further improvement of GONAD that is easily applicable to both mice and rats. It is a rapid, low‐cost, and ethical approach fulfilling the 3R principles of animal experimentation: Reduction, Replacement, and Refinement. This method has been reconstructed and renamed as “improved GONAD (i‐GONAD)” for mice, and “rat improved GONAD (rGONAD)” for rats.


| BACKG ROUND AND FE ATURE S
The recently developed CRISPR/Cas9 system is the most convenient and reliable method for generating animals carrying modified genomes. This system efficiently generates animals with the need for "knock-out" and "knock-in" of target sequences (Atanur et al., 2013;Gurumurthy, Grati, et al., 2016). However, for most laboratories, the genome engineering of mammals is a difficult and laborious task because the most common procedures for creating genome-edited mammals include three major ex vivo embryo handling steps (Li et al., 2013), namely, (1) the isolation of zygotes, (2) the microinjection of zygotes, and (3) the transfer of microinjected zygotes into the oviducts of pseudopregnant females. These three steps require the researchers and technicians performing the procedures to be proficient in highly technical skills, and necessitate the use of expensive apparatus such as micromanipulators.
To simplify these complex and laborious processes, we established a novel genome engineering method, named Genome-editing via Oviductal Nucleic Acids Delivery (GONAD), for mice (Gurumurthy, Takahashi, et al., 2016;Gurumurthy et al., 2019;Ohtsuka et al., 2018;Takahashi et al., 2015). In this method, in vivo genome editing of early preimplantation embryos present in the oviducts of pregnant females is performed. Therefore, GONAD does not require the aforementioned ex vivo handling of embryos. In the first GONAD trial with Cas9 mRNA and sgRNA, genome-editing efficiency was approximately 25% (Takahashi et al., 2015). In 2018, we developed a novel method for mice (i-GONAD), and its efficiency was substantially improved with the use of Cas9 protein and crRNA/tracrRNA complex (knock-out, 50-100%;knock-in, 15-40%;Ohtsuka et al., 2018). Moreover, we established a method to produce knock-out and knock-in rats with high efficiency, named rat improved GONAD (rGONAD) (Kobayashi et al., 2018).
In studies involving animals, it is essential to meet the 3R principles of animal experimentation: Reduction, Replacement, and Refinement. Unlike traditional approaches, GONAD does not require the sacrifice of pregnant females to isolate zygotes; many females that undergo GONAD can deliver pups. Moreover, GONAD does not require the preparation of pseudopregnant females (obtained by mating females with vasectomized males); therefore, we can reduce the number of animals used. For these reasons, the production of genome-edited animals with GONAD meets the 3R principles.
Here, we provide a step-by-step protocol for GONAD with some examples of genome-edited mice and rats. We detail rGO-NAD and electroporation conditions and discuss the possibility of more efficient production with biallelic disruptions than with the previous protocol (Gurumurthy et al., 2019). GONAD is simple and rapid (Figure 1), and we strongly believe that it will facilitate the production of genome-engineered rodents in any laboratory.
Furthermore, GONAD can be readily applied in mammals such as guinea pigs, hamsters (Hirose et al., 2020), cows, and pigs, in which traditional gene targeting approaches using embryonic stem cells or ex vivo embryo culture methods are not well established.
Nonetheless, it is still difficult to use GONAD to introduce larger mutations requiring a long DNA donor (>2 kb). If even larger knockins need to be engineered, other methods such as microinjection and CRISPR-READI may be more suitable (Chen et al., 2019;Chenouard et al., 2021).

| Micropipette (Figures 2e and 3a)
We usually use a puller and glass capillaries to prepare micropipettes for microinjection into the oviduct. Conditions for preparing the injection capillary are as follows: Puller, Narishige PN-31; glass capillary, Drummond #1-000-0300, HEATER 80, MAGNET-SUB 30, or MAGNET-MAIN 60-80, depending on the season and humidity. Before injection, cut off the tip of the capillary with scissors ( Figure 3a). -Tip 1.

| Mouth pipette (Figure 3b)
Many types of mouth-pipetting devices can be used. In our laboratory, we connect the aspirator tube assembly (for the calibrated micropipette) and the glass capillary. -Tip 2.  3.3.1 | Surgical procedure (Movie S1)

| Design of the specific guide RNA
1. Measure the body weight of the females.
3. Confirm anesthesia based on whether the mice or rats respond.
4. Spray rubbing alcohol on the dorsal skin.
5. Make a dorsal incision at the central portion of the back skin.
6. Make an incision in the muscle layer on the left side.
12. Inject the solution into the oviductal lumen upstream of the ampulla with a mouth pipette (Figure 5b). (Figure 5c,d, Movie S3)

13.
Cover the oviduct with a piece of a wet KimWipe towel soaked in PBS (Figure 5c). 14.
Dip the tweezer-type electrodes in PBS. Hold the oviduct between the electrodes (Figure 5d).
Remove the KimWipe and aorta clamp.

19.
Return the tissues to their original position.

20.
Repeat steps 6-19 on the other side of the oviduct.

21.
Close the skin using a suture wound clip.

22.
Maintain the mice/rats in a cage at 37°C until they are awake.
1. Euthanize the pregnant mice or rats at 1 or 2 days after in vivo electroporation, and collect the organs connecting the ovary, oviducts, and upper portion of the uterus.
2. Wash embryos with PBS, and place them in a 60-mm dish.
3. Insert a 30 G needle attached to a 1-ml syringe containing 10% FBS/PBS into the oviductal lumen.

Flush out 10% FBS/PBS while under microscopic observation.
5. Collect the embryos with a pipette.
6. Observe the embryos with a fluorescence inverted microscope ( Figure 7).

| TIPS
1. The glass capillary tube used for GONAD does not need to be of high quality, unlike those required for embryo microinjections.
2. This mouth pipette is also used for transferring embryos on 1 or 2 days after electroporation. 7. Caution! In our initial trial, we could hardly obtain pups from the super-ovulated female mice (C57BL/6) and rats. Therefore, we used estrous female mice and rats without super-ovulation.
8. Point: Mix CRISPR/Cas9 reagents in a 1.5-ml tube on the inner wall of the "cap" (Figure 5). It is very easy to load this into the capillary when the solution is injected.

Troubleshooting:
No pups or a low number of pups: Apply higher current voltage. Optimize electroporation conditions for mice or rats. See also Figure 8.
Use super-ovulated females. Use estrous female mice or rats without super-ovulation.
Low genome-editing efficiency: Failure of injection and/or electroporation. Practice GONAD with tetramethylrhodamine-labeled dextran after detecting fluorescence with more than 50% efficiency.
The chosen guide RNA is wrong. Change to the other guide RNA. See also section 3.5.3.
Homozygous mutant mice or rats are lethal. Efficiency of genome editing by GONAD is often high, and therefore, both alleles may be modified in these mice or rats. Lower the current voltage to edit only one allele.

| Electroporation conditions
We examined six different conditions for electroporation (poring pulse voltage: 50, 40, or 30 V; number of transfer pulses: 3 or 6; see Although in mice the efficiency did not appear to differ between three and six transfer pulses, in rats six transfer pulses were more efficient than three (Figure 8c,d), as described in our previous report (Kobayashi et al., 2018).
These data suggest that the most suitable conditions are as follows: • Mouse: Poring pulse voltage, 30-50 V; number of transfer pulses: 3 • Rat: Poring pulse voltage, 40-50 V; number of transfer pulses: 6

| Gene editing
In our laboratory, the Cas9 protein, guide RNA, and ssDNA of 20 bases including three tandem stop codons were injected for producing gene-disrupted mice and rats via the knock-in approach (Figure 9a,b) (Koyano et al., 2019;Namba et al., 2021). As shown in Figure 8c,d, we were able to obtain gene-edited mice (p21 gene, four pups, 31%; p16/19 gene, four pups, 40%). We also obtained double-mutant mice by using a mixture of two CRISPR/Cas9 reagents ( Figure 9e). Fewer than five females were used to produce these knock-in mice, suggesting that i-GONAD is a useful method from the perspective of the 3R principles.
The rGONAD method is also highly efficient in both knock-out and knock-in trials, as shown in our previous report (Kobayashi et al., 2018), and we succeeded in generating a novel Alport syndrome rat model with this method (Namba et al., 2021). In addition, many pups obtained by rGONAD had mutations in both alleles ( Figure 10, Table 1), suggesting that this method allows efficient production of rats with biallelic disruptions, which is highly useful for the study of gene functions using F0 pups.

ACK N OWLED G M ENTS
We are grateful to Chieko Takahashi