SCR7 | Tie2 Signal

Introduction of a point mutation in the KRAS gene of in vitro fertilized porcine zygotes via electroporation of the CRISPR/ Cas9 system with single-stranded oligodeoxynucleotides

Abstract
This study aimed to investigate the efficiency of KRAS gene editing via CRISPR/Cas9 delivery by electroporation and analyzed the effects of the non-homologous end- joining pathway inhibitor Scr7 and single-stranded oligodeoxynucleotide (ssODN) ho- mology arm length on introducing a point mutation in KRAS. Various concentrations (0–2 µM) of Scr7 were evaluated; all concentrations of Scr7 including 0 µM resulted in the generation of blastocysts with a point mutation and the wild-type sequence or indels. No significant differences in the blastocyst formation rates of electroporated zygotes were observed among ssODN homology arm lengths, irrespective of the gRNA (gRNA1 and gRNA2). The proportion of blastocysts carrying a point mutation with or without the wild-type sequence and indels was significantly higher in the ssODN20 group (i.e., the group with a ssODN homology arm of 20 bp) than in the ssODN60 group (gRNA1: 25.7% vs. 5.4% and gRNA2: 45.5% vs. 5.9%, p < .05). In
conclusion, the CRISPR/Cas9 delivery with ssODN via electroporation is feasible for the generation of point mutations in porcine embryos. Further studies are required to improve the efficiency and accuracy of the homology-directed repair.

K E Y WO R D S
CRISPR/Cas9, KRAS gene, pig, point mutation, ssODN
1Faculty of Bioscience and Bioindustry,
Tokushima University, Tokushima, Japan
2Faculty of Veterinary Science, Prince of
Songkla University, Songkhla, Thailand
3College of Agricultural Science, Guangdong Ocean University, Guangdong, China
4School of Biological Science, Tokai
University, Sapporo, Japan

Correspondence
Maki Hirata, Faculty of Bioscience and Bioindustry, Tokushima University, 2272-1 Ishii, Myozai-gun, Tokushima 779-3233, Japan.
Email: [email protected]

Funding information
Japan Science and Technology Agency, Grant/Award Number: JPMJOP1613; JSPS KAKENHI, Grant/Award Number: 18K12062 and JP19K16014; Tokushima University

1 | INTRODUC TION

In humans, oncogenic mutations in KRAS play a crucial role in the pathogenesis of cancer (Li et al., 2015; Valtorta et al., 2013). Mutant KRAS could initiate the activation of cell signaling mole- cules leading to cell differentiation, growth, chemotaxis, and apop- tosis (Jancik et al., 2010). Transgenic animal models with point mutations in KRAS have been generated using conventional pro- moter and gene trap targeting vectors together with somatic cell nuclear transfer techniques to clarify the impact of distinct KRAS nucleotide changes (LaConti et al., 2011; Li et al., 2015). However,
this approach requires the time-consuming and labor-intensive screening of correctly modified cells (Capecchi, 1989; Rong & Xu, 2018). Accordingly, many recent studies have focused on al- ternative strategies for the efficient generation of animal models harboring point mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, a fast and simple gene editing system, has been applied to several taxa, such as Caenorhabditis elegans (Zhao et al., 2014), Drosophila (Port et al., 2014), zebrafish (Armstrong et al., 2016), mouse (Inui et al., 2014), and pig (Wang et al., 2016), for the introduction of precise nucleotide changes in specific genes.

Anim Sci J. 2021;92:e13534. wileyonlinelibrary.com/journal/asj

https://doi.org/10.1111/asj.13534

CRISPR/Cas9 with newly designed single-stranded oligodeoxy- nucleotides (ssODNs) can be used for specific gene editing to obtain point mutations, defined sequence changes, or insertions of a small tag. Traditional microinjection methods have been employed to introduce the Cas9 nuclease and ssODN into embryos (Armstrong et al., 2016; Inui et al., 2014). However, embryonic death by mechanical damage or high concentrations of CRISPR/Cas9 constructs as well as low suc- cess rates are still major limitations of this method (Kaneko et al., 2014; Moody, 2018). We previously used electroporation to transfer the CRISPR/Cas9 system into embryos to generate blastocysts with dif- ferent types of mutations with acceptable output quality (Hirata, Tanihara, et al., 2019; Tanihara et al., 2019). Electroporation may be an alternative tool to generate embryos with defined point mutations in specific genes by the CRISPR/Cas9 system with ssODN.

When double-strand breaks (DSBs) are induced by gene editors, cells repair DNA by the non-homologous end-joining (NHEJ) or homology-directed repair (HDR) pathways (Kanaar et al., 1998). Point or small mutations can be artificially introduced by CRISPR/ Cas9-mediated HDR using ssODN with homology arms as donor DNA with the desired point mutation (Inui et al., 2014). However, the efficiency of CRISPR/Cas9-mediated HDR is low because DSBs caused by Cas9 can be repaired by the NHEJ pathway (Frit et al., 2014; Liu et al., 2018). A key enzyme in the NHEJ pathway, 5,6-Bis-(benzylideneamino)-2-mercaptopyrimidin-4-ol (Scr7) is an inhibitor of DNA ligase IV; therefore, supplementation with Scr7 may improve the efficiency of HDR by inhibiting the NHEJ pathway (Maruyama et al., 2015; Srivastava et al., 2012).

Since domestic pigs share similar anatomical and physiological properties to those of humans and are advantageous with respect to breeding and handling, they are considered a promising animal model in biomedical research (Vodicka et al., 2005). A previous study using transgenic pigs with inducible oncogenic KRASG12D and dominant-negative TP53, known as a vital tumor suppressor, showed that the inducible expression of these genes is tumorigenic (Schook et al., 2015). Therefore, the establishment of pigs with mutant KRAS will improve our understanding of the pathogenesis of cancer and accelerate the development of novel therapeutic approaches under in vivo conditions. In humans, KRASG12D and KRASG12A are common mutations in KRAS codon 12 (Peeters et al., 2013). Therefore, the objective of this study was to investigate the efficacy of CRISPR/ Cas9 with ssODN for the generation of KRASG12A point mutations in porcine blastocysts via electroporation. Various components of the system were evaluated, including the effects of the concentration of the DNA ligase IV inhibitor Scr7 as well as the homology arm length of the ssODN on the production of a point mutation.

2 | MATERIAL S AND METHODS

2.1 | General

In this study, we did not use live animals, so there was no ethical ap- proval required.

2.2 | Oocyte collection and in vitro maturation

Pig ovaries were obtained from prepubertal gilt crosses (Landrace × Large White × Duroc breeds) at a local slaughterhouse and were transported in physiological saline within 1 hr to the labo- ratory at 30°C. Ovaries were washed three times with prewarmed physiological saline solution supplemented with 100 IU/ml penicil- lin G potassium (Meiji) and 0.1 mg/ml streptomycin sulfate (Meiji). Follicles with diameters of 3–6 mm on the ovarian surface were sliced on a sterilized dish using a surgical blade, and cumulus–oocyte complexes (COCs) were visualized and collected under a stereomi- croscope. Approximately 50 COCs were cultured in 500 µl of matu- ration medium consisting of tissue culture medium 199 with Earle's salts (TCM 199; Thermo Fisher Scientific) supplemented with 10% (v/v) porcine follicular fluid, 0.6 mM cysteine (Sigma-Aldrich), 50 µM β-mercaptoethanol (Wako Pure Chemical Industries Ltd.), 50 µM sodium pyruvate (Sigma-Aldrich), 2 mg/ml D-sorbitol (Wako Pure Chemical Industries Ltd.), 10 IU/ml equine chorionic gonadotropin (Asuka Pharmaceutical), 10 IU/ml human chorionic gonadotropin (Nippon Zenyaku Kogyo), and 50 µg/ml gentamicin (Sigma-Aldrich), then covered with mineral oil (Sigma-Aldrich) for 22 hr in 4-well dishes (Nunc A/S, Roskilde). The COCs were transferred into a matu- ration medium without hormones for an additional 22 hr. COCs were incubated at 39°C in a humidified incubator containing 5% CO2 in air.

2.3 | In vitro fertilization

Mature oocytes were subjected to in vitro fertilization (IVF) as de- scribed previously (Nguyen et al., 2017). Briefly, frozen-thawed ejaculated spermatozoa were transferred to 5 ml of fertilization me- dium (PFM; Research Institute for the Functional Peptides Co.) and washed by centrifugation at 500 × g for 5 min. The pelleted sper- matozoa were resuspended in fertilization medium and adjusted to 1 × 106 cells/ml. Then, approximately 50 oocytes were transferred to 500 µl of sperm-containing fertilization medium, covered with mineral oil in 4-well dishes, and co-incubated for 5 hr at 39°C in a humidified incubator containing 5% CO2, 5% O2, and 90% N2. After co-incubation, the putative zygotes were denuded from the cumulus cells and the attached spermatozoa by mechanical pipetting, trans- ferred to porcine zygote medium (PZM-5; Research Institute for the Functional Peptides Co.) and cultured for 7 hr until electroporation.

2.4 | Electroporation

Electroporation was performed as described previously (Tanihara et al., 2016). Briefly, an electrode (LF501PT1-20; BEX) was con- nected to a CUY21EDIT II electroporator (BEX) and placed under a stereoscopic microscope. Approximately 50 putative zygotes were washed with Opti-MEM I solution (Gibco/Invitrogen Co.) and placed in a line in the electrode gap on the chamber slide filled with 10 µl of Nuclease-Free Duplex Buffer (Integrated DNA Technologies (IDT))with 100 ng/μl gRNA (Alt-RTM CRISPR crRNAs and tracrRNA, chemi- cally modified and length optimized variants of the native guide RNA purchased from IDT) targeting porcine KRAS (Figure 1a), 100 ng/μl Cas9 protein (Takara Bio, Inc.), and ssODN. gRNAs were designed near the site of the point mutation by using the CRISPRdirect webt- ool (https://crispr.dbcls.jp/; Naito et al., 2015). Four ssODNs with different homology arm lengths were designed (Figure 1b). After electroporation (five 1-msec square pulses at 25 V), zygotes were washed with PZM-5 and cultured for 3 days. The embryos were sub- sequently cultured in porcine blastocyst medium (PBM; Research Institute for the Functional Peptides Co.) for 4 days to evaluate the ability to develop to the blastocyst stage and the blastocyst geno- types. Zygotes and embryos were incubated at 39°C in a humidified incubator containing 5% CO2, 5% O2, and 90% N2.

2.5 | Analysis of targeted gene sequences after electroporation

Genomic DNA was isolated from blastocysts collected individually by boiling in a 50 mM NaOH solution at 98°C for 10 min, followed by neutralization in 100 mM Tris HCl. The genomic regions flanking the gRNA target sequences were PCR-amplified using the follow- ing specific primers: 5′-GGCGTTTTCATCGCTAAACT-3′ (forward) and 5′-GGCTCTCCATGTCTGTGGTT-3′ (reverse). The PCR prod- ucts were extracted by agarose gel electrophoresis using a Fast Gene Gel/PCR Extraction Kit (Nippon Genetics, Tokyo, Japan). The targeted genomic regions of the PCR products were directly se- quenced by Sanger sequencing using the BigDye Terminator Cycle Sequencing Kit version 3.1 (Thermo Fisher Scientific K.K.) and an ABI 3500 genetic analyzer (Applied Biosystems). The TIDE (tracking of indels by decomposition) and TIDER (tracking of indels and re- combination events) bioinformatics package (Brinkman et al., 2014, 2018) were used to determine the genotype of each blastocyst. Blastocysts were classified according to genotype as follows: carry- ing the wild-type (WT) sequence only (WT), carrying the target point mutation only (PM), carrying the desired point mutation as well as the WT sequence (PM + WT), carrying the desired point mutation with an insertion and/or deletion around the gRNA targeting site (PM + Indels), carrying more than one type of mutation (insertion and/or deletion) near the gRNA targeting site without the desired point mutation (Indels). Representative genomic sequences and the output of TIDER analysis of porcine blastocysts formed after elec- troporation of zygotes with Cas9, gRNA, and ssODN were shown in Figure S1.

2.6 | Experimental design

2.6.1 | Experiment 1: Efficiency of Scr7 for
introducing a point mutation A mixture of the gRNA targeting porcine KRAS, Cas9 protein, ssODN as a donor template, and Scr7 (Xcessbio Biosciences, Inc.) was used Introduction of a point mutation into porcine KRAS by zygote electroporation. (a) gRNA sequences targeting KRAS. (b) Single- stranded oligodeoxynucleotide (ssODN) sequences with various homology arm lengths. (c) Schematic illustration of the experimental design. The Cas9 protein, gRNA1 or 2 targeting porcine KRAS, and ssODN were introduced into porcine zygotes by electroporation. ssODN had homology arms of 20, 40, 60, or 80 bp. In this illustration, the ssODN40 sequence is shown. Two mutant nucleotides are indicated in red. The original two nucleotides in porcine KRAS are indicated in blue. Amino acid sequences flanking the target site before and after the introduction of the point mutation are also shown. The replaced amino acid (Alanine) is indicated in red to induce the desired point mutation in porcine zygotes by elec- troporation (Figure 1c). To evaluate the effects of the Scr7 concen- tration on the production of a point mutation, gRNA1 (Figure 1a), Cas9 protein, and 16 pmol/μl (412.6 µg/ml) ssODN40 (Figure 1b) with Scr7 (1 or 2 µM) or without Scr7 (0 µM) were electroporated into porcine putative zygotes at 12 hr after the start of IVF. After in vitro culture for 7 days, the resulting blastocysts were collected and genotyped, as described above.

2.6.2 | Experiment 2: Effect of the homology arm length of ssODN on the generation of a point mutation by electroporation

A previous study in mice demonstrated that the cutting site of gRNA affects the frequency of point mutation (Inui et al., 2014); therefore, gRNA2 was additionally designed with a targeting site different from that of gRNA1 (Figure 1a,c). To evaluate the effects of homology arm length of ssODN using two gRNAs on blastocyst formation and the production of a point mutation, gRNA1 or gRNA2, Cas9 protein, and 1 µM Scr7 with 16 pmol/μl ssODN20 (208.9 µg/ml), ssODN40 (412.6 µg/ml), ssODN60 (605.4 µg/ml), or ssODN80 (803.1 µg/ml; i.e., ssODN with homology arms of 20, 40, 60, or 80 bp; Figure 1b) were electroporated into porcine putative zygotes at 12 hr after the start of IVF. The Scr7 concentration that was effective for the pro- duction of a point mutation in Experiment 1 was used in this experi- ment. After in vitro culture for 7 days, the resulting blastocysts were collected and genotyped, as described above. As a control, some zygotes were cultured with PZM-5 and PBM for 7 days without electroporation.

2.7 | Statistical analysis

All percentages were subjected to arcsine transformation before performing analysis of variance (ANOVA). The transformed data were tested by ANOVA, followed by Fisher's protected least sig- nificant difference (LSD) test, using StatView software (Abacus Concepts). The percentages of blastocysts carrying mutation in the total number of blastocysts were analyzed by chi-squared analysis with Fisher's exact test. Differences with a probability value (p) of 0.05 or less were considered statistically significant.

3 | RESULTS

Various Scr7 concentrations were evaluated for the production of a point mutation in porcine zygotes by electroporation. There were no significant differences in the percentages of blastocysts carrying mu- tations with respect to the Scr7 concentration (Figure 2). Of zygotes electroporated with Scr7, 20.0% (1 µM Scr7) and 26.4% (2 µM Scr7) of examined blastocysts carried a point mutation as well as the WT sequence or indels. Of zygotes electroporated without Scr7, 13.3% Genotypes of blastocysts derived from zygote electroporated Cas9 protein, gRNA targeting KRAS, and ssODN40 with different concentrations of Scr7. The percentage of blastocysts carrying each mutation type is shown. Numbers in parentheses indicate the total number of blastocysts examined. PM + WT: blastocyst carrying a point mutation with the wild-type sequence, PM + Indels; blastocyst carrying a point mutation with an insertion and/or deletion around the gRNA target site, Indels: blastocyst carrying an insertion and/or deletion around the gRNA target site of examined blastocysts carried a point mutation, as well as the WT sequence or indels. The Sanger sequencing and TIDE analysis showed that most of the resulting blastocysts had multiple alleles, indicating that they had mosaic mutations. The percentages of mosaic blasto- cysts carrying more than three kinds of alleles from zygotes elec- troporated with 1 µM Scr7, with 2 µM Scr7, and without Scr7 were 28.6%, 29.4%, and 33.3%, respectively. There were no significant dif- ferences in the percentages of mosaic blastocysts carrying more than three kinds of alleles with respect to the Scr7 concentration. Based on these observations, 1 µM of Scr7 was used in Experiment 2.

There were no significant differences in the blastocyst formation rates of electroporated zygotes between groups with different homol- ogy arm lengths of ssODN, irrespective of the gRNA (Figures 3a and 4a). However, electroporation treatment with ssODN significantly de- creased (p < .05) the blastocyst formation rates of zygotes compared with that in the control group without electroporation, irrespective of the homology arm length of ssODNs and target site of gRNAs. Using gRNA1, 2.9% and 3.1% of blastocysts from zygotes electro- porated with ssODN20 and ssODN40, respectively, had only the desired point mutation (Figure 3b). Using gRNA2, 3.0%, 2.9%, 2.9%, and 3.1% of blastocysts from zygotes electroporated with ssODN20, ssODN40, ssODN60, and ssODN80, respectively, had only the point mutation (Figure 4b). Using gRNA1, the rates of blastocysts carrying a point mutation with or without the WT sequence and indels were significantly higher (p < .05) in the ssODN20 group (25.7%) than in the ssODN60 group (5.4%). The percentages of mosaic blastocysts

Blastocyst formation rates (a) and genotypes (b) after zygote electroporation of Cas9 protein, ssODN with different homology arm lengths, Scr7 and gRNA1 targeting KRAS. Numbers in parentheses indicate the total number of oocytes and embryos examined. PM; blastocyst carrying only a point mutation, PM + WT: blastocyst carrying a point mutation with the wild-type sequence, PM + Indels; blastocyst carrying a point mutation with an insertion and/or deletion around the gRNA target site, Indels: blastocyst carrying an insertion and/or deletion around the gRNA target site. Error bars indicate mean ± SEM (a). a,b,*, p < .05

Blastocyst formation rates (a) and genotypes (b) after zygote electroporation of Cas9, ssODN with different homology arm lengths, Scr7 and gRNA2 targeting KRAS. Numbers in parentheses indicate the total number of oocytes and embryos examined. PM; blastocyst carrying only a point mutation, PM + WT: blastocyst carrying a point mutation with the wild-type sequence, PM + Indels; blastocyst carrying a point mutation with an insertion and/or deletion around the gRNA target site, Indels: blastocyst carrying an insertion and/or deletion around the gRNA target site. Error bars indicate mean ± SEM (A). a,b,*, p < .05 carrying more than three kinds of alleles from zygotes electroporated with ssODN20, ssODN40, ssODN60, and ssODN80 were 28.6%, 18.8%, 13.5%, and 25.0%, respectively. Using gRNA2, the rates of blastocysts carrying a point mutation with or without the WT se- quence and indels were significantly higher (p < .05) in the ssODN20 group (45.5%) than in the ssODN60 (5.9%) and ssODN80 (15.6%) groups. The percentages of mosaic blastocysts carrying more than three kinds of alleles from zygotes electroporated with ssODN20, ssODN40, ssODN60, and ssODN80 were 6.1%, 14.7%, 2.9%, and 3.1%, respectively. Using gRNA1 and 2, there were no significant dif- ferences in the percentages of mosaic blastocysts carrying more than three kinds of alleles with respect to the ssODN group.

4 | DISCUSSION

In this study, we demonstrated the utility of the CRISPR/Cas9 sys-
tem with ssODN via electroporation for generating specific point mutations in KRAS in porcine blastocysts. Theoretically, the intro- duction of DSBs by the CRISPR/Cas9 system is restored by the major DNA repair pathways, NHEJ and HDR (Hu et al., 2018). The NHEJ pathway has been characterized as template-independent and di- rectly brings two DNA ends together via DNA ligase IV, often resulting in indel mutations by repair errors (Brissett et al., 2011; Lieber, 2010). In contrast, HDR involves a series of pathways with high-fidelity in a template-dependent manner (Li & Heyer, 2008; Nardi et al., 2020) where double-stranded DNA fragments or the ssODN can work as templates, leading to a change in the nucleotide sequence or point mutation (Wu et al., 2013; Yang, Wang, et al., 2013). Competition between the NHEJ and HDR pathways for DSB repair has been ob- served (Shrivastav et al., 2008). Moreover, the presence of a DNA ligase IV inhibitor and ssODN could favor the HDR pathway over the NHEJ pathway (Hu et al., 2018). Therefore, it is necessary to optimize the factors affecting the efficiency of CRISPR/Cas9 systems, such as the concentration of the DNA ligase IV inhibitor and the homology arm length of ssODN, as we did in this study.

The use of Scr7, a specific inhibitor of DNA ligase IV, is a re- cent advance in gene editing technology (Hu et al., 2018; Vartak & Raghavan, 2015). In our study, Scr7 could not significantly improve the ratio of blastocysts carrying a point mutation, irrespective of the concentration. These findings are in an agreement with those of a previous study showing that HDR efficiency in Chinese ham- ster ovary cells is not affected by Scr7 treatment (Lee et al., 2016). This may be explained by protective mechanisms against chemical manipulation or the occurrence of spontaneous alternative NHEJ pathways (Ceccaldi et al., 2015; Lee et al., 2016; Mateos-Gomez et al., 2015). However, another study has demonstrated that the op- timal concentration of Scr7 for HDR pathway activity in mammalian cells is 1 µM (Chu et al., 2015; Maruyama et al., 2015). When consid- ering the genotypes of individual blastocysts in this study, it should be noted that 20.0% and 26.4% of examined blastocysts showed a point mutation in KRAS when the concentrations of Scr7 were 1 and 2 µM, respectively, even though they contained cells harboring ei- ther the WT sequence or indel mutation (Figure 2). This suggests that the inhibition of DNA ligase IV by optimizing the dosage of Scr7 still has the potential to improve HDR pathway activity in gene- edited porcine zygotes, although further studies are needed. Even though there were no significant differences in the percentages of blastocysts carrying point mutations with respect to the Scr7 con- centration, a Scr7 concentration of 1 µM was chosen for subsequent experiments in this study based on previous studies in mammalian cells and mouse zygotes (Chu et al., 2015; Maruyama et al., 2015).

The other crucial determinant of HDR efficiency is the length of ssODN, which can influence the homology between the template and target site (Wang et al., 2016). To assess the effect of the homology arm length of ssODN using two gRNAs on embryonic development and the mutation frequency, four ssODN templates with different lengths of homology arms were electroporated into the porcine pu- tative zygotes. We found that electroporation treatment with ssODN significantly decreased the blastocyst formation rate, irrespective of the homology arm length of ssODNs or the target site of gRNAs. In our previous study, we reported that the target site of gRNA af- fects the rate of blastocyst formation in zygotes (Hirata, Wittayarat, et al., 2019), whereas the concentration of Cas9 protein has no appar- ent effects on embryonic development (Le et al., 2020). Moreover, we reported that the electroporation condition used in this study (five 1-ms square pulses at 25 V) has no negative effect on embry- onic development (Nishio et al., 2018). However, to our knowledge, the effect of KRAS mutation on embryonic development until blas- tocyst formation has not been reported in pigs.

In mice, homozygous K-ras−/− embryos die progressively by embryonic day 12.5 (Esteban et al., 2001), indicating that the effects of KRAS mutation on embry- onic development may appear following implantation. Therefore, the addition of ssODN may affect the development of electroporated zy- gotes, as evidenced by the decrease in the blastocyst formation rate, irrespective of the target site of gRNA. However, the frequencies of blastocysts harboring a point mutation with or without the WT se- quence and/or indels were significantly higher in the ssODN20 group than in the ssODN60 group when gRNA1 was used (Figure 3b), andthose were also significantly higher in the ssODN20 group than in the ssODN60 and ssODN80 groups when gRNA2 was used (Figure 4b). Although the elongation of ssODNs arms is thought to prevent the loss of homology between the template and target site due to exonu- clease degradation activity, it may affect secondary structures of the oligonucleotides and lead to the depletion of donor resources avail- able for HDR (Howden et al., 2015; Wang et al., 2016; Yang, Guell, et al., 2013). Indeed, using the ssODN20 and ssODN40 templates with gRNA1, we obtained point mutations without the WT sequence and indels in porcine KRAS at HDR frequencies of 2.9% and 3.1%, respectively, whereas further length increases failed to improve the HDR efficiency. Similar to our study, previous reports have revealed that further elongation of ssODN homology arms does not increase the HDR frequency in gene-modified pigs (Song et al., 2016; Wang et al., 2016). When gRNA2 was used, the numbers of blastocysts carrying point mutations without the WT sequence and indels were not affected by the length of the ssODN. Nevertheless, the higher frequency of blastocysts harboring a point mutation with or without the WT sequence and/or indels observed in the ssODN20 group is consistent with that observed in the previous studies. These results indicate that an adequate homology arm length of ssODN is a key parameter for promoting HDR pathway activity.

In conclusion, we showed the possibility that the DNA ligase IV inhibitor Scr7 and a ssODN homology arm with an adequate length could improve the outcome of CRISPR/Cas9-assisted ssODN- mediated point mutation via electroporation in pigs. Although we achieved the generation of KRAS point mutations, most blastocysts still showed incomplete HDR repair, including the WT sequence or indel mutations. To improve the efficacy of this system, further investigations are thus required, including the optimization of the ssODN concentration.

ACKNOWLEDG MENTS
We thank the Nippon Food Packer, K. K. Shikoku (Tokushima, Japan), for supplying pig ovaries. This work was supported in part by the Program of Open Innovation Platform with Enterprises, Research Institute and Academia (OPERA) grant number JPMJOP1613 from the Japan Science and Technology Agency (JST), the JSPS KAKENHI Grant Numbers 18K12062 and JP19K16014, and the Research Clusters program of Tokushima University.

CONFLIC T OF INTEREST
The authors declare no Conflict of Interests for this article.

DATA AVAIL ABILIT Y STATEMENT
All data generated or analyzed during this study are included in this published article.

ImageImageORCID
ImageImageMaki Hirata https://orcid.org/0000-0003-4673-1581 Yoko Sato https://orcid.org/0000-0003-1683-8505 Nhien T. Nguyen https://orcid.org/0000-0001-9361-3217
Fuminori Tanihara https://orcid.org/0000-0002-2158-5808

R EFER EN CE S
Armstrong, G. A., Liao, M., You, Z., Lissouba, A., Chen, B. E., & Drapeau,
P. (2016). Homology directed knockin of point mutations in the zebrafish tardbp and fus genes in ALS using the CRISPR/Cas9 system. PLoS One, 11, e0150188. https://doi.org/10.1371/journ al.pone.0150188
Brinkman, E. K., Chen, T., Amendola, M., & van Steensel, B. (2014). Easy quantitative assessment of SCR7 genome editing by sequence trace decomposition. Nucleic Acids Research, 42, e168. https://doi. org/10.1093/nar/gku936
Brinkman, E. K., Kousholt, A. N., Harmsen, T., Leemans, C., Chen, T., Jonkers, J., & van Steensel, B. (2018). Easy quantification of template-directed CRISPR/Cas9 editing. Nucleic Acids Research, 46, e58. https://doi.org/10.1093/nar/gky164
Brissett, N. C., Martin, M. J., Pitcher, R. S., Bianchi, J., Juarez, R., Green,
A. J., Fox, G. C., Blanco, L., & Doherty, A. J. (2011). Structure of a preternary complex involving a prokaryotic NHEJ DNA poly- merase. Molecular Cell, 41, 221–231. https://doi.org/10.1016/j. molcel.2010.12.026
Capecchi, M. R. (1989). Altering the genome by homologous recombi- nation. Science, 244, 1288–1292. https://doi.org/10.1126/scien ce.2660260
Ceccaldi, R., Liu, J. C., Amunugama, R., Hajdu, I., Primack, B., Petalcorin,
M. I., & D’Andrea, A. D. (2015). Homologous-recombination-deficient tumours are dependent on Poltheta-mediated repair. Nature, 518, 258–262. https://doi.org/10.1038/nature14184
Chu, V. T., Weber, T., Wefers, B., Wurst, W., Sander, S., Rajewsky, K., & Kuhn, R. (2015). Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nature Biotechnology, 33, 543–548. https://doi.org/10.1038/ nbt.3198
Esteban, L. M., Vicario-Abejón, C., Fernández-Salguero, P., Fernández- Medarde, A., Swaminathan, N., Yienger, K., Lopez, E., Malumbres, M., McKay, R., Ward, J. M., Pellicer, A., & Santos, E. (2001). Targeted ge- nomic disruption of H-ras and N-ras, individually or in combination, reveals the dispensability of both loci for mouse growth and devel- opment. Molecular and Cellular Biology, 21, 1444–1452. https://doi. org/10.1128/MCB.21.5.1444-1452.2001
Frit, P., Barboule, N., Yuan, Y., Gomez, D., & Calsou, P. (2014). Alternative end-joining pathway(s): Bricolage at DNA breaks. DNA Repair, 17, 81– 97. https://doi.org/10.1016/j.dnarep.2014.02.007
Hirata, M., Tanihara, F., Wittayarat, M., Hirano, T., Nguyen, N. T., Le,
Q. A., Namula, Z., Nii, M., & Otoi, T. (2019). Genome mutation after introduction of the gene editing by electroporation of Cas9 pro- tein (GEEP) system in matured oocytes and putative zygotes. Vitro Cellular & Developmental Biology Animal, 55, 237–242. https://doi. org/10.1007/s11626-019-00338-3
Hirata, M., Wittayarat, M., Hirano, T., Nguyen, N. T., Le, Q. A., Namula, Z., Fahrudin, M., Tanihara, F., & Otoi, T. (2019). The relationship between embryonic development and the efficiency of target mutations in porcine endogenous retroviruses (PERVs) Pol genes in porcine em- bryos. Animals, 9, 593. https://doi.org/10.3390/ani9090593
Howden, S. E., Maufort, J. P., Duffin, B. M., Elefanty, A. G., Stanley, E. G., & Thomson, J. A. (2015). Simultaneous reprogramming and gene correction of patient fibroblasts. Stem Cell Reports, 5, 1109–1118. https://doi.org/10.1016/j.stemcr.2015.10.009
Hu, Z., Shi, Z., Guo, X., Jiang, B., Wang, G., Luo, D., Chen, Y., & Zhu, Y.-S. (2018). Ligase IV inhibitor SCR7 enhances gene editing directed by CRISPR-Cas9 and ssODN in human cancer cells. Cell & Bioscience, 8, 12. https://doi.org/10.1186/s13578-018-0200-z
Inui, M., Miyado, M., Igarashi, M., Tamano, M., Kubo, A., Yamashita, S., Asahara, H., Fukami, M., & Takada, S. (2014). Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system. Scientific Reports, 4, 5396. https://doi.org/10.1038/srep0
5396
Jancik, S., Drabek, J., Radzioch, D., & Hajduch, M. (2010). Clinical rel- evance of KRAS in human cancers. Journal of Biomedicine and Biotechnology, 2010, 1–13. https://doi.org/10.1155/2010/150960
Kanaar, R., Hoeijmakers, J. H., & van Gent, D. C. (1998). Molecular mech- anisms of DNA double strand break repair. Trends in Cell Biology, 8, 483–489. https://doi.org/10.1016/S0962-8924(98)01383-X
Kaneko, T., Sakuma, T., Yamamoto, T., & Mashimo, T. (2014). Simple knockout by electroporation of engineered endonucleases into in- tact rat embryos. Scientific Reports, 4, 6382. https://doi.org/10.1038/ srep06382
LaConti, J. J., Shivapurkar, N., Preet, A., Deslattes Mays, A., Peran, I., Kim,
S. E., Marshall, J. L., Riegel, A. T., & Wellstein, A. (2011). Tissue and serum microRNAs in the Kras(G12D) transgenic animal model and in patients with pancreatic cancer. PLoS One, 6, e20687. https://doi. org/10.1371/journal.pone.0020687
Le, Q. A., Hirata, M., Nguyen, N. T., Takebayashi, K., Wittayarat, M., Sato, Y., & Otoi, T. (2020). Effects of electroporation treatment using different concentrations of Cas9 protein with gRNA target- ing Myostatin (MSTN) genes on the development and gene editing of porcine zygotes. Animal Science Journal, 91, e13386. https://doi. org/10.1111/asj.13386
Lee, J. S., Grav, L. M., Pedersen, L. E., Lee, G. M., & Kildegaard, H. F. (2016). Accelerated homology-directed targeted integration of transgenes in Chinese hamster ovary cells via CRISPR/Cas9 and flu- orescent enrichment. Biotechnology and Bioengineering, 113, 2518– 2523. https://doi.org/10.1002/bit.26002
Li, S., Edlinger, M., Saalfrank, A., Flisikowski, K., Tschukes, A., Kurome, M., Zakhartchenko, V., Kessler, B., Saur, D., Kind, A., Wolf, E., Schnieke, A., & Flisikowska, T. (2015). Viable pigs with a conditionally-activated oncogenic KRAS mutation. Transgenic Research, 24, 509–517. https:// doi.org/10.1007/s11248-015-9866-8
Li, X., & Heyer, W. D. (2008). Homologous recombination in DNA repair and DNA damage tolerance. Cell Research, 18, 99–113. https://doi. org/10.1038/cr.2008.1
Lieber, M. R. (2010). The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annual Review of Biochemistry, 79, 181–211. https://doi.org/10.1146/annurev.bioch em.052308.093131
Liu, M., Rehman, S., Tang, X., Gu, K., Fan, Q., Chen, D., & Ma, W. (2018). Methodologies for improving HDR efficiency. Frontiers in Genetics, 9, 691. https://doi.org/10.3389/fgene.2018.00691
Maruyama, T., Dougan, S. K., Truttmann, M. C., Bilate, A. M., Ingram, J. R., & Ploegh, H. L. (2015). Increasing the efficiency of precise genome ed- iting with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nature Biotechnology, 33, 538–542. https://doi.org/10.1038/nbt.3190 Mateos-Gomez, P. A., Gong, F., Nair, N., Miller, K. M., Lazzerini-Denchi, E., & Sfeir, A. (2015). Mammalian polymerase theta promotes alter-
native NHEJ and suppresses recombination. Nature, 518, 254–257s Moody, S. A. (2018). Microinjection of mRNAs and oligonucleotides.
Cold Spring Harbor Protocols, 2018(12), pdb.prot097261. https://doi. org/10.1101/pdb.prot097261
Naito, Y., Hino, K., Bono, H., & Ui-Tei, K. (2015). CRISPRdirect: Software for designing CRISPR/Cas guide RNA with reduced off-target sites. Bioinformatics, 31, 1120–1123. https://doi.org/10.1093/bioinforma tics/btu743
Nardi, I. K., Stark, J. M., Larsen, A., Salgia, R., & Raz, D. J. (2020). USP22 interacts with PALB2 and promotes chemotherapy resistance via homologous recombination of DNA double-strand breaks. Molecular Cancer Research, 18, 424–435. https://doi.org/10.1158/1541-7786. MCR-19-0053
Nguyen, T.-V., Tanihara, F., Do, L., Sato, Y., Taniguchi, M., Takagi, M., Van Nguyen, T., & Otoi, T. (2017). Chlorogenic acid supplementa- tion during in vitro maturation improves maturation, fertilization and developmental competence of porcine oocytes. Reproduction in Domestic Animals, 52, 969–975. https://doi.org/10.1111/rda.13005

Nishio, K., Tanihara, F., Nguyen, T.-V., Kunihara, T., Nii, M., Hirata, M., Takemoto, T., & Otoi, T. (2018). Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos. Reproduction in Domestic Animals, 53, 313–318. https://doi.org/10.1111/rda.13106
Peeters, M., Douillard, J. Y., Van Cutsem, E., Siena, S., Zhang, K., Williams, R., & Wiezorek, J. (2013). Mutant KRAS codon 12 and 13 alleles in patients with metastatic colorectal cancer: Assessment as prognos- tic and predictive biomarkers of response to panitumumab. Journal of Clinical Oncology, 31, 759–765.
Port, F., Chen, H. M., Lee, T., & Bullock, S. L. (2014). Optimized CRISPR/ Cas tools for efficient germline and somatic genome engineering in Drosophila. Proceedings of the National Academy of Sciences of the United States of America, 111, E2967–E2976. https://doi.org/10.1073/ pnas.1405500111
Rong, Z., & Xu, Y. (2018). Genome editing of pluripotent stem cells. In
K. Appasani (Ed.), Genome editing and engineering: From TALENs, ZFNs and CRISPRs to molecular surgery (pp. 270–284). Cambridge University Press.
Schook, L. B., Collares, T. V., Hu, W., Liang, Y., Rodrigues, F. M., Rund, L. A., Schachtschneider, K. M., Seixas, F. K., Singh, K., Wells, K. D., Walters,
E. M., Prather, R. S., & Counter, C. M. (2015). A genetic porcine model of cancer. PLoS One, 10, e0128864. https://doi.org/10.1371/journ al.pone.0128864
Shrivastav, M., De Haro, L. P., & Nickoloff, J. A. (2008). Regulation of DNA double-strand break repair pathway choice. Cell Research, 18, 134–147. https://doi.org/10.1038/cr.2007.111
Song, J., Yang, D., Xu, J., Zhu, T., Chen, Y. E., & Zhang, J. (2016). RS-1
enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nature Communications, 7, 10548. https://doi.org/10.1038/ncomm s10548
Srivastava, M., Nambiar, M., Sharma, S., Karki, S. S., Goldsmith, G., Hegde, M., Kumar, S., Pandey, M., Singh, R. K., Ray, P., Natarajan, R., Kelkar, M., De, A., Choudhary, B., & Raghavan, S. C. (2012). An inhibitor of nonhomologous end-joining abrogates double-strand break repair and impedes cancer progression. Cell, 151, 1474–1487. https://doi. org/10.1016/j.cell.2012.11.054
Tanihara, F., Hirata, M., Nguyen, N. T., Le, Q. A., Hirano, T., Takemoto, T.,
& Otoi, T. (2019). Generation of PDX-1 mutant porcine blastocysts by
S., Scala, E., Veronese, S., Laurent-Puig, P., Siena, S., Tejpar, S., Mottolese, M., Punt, C. J. A., Gambacorta, M., … Di Nicolantonio, F. (2013). KRAS gene amplification in colorectal cancer and impact on response to EGFR-targeted therapy. International Journal of Cancer, 133, 1259–1265. https://doi.org/10.1002/ijc.28106
Vartak, S. V., & Raghavan, S. C. (2015). Inhibition of nonhomologous end joining to increase the specificity of CRISPR/Cas9 genome editing. FEBS Journal, 282, 4289–4294. https://doi.org/10.1111/febs.13416
Vodička, P., Smetana, K., Dvořánková, B., Emerick, T., Xu, Y. Z., Ourednik, J., Ourednik, V., & Motlík, J. (2005). The miniature pig as an animal model in biomedical research. Annals of the New York Academy of Sciences, 1049, 161–171. https://doi.org/10.1196/annals.1334.015
Wang, K., Tang, X., Liu, Y., Xie, Z., Zou, X., Li, M., Yuan, H., Ouyang, H., Jiao, H., & Pang, D. (2016). Efficient generation of orthologous point mutations in pigs via CRISPR-assisted ssODN-mediated homology- directed repair. Molecular Therapy – Nucleic Acids, 5, e396. https://doi. org/10.1038/mtna.2016.101
Wu, Y., Liang, D., Wang, Y., Bai, M., Tang, W., Bao, S., Yan, Z., Li, D., & Li, J. (2013). Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell, 13, 659–662. https://doi.org/10.1016/j. stem.2013.10.016
Yang, H., Wang, H., Shivalila, C. S., Cheng, A. W., Shi, L., & Jaenisch, R. (2013). One-step generation of mice carrying reporter and condi- tional alleles by CRISPR/Cas-mediated genome engineering. Cell, 154, 1370–1379. https://doi.org/10.1016/j.cell.2013.08.022
Yang, L., Guell, M., Byrne, S., Yang, J. L., De Los Angeles, A., Mali, P.,
Aach, J., Kim-Kiselak, C., Briggs, A. W., Rios, X., Huang, P.-Y., Daley, G., & Church, G. (2013). Optimization of scarless human stem cell genome editing. Nucleic Acids Research, 41, 9049–9061. https://doi. org/10.1093/nar/gkt555
Zhao, P., Zhang, Z., Ke, H., Yue, Y., & Xue, D. (2014). Oligonucleotide- based targeted gene editing in C. elegans via the CRISPR/Cas9 sys- tem. Cell Research, 24, 247–250. https://doi.org/10.1038/cr.2014.9