Summary of production of genome-edited pigs.
Recently, a series of genome editing technologies including ZFNs, TALENs, and CRISPR/Cas9 systems have enabled gene modification in the endogenous target genes of various organisms including pigs, which are important for agricultural and biomedical research. Owing to its simple application for gene knockout and ease of use, the CRISPR/Cas9 is now in common use worldwide. The most important aspect of this process is the selection of the method used to deliver genome editing components to embryos. In earlier stages, zygote microinjection of these components [single guide RNA (sgRNA) + DNA/mRNA for Cas9] into the cytoplasm and/or nuclei of a zygote has been frequently employed. However, this method is always associated with the generation of mosaic embryos in which genome-edited and unedited cells are mixed together. To avoid this mosaic issue, in vitro electroporation of zygotes in the presence of sgRNA mixed with Cas9 protein, referred to as a ribonucleoprotein (RNP), is now in frequent use. This review provides a historical background of the production of genome-edited pigs and also presents current research concerning how genome editing is induced in somatic cell nuclear transfer-derived embryos that have been reconstituted with normal nuclei.
- genome editing
- gene modification
- somatic cell nuclear transfer
- knock out
- knock in
The domestic pig has been widely used as a large animal model in biomedical research, as it is similar to humans with respect to the size of body and internal organs, longevity, anatomy, physiology, and metabolic profile . Modification of the porcine genome is also important for studying the mechanisms underlying genetic disorders, developing therapeutic drugs, and improving pig meat production yields [2, 3]. Over the past three decades, attempts have been made to modify the porcine genome using genetic engineering technology, starting after Gordon et al.  first reported DNA microinjection (MI)-based production of transgenic (Tg) mice. Hammer et al.  first reported the successful production of Tg piglets using the technique reported by Gordon et al. , but attaining this result was more difficult than for rodents, where pronuclei are clearly visible using an optical microscope. In the case of porcine zygotes, pronuclei are difficult to see due to the presence of high lipid content in the cytoplasm. Researchers must briefly centrifuge zygotes to visualize the pronuclei prior to MI , which is labor-intensive and requires skill. Moreover, MI-mediated transgene integration into host chromosomes occurs randomly, which often causes gene silencing . However, for precise and efficient genetic modification in the porcine genome, homologous recombination (HR)-based gene targeting technology may be recommended, which was first developed by Smithies’ group in mice . In this case, the use of germline-competent embryonic stem (ES) cells is a prerequisite. These ES cells are first transfected with a targeting vector and then recombinant ES clones showing successful targeting are obtained. This vector usually contains a gene of interest (GOI) to be integrated into the target locus, together with a selection marker gene such a neomycin resistance gene (
In 1996, scientists at
However, this situation drastically changed when new gene-targeting technologies emerged for precisely manipulating mammalian genomes, called “second-generation genome editing.” These technologies require the design of site-specific engineered nucleases which can be zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) nucleases, all of which induce a double-stranded break (DSB) at a specific site in the genome. This DSB facilitates genetic modification such as nonhomologous end-joining (NHEJ) and homology directed repair (HDR) , as described below. Using these genome-editing systems, many GE piglets have been produced using SCNT of genome-edited cells, or direct microinjection of genome-editing components (including engineered endonucleases) into the cytoplasm of zygotes, as described below in more detail.
2. Background of second-generation genome editing
As mentioned above, site-specific engineered nucleases are used in these genome-editing techniques. ZFNs, TALENs, and CRISPR/Cas9 can all bind to DNA and induce DSB, which triggers endogenous DNA repair. If the template DNA is absent, the DSB is repaired via the NHEJ pathway where insertion or deletion of nucleotides (hereinafter called “indels”) can happen in the cleaved area. These indels often cause frameshift of the amino acid sequence, leading to the generation of abnormal proteins or formation of a premature stop codon leading to cessation of protein synthesis. If template DNA homologous to the target site is present, it is inserted into the cleaved area via a site-specific HR event which is called HDR. Generally, NHEJ occurs in cells independent of its cell cycle, but HDR occurs primarily in dividing cells .
The ZFN technique uses the ZF protein (which binds to the target DNA) and the endonuclease
The TALEN technique uses proteins, termed transcription activator-like effectors (TALEs), which contain 33–35 amino acid repeats that flank a central DNA binding region (amino acids 12 and 13), and
CRISPR/Cas9 employs a short (20 bp) RNA sequence called single-guide RNA (sgRNA) which can bind to the specific chromosomal DNA site together with the Cas9 endonuclease [37, 38, 39, 40]. Once bound, two independent nuclease domains in Cas9 each cleave one of the DNA strand’s three bases upstream of the protospacer adjacent motif (PAM), introducing DSB at the target site of the host chromosome, which is then repaired by NHEJ. This system is different from the other genome editing tools such as ZFNs and TALENs, and thus synthesis of sgRNA is a
3. History of GE in pigs
Table 1 lists instances of production of GE piglets with genome editing technology from 2011 to 2018. This section provides a brief explanation on the background of GE pig production.
|Method||Genome editing tool (mode for gene modification)||Method for gene modification||Outcome||Target gene||References|
|SCNT||ZFN (indels)||Using adult porcine ear fibroblasts hemizygous for the eGFP transgene||Seven of nine embryos (Day 12) exhibited loss of fluorescence|||
|SCNT||ZFN (indels)||Using porcine fibroblasts transfected with ZFN plasmid||Of 10 live piglets delivered, two carried the predicted ZFN-induced mutation; lower expression of both |||
|SCNT||ZFN (indels)||Using porcine fetal fibroblasts transfected with ZFN plasmid||Of six fetuses, all completely lacked α-Gal epitopes|||
|MI||TALEN (indels)||Cytoplasmic MI of TALEN mRNA toward IVF-derived zygotes||CI of TALEN mRNAs inducing gene KO in up to 75% of embryos; Of the 18 live-born clones, eight contained monoallelic mutations and 10 contained biallelic modifications of the |||
|MI||ZFN, TALEN (indels)||Cytoplasmic MI of ZFN or TALEN mRNA toward ||Of 39 piglets produced, eight carried TALEN-derived editing events (21%); of nine piglets produced, one carried an editing event at the ZFN target site (11%)|||
|SCNT||TALENs (indels)||Using porcine fetal fibroblasts transfected with TALEN plasmid||Three piglets with biallelic mutations of the |||
|SCNT||TALENs (indels/KI)||Using porcine fibroblasts transfected with TALEN mRNA + ssODN||Of eight piglets born from |||
|SCNT||ZFNs (indels)||Using porcine fetal fibroblasts transfected with ZFN mRNA||The resulting |||
|SCNT||ZFN (indels)||Using porcine adult liver-derived cells transfected with ZFN plasmid through the two-steps||Four viable and healthy cloned pigs obtained exhibited disruption of the |||
|SCNT||ZFN (KI)||Using porcine fibroblast cells transfected with ZFN plasmid and donor DNA||Successfully produced healthy monoallelic/biallelic CMAH KO pigs|||
|SCNT/MI||CRISPR/Cas9 (KI/indels)||Using porcine fetal fibroblasts transfected with sgRNA-Cas9 plasmids + donor DNA/cytoplasmic MI of Cas9 mRNA/sgRNA toward IVF-derived zygotes||Of the |||
|MI||CRISPR/Cas9 (indels)||Cytoplasmic MI of Cas9 mRNA/sgRNA toward ||Ten of 16 resulting piglets had indels with an efficiency of 63% and were comprised by cells with monoallelic mutant; they can be a model for von Willebrand disease|||
|SCNT||CRISPR/Cas9 (indels)||Using porcine fetal fibroblasts transfected with Cas9/sgRNA expression plasmid||A total of three piglets were obtained; fibroblasts from all three animals were negative for class I SLA cell surface expression|||
|SCNT||TALENs (indels)||Using pig fetal fibroblasts transfected with TALEN plasmid||Of 27 live cloned piglets obtained, nine were targeted with biallelic mutations in |||
|SCNT||ZFN (indels)||Using pig fetal fibroblasts transfected with ZFN plasmid||Three |||
|SCNT||CRISPR/Cas9 (indels)||Using pig liver-derived cells transfected with two or three plasmids expressing Cas9 and sgRNA targeting to GGTA1, CMAH, or putative iGb3S genes||Of 10 fetuses obtained, five had mutations in both the |||
|SCNT||ZFNs (indels)||Using pig fetal fibroblasts transfected with ZFN plasmid||The |||
|SCNT||TALENs (indels)||Using pig liver-derived cells transfected with TALEN plasmid||Livers from |||
|SCNT||ZFN/TALEN (indels)||Using pig fetal fibroblasts transfected with TALEN or ZFN mRNA||One of the cloned pigs generated GalT/CMAH-double homozygous KO pigs|||
|SCNT||CRISPR/Cas9 (KI)||Using pig fetal fibroblasts transfected with targeting donor vector and two expression vectors for sgRNA and Cas9||Highly efficient KI (up to 54%) was achieved after drug selection; one cloned piglet obtained showed correct targeting|||
|SCNT||CRISPR/Cas9||Using pig fetal fibroblasts transfected with sgRNA, Cas9 expression plasmids||Four cloned double KO piglets showing loss of expression for both |||
|MI||CRISPR/Cas9 (KI)||Cytoplasmic MI of Cas9 mRNA + sgRNA + ssODN toward ||Two live-born piglets obtained showed the white coat-color phenotype over its entire body|||
|MI||CRISPR/Cas9 (KI)||Cytoplasmic MI of Cas9 mRNA + sgRNA + circular vector toward ||All 16 piglets born were healthy and carried the expected KI allele; the KI allele was successfully transmitted through germline|||
|MI||CRISPR/Cas9 (indels)||Cytoplasmic MI of Cas9 mRNA and sgRNA toward ||Bi-allelic modifications of pig |||
|SCNT||CRISPR/Cas9 (indels)||Using pig fetal fibroblasts transfected with sgRNA-Cas9 encoding vector||Of eight marker-gene-free cloned pigs with biallelic mutations obtained, some showed phenotypes similar to DM|||
|SCNT||CRISPR/Cas9 (indels)||Using liver-derived cells transfected with sgRNA-Cas9 encoding vectors||One triple knockout pig was obtained; Cells from this cloned pig exhibited reduced human IgM and IgG binding|||
|SCNT||CRISPR/Cas9 (indels)||Using pig fetal fibroblasts transfected with sgRNA-Cas9 encoding vector||Three cloned piglets with biallelic mutation produced showed no antibody-producing B cells|||
|EP||CRISPR/Cas9 (indels)||Using Cas9 protein and sgRNA (RNP) toward IVF-derived zygotes||The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and efficient production of |||
|MI||CRISPR/Cas9 (indels)||Cytoplasmic MI of Cas9 mRNA and sgRNA toward ||Of two piglets obtained, one piglets exhibited |||
|SCNT||ZFN (indels)||Using pig fetal fibroblasts transfected with ZFN-encoding mRNA||The heterozygous |||
|SCNT||CRISPR/Cas9 (KI)||Using pig fetal fibroblasts transfected with Cas9-sgRNA expression vector + donor DNA containing Cre/loxP system||Two male live piglets with mono-allelic |||
|SCNT||TALENs (indels)||Using pig fetal fibroblasts transfected with TALEN plasmids||In total, 12 live and two stillborn piglets were collected; all fetuses and piglets exhibited homozygous |||
|SCNT||TALENs (indels)||Using porcine fetal fibroblasts co-transfected with TALEN and hDAF expression plasmids||Six live-born piglets and three stillborn piglets were obtained; the piglets showed eight base mono-allelic mutations of |||
|SCNT||CRISPR/Cas9 (indels)||Using fetal fibroblast cells transfected with sgRNA-Cas9 encoding vector||Four live |||
|SCNT||TALENs (indels)||Using dermal fibroblasts transfected with TALEN plasmids|||
|SCNT||TALENs/Cas9 (KI)||Using fetal fibroblast cells transfected with Cas9/sgRNA or TALEN vector + ssODN||Of seven cloned piglets, some expressed human insulin|||
|SCNT||CRISPR/Cas9 (KI)||Using fetal fibroblast cells transfected with sgRNA-Cas9 encoding vector + ssODN||One cloned stillborn piglet harbored the orthologous p.C313Y mutation at the |||
|SCNT||TALENs (indels)||Using ear fibroblasts transfected with TALEN vectors||Thirty |||
|MI||CRISPR/Cas9 (KI)||Cytoplasmic MI of Cas9 mRNA + sgRNA + ssODN toward ||Of five piglets delivered alive, three exhibited pigmentary disorders with light-colored iris in eye, which was observed in patients harboring |||
|MI||CRISPR/Cas9 (indels)||Cytoplasmic MI of sgRNA-Cas9 encoding vector toward ||Of six healthy fetuses recovered, four exhibited loss of α-Gal epitope expression, indicating a biallelic KO of |||
|MI||CRISPR/Cas9 (indels)||Cytoplasmic MI of Cas9 mRNA + three types of sgRNAs toward ||Of two live-born piglets delivered, one piglet showed biallelic modification of all three genes, and another showed biallelic modification of the |||
|SCNT-MI||CRISPR/Cas9 (indels)||Cytoplasmic MI of RNP toward SCNT embryos||Six fetuses recovered revealed that all fetuses carried biallelic edits for the |||
|SCNT||CRISPR/Cas9 (indels)||Using kidney fibroblasts transfected with ZFN vectors||Two healthy normal females with |||
|MI||CRISPR/Cas9 (indels)||Cytoplasmic MI of Cas9 mRNA + sgRNA toward IVF-derived zygotes||Seventeen live piglets and two stillborn were produced; all had mutations in both genes (no pigs with wild-type sequence)|||
|MI||CRISPR/Cas9 (indels)||Cytoplasmic MI of Cas9 mRNA + sgRNA toward ||Eighteen piglets recovered showed either mono- or bi-allelic modifications and no wild-type animals; |||
|SCNT||CRISPR/Cas9 (indels)||Using fetal fibroblasts transfected with sgRNA and Cas9 expression vectors||Six biallelic KO pigs with mutations in |||
|SCNT||TALENs||Using fetal fibroblasts transfected with TALEN plasmid||All six live piglets obtained carried biallelic mutations in the |||
|SCNT||TALEN (indels)||Using fetal fibroblasts transfected with TALEN plasmid||A total of 18 live piglets were obtained; they showed hypermuscular characteristics|||
|SCNT||CRISPR/Cas9 (indels)||Using fetal fibroblasts transfected with sgRNA-Cas9 encoding vectors||A total of 37 PERV-inactivated piglets were generated; 15 piglets remain alive|||
|SCNT||CRISPR/Cas9 (indels)||Using fetal fibroblasts transfected with sgRNA-Cas9 encoding vectors||Of 26 female piglets delivered, 23 piglets carried mutations in the |||
|SCNT||CRISPR/Cas9 (KI)||Using fetal fibroblasts transfected with Cas9-gRNA plasmid and targeting vector||Twelve male piglets were born and expressed |||
|MI||CRISPR/Cas9 (KI)||Cytoplasmic MI of RNP toward ||A total of 18 fetuses/born piglets were obtained; successful insertion of pseudo |||
|MI||CRISPR/Cas9 (indels)||Cytoplasmic MI of Cas9 mRNA + sgRNAs toward ||Indels in 92–100% of the embryos analyzed; all resulting 12 piglets had biallelic edits of |||
|MI||CRISPR/Cas9 (indels)||Direct pronuclear microinjection of Cas9-gRNA plasmid||Of seven born piglets, one exhibited biallelic KO phenotype and one did monoallelic KO one|||
|MI||CRISPR/Cas9 (indels)||Cytoplasmic MI of Cas9 mRNA and dual sgRNAs toward ||Of nine fetuses examined, three exhibited bi-allelic mutations at the |||
|SCNT (Handmade cloning)||CRISPR/Cas9 (indels)||Using fetal fibroblasts transfected with sgRNA and Cas9 expression vectors||Eleven live bi-allelic |||
|EP||CRISPR/Cas9||Using Cas9 protein and sgRNA (RNP) toward IVF-derived zygotes||Of 11 piglets born, nine survived; six of nine carried mutations in |||
|SCNT||CRISPR/Cas9 (KI)||Using fetal fibroblasts transfected with sgRNA- Cas9 plasmid + donor DNA||Three piglets born grew and developed normally; all these piglets had |||
|SCNT||CRISPR/Cas9 (KI)||Using fetal fibroblasts transfected with sgRNA- Cas9 plasmid + donor DNA||Of seven naturally delivered piglets, six showed successful KI; the KI allele was successfully transmitted through germline|||
|SCNT||CRISPR/Cas9 (indels)||Using fetal fibroblasts transfected with sgRNA- Cas9 plasmid||Of a total of 17 piglets obtained, 12 appeared healthy; all had mutations at the target locus|||
In 2011, three types of GE piglets were produced using ZFNs from different laboratories. All of these piglets were produced by SCNT using GE cells as a SCNT donor. The first report showing successful production of GE piglets involved the disruption of enhanced green fluorescent protein (
The successful production of genome-edited piglets with bi-allelic KO genotype obtained after cytoplasmic MI of
Hai et al.  first demonstrated that GE pigs can be produced using the CRISPR/Cas9 system. They performed cytoplasmic MI with Cas9 mRNA and sgRNA targeted to
Successful knock-in (KI) of a GOI into the target locus was first reported in pigs by Ruan et al.  and Peng et al. . Zhou et al.  demonstrated the production of SCNT-treated piglets with mutations in multiple genes after a single transfection.
Fischer et al.  first succeeded in producing GE pigs by cytoplasmic MI of a Cas9 protein/gRNA complex called a ribonucleoprotein (RNP). Furthermore, GE pigs could be efficiently produced by
4. Delivery method
For the production of GE pigs, the choice of delivery method for genome editing components in porcine zygotes is important. As shown in Table 1, the methods for the production of GE pigs achieved by delivering genome editing reagents at earlier stages of development can be largely divided into four groups: the first is MI of genome editing reagents (in a form of DNA, mRNA or protein) into zygotes (Figure 1A); the second is SCNT using GE cells as the SCNT donor (Figure 1B); the third is
MI is an important tool in the creation of GE piglets. To date, about 30% (17/60) of studies (Table 1) have employed this approach. For example, in the case of MI with CRISPR/Cas9-related mRNA, a single cytoplasmic MI of 2–10 pL containing 125 ng/μL Cas9 mRNA and 12.5 ng/μL sgRNA was adopted . Yu et al.  employed Cas9 mRNA (20 ng/μL) and sgRNA (10 ng/μL) mixtures for cytoplasmic MI.
Is MI of these components deleterious to the development of porcine zygotes? According to Hai et al. , the
Selecting appropriate zygotes also appears to be an important factor in the production of GE pigs. For acquisition of viable zygotes, there are at least two methods. One is isolation of zygotes from oviducts of a female that has been inseminated, hereinafter called “
The frequent generation of individuals with mosaic genotypes is also a serious problem associated with MI-based GE pig production. Sato et al.  demonstrated that cytoplasmic MI of parthenogenetically activated porcine embryos (hereinafter called “parthenotes”) with Cas9 mRNA + sgRNA caused frequent mosaicism in the offspring (blastocysts) with cells with mixed genotype, so-called normal wild-type cells and mutated cells, when they were subjected to cytoplasmic MI immediately after oocyte activation. Notably, Carlson et al.  suggested that 100% of bovine embryos exhibited fluorescence expression after cytoplasmic MI of
Interestingly, Petersen et al.  demonstrated that cytoplasmic MI of DNA vectors coding for CRISPR/Cas9 targeting the porcine
SCNT using GE cells as an SCNT donor is another way to produce GE pigs. The merit of this approach is the use of
EP is known to be a useful and powerful gene delivery tool enabling transfer of exogenous substances (i.e., DNA) into a cell and was first applied to rat zygotes for genome editing by Kaneko et al. . Since then, many researchers have successfully induced gene edits by using this technology in mice [135, 136], bovines  and pigs . The merit of this technology is that it is simple, rapid and convenient for genome editing in zygotes, compared to the previous MI-based technique. Notably, about 30–50 zygotes can be edited with one pulse of EP. Furthermore, EP only requires a square pulse generator called an electroporator, and not a more expensive micromanipulator system.
As mentioned previously, Tanihara et al.  first applied EP to porcine IVF-derived zygotes and produced genome-edited pigs. They used CRISPR/Cas9-based RNP for knock-in of a target gene, and achieved reduced mosaicism and higher efficiency of genome-edited pig production with EP (30 V, square pulse 1.0 ms in duration repeated five times) using an electrode (#LF501PT1-20; BEX Co. Ltd., Tokyo, Japan) connected to a CUY21EDIT II electroporator (BEX Co. Ltd.). Notably, they reported no appreciable reduction in the developmental ability of the EP-treated embryos.
4.4 MI after SCNT
Although direct modification of zygotic genomes provides some advantages, SCNT also provides a significant advantage by permitting the isolation of cells containing precise modifications before the expense of animal production is incurred. As mentioned previously, Sheets et al.  successfully produced genome-edited cloned pigs by combining SCNT with CRISPR/Cas9 MI, which is beneficial for researchers as they do not need to manage a founder herd, and can eliminate the need for laborious
4.5 EP after SCNT
Similar to the approach shown by Sheets et al. , we tried to obtain cloned GE piglets through
SCNT-derived embryos were obtained by inserting fetal fibroblasts derived from microminiature pigs (MMP)  into the perivitelline space between enucleated porcine oocytes (derived from ovaries obtained from a slaughterhouse) and zona pellucida, according to the method described by Miyoshi et al.  (Figure 2B). The resulting SCNT-derived embryos were then subjected to electric activation following electric fusion between an egg and a cell (Figure 2B). Six or 12 h after activation, the SCNT-treated embryos were subjected to
First, we examined whether the
Second, we performed CRISPR/Cas9-based genome editing (targeted to the endogenous
|Stage at EP after activation of SCNT-treated embryos||EP condition2||Total number of SCNT-treated embryos examined||No. of embryos cleaved to the two-cell stage (%)||No. of embryos developed to blastocysts (%)|
|6 h||0.5||12||8 (66.7)||1 (8.3)|
|1.0||12||11 (91.7)||2 (16.7)|
|12 h||0.5||33||21 (63.6)||5 (15.2)|
|1.0||35||23 (65.7)||3 (8.6)|
5. Other techniques and factors affecting efficacy of the genome editing system
As shown above, genome editing tools such as ZFNs, TALENs and CRISPR/Cas9 are considered useful in enabling site-specific gene modification in livestock such as pigs. However, there are still several techniques and factors that influence performance which must be addressed. These include the single embryo assay, off-target cutting, multiplexed genome engineering, KI, and Cas9 pigs. In this section, these techniques or factors are described in greater detail.
5.1 Single embryo assay
To increasing the efficiency of genome editing systems, it is important to select suitable sets of ZFNs (or TALENs) or sgRNA (in the case of CRISPR/Cas9). Researchers therefore must check the efficiency of these reagents by introducing them into cultured cells, but at this point it remains unknown whether they will function
It may be required to confirm at a molecular level whether the genome-edited embryos have mutations. In this case, WGA has often been employed for amplifying the whole genome of an embryo (blastocyst) using genomic DNA isolated from a single embryo as the DNA template [140, 141], since the blastocyst DNA is often too small to generate a sufficient amount of PCR product. The effectiveness of WGA-based amplification of blastocyst DNA has already been confirmed by ours  and others . The resulting products obtained after PCR using WGA-derived DNA as the template are then subjected to direct sequencing for identification of possible mutations in the target gene, as shown in Figure 4B.
5.2 Off-target cleavage
Since sgRNA used in the CRISPR/Cas9 system can recognize only a short sequence (20 bp) at the target gene where Cas9 cleaves, other genes with a similar sequence to the sgRNA may be susceptible to Cas9-mediated DNA cleavage, which leads to the occasional generation of off-target cutting [29, 143]. This unintended cutting is considered a serious problem to be resolved.
Several strategies to minimize off-target cutting have been employed including the use of the double nickase mutant form of Cas9, which induces a single-strand break instead of DSB ; the use of RNP, whose half-life is shorter than the duration of transcription of plasmid or viral nucleic acids [110, 145]; or the fusion of catalytically inactive Cas9 with
Notably, in the case of GE pigs and embryos, there have been no reports of off-target mutagenesis as shown by the following papers: [43, 50, 61, 62, 63, 64, 69, 74, 77, 78, 80, 86, 100, 105]. This suggests a very low probability of off target-cleavage in GE pigs.
5.3 Multiplexed genome engineering
The CRISPR/Cas9 system can confer multigene KO in one shot of gene delivery [152, 153]. This property is especially beneficial for the purpose of creating disease model animals, as certain types of diseases are known to be caused by multigene defects. Interestingly, Sakurai et al.  demonstrated that at least nine endogenous genes can be knocked out simultaneously through a single shot of cytoplasmic MI of 12 sgRNAs together with Cas9 mRNA into murine zygotes. In pigs, Zhou et al.  demonstrated successful generation of PARK2 (
As shown in Table 1, in 2015 successful KI in pigs was reported by several groups. For example, Wang et al.  performed MI with
Ruan et al.  demonstrated production of GE pigs with successful KI of GOI into the target
Generally, it is believed that HDR-mediated KI is more difficult than NHEJ-based indels. For example, in proliferating human cells, NHEJ has been reported to repair 75% of DSBs, while HDR repaired the remaining 25% . To enhance the HDR efficiency, several approaches are now being attempted. For examples, co-injection of murine zygotes with a mixture containing Cas9 mRNA, sgRNA, template ssODNs and Scr7 (an inhibitor for DNA ligase IV) significantly improved the efficiency of HDR-mediated insertional mutagenesis . Chu et al.  also demonstrated usefulness of Scr7 for abolishing NHEJ activity and increasing HDR in both human and mouse cell lines. However, the function of Scr7 in promoting HDR remains controversial. Some researchers demonstrated that Scr7 failed to increase HDR rates in rabbit embryos  and porcine fetal fibroblasts . On the contrary, Li et al.  demonstrated that Scr7 promoted HDR efficiency in porcine fetal fibroblasts. The same group also showed that other reagents L755507 (β-3 adrenergic receptor agonist) and resveratrol (small-molecule compound found in grapes) also showed similar effects (promotion of HDR efficiency) in porcine cells.
5.5 Cas9 pigs
As mentioned previously, the current generation of gene-edited pigs has mostly been produced through either MI or SCNT approaches, which are both expensive and time-consuming. In mice, several Tg lines carrying a Cas9-expressing cassette have been created [154, 161, 162]. These Tg mice are thought to be useful animals for direct
Because pigs are similar to humans in physiological, anatomical, and genetic aspects, they are now seen as a leading animal model for biomedical research. Recent advances in genome editing technology have led to accelerated production of GE pigs within a relatively short time period, which is beneficial due to cost savings in propagation of GE animals and maintaining animals for breeding. Production of GE pigs can be largely categorized into two approaches, so-called MI/EP-mediated production of GE zygotes and SCNT using GE cells as the SCNT donor. There are advantages and drawbacks for both these approaches. For example, the former is simpler, more convenient, and cost-effective than the latter. However, the available genetic background is limited. In this context, the latter is beneficial for the flexibility of choosing any type of genetic background, because the genetic background of SCNT-derived cloned pigs is determined by that of donor cells used for SCNT. Unfortunately, the efficiency of SCNT is extremely low at present. MI/EP with SCNT-treated embryos may compensate for these disadvantages associated with MI/EP or SCNT-mediated production of GE piglets, if the efficiency of SCNT is greatly improved in future.
We thank Shogo Matsunaga for their support in the GENTEP-related experiment, shown in Figures 3 and 4. This study was partly supported by a grant (no. 19K06372 for Masahiro Sato; nos. 25450475 and 16K08085 for Kazuchika Miyoshi; no. 18K09839 for Emi Inada; no. 17H04412 for Issei Saitoh; no. 16H05176 for Akihide Tanimoto) from the Ministry of Education, Science, Sports, and Culture, Japan.
Conflicts of interest
The founding sponsors had no role in the design of the study, collection, analyses, or interpretation of data, writing of the manuscript, and decision to publish the results.
Masahiro Sato designed the study and drafted the manuscript; Kazuchika Miyoshi and Hiroaki Kawaguchi involved in the GENTEP-related experiment; Emi Inada and Issei Saitoh critically revised the manuscript; Akihide Tanimoto supervised the manuscript.