Open access peer-reviewed chapter

Recent Advance in Genome Editing-Based Gene Modification in Pigs

Written By

Masahiro Sato, Kazuchika Miyoshi, Hiroaki Kawaguchi, Emi Inada, Issei Saitoh and Akihide Tanimoto

Submitted: January 21st, 2019 Reviewed: June 13th, 2019 Published: July 22nd, 2019

DOI: 10.5772/intechopen.88022

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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
  • CRISPR/Cas9
  • ZFNs
  • TALENs
  • pigs
  • gene modification
  • microinjection
  • electroporation
  • somatic cell nuclear transfer
  • knock out
  • knock in
  • gene-engineered
  • ribonucleoprotein

1. Introduction

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 [1]. 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. [4] first reported DNA microinjection (MI)-based production of transgenic (Tg) mice. Hammer et al. [5] first reported the successful production of Tg piglets using the technique reported by Gordon et al. [4], 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 [5], which is labor-intensive and requires skill. Moreover, MI-mediated transgene integration into host chromosomes occurs randomly, which often causes gene silencing [6]. 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 [7]. 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 (neo), and DNA sequences of appropriate length, termed homology arms (HA), that correspond to the endogenous target gene, are placed at both ends of the DNA containing the GOI and a marker gene. Chimeric mice can be obtained through blastocyst injection with the targeted ES clones, and the resulting chimeric mice would contribute to produce heterozygous mice carrying mutated traits (GOI/selection marker gene) in the target locus [8]. Unfortunately, there are no germline-competent porcine ES cells, despite extensive efforts [9, 10, 11, 12, 13]. Thus, to date, production of gene-targeted pigs derived from recombinant porcine ES cells has not yet been successful.

In 1996, scientists at the Roslin Institute(Wilmut and colleagues) first succeeded in producing cloned sheep using somatic cell nuclear transfer (SCNT) technology. They used fetal fibroblasts [14] or adult mammary gland-derived fibroblasts [15] as SCNT donors. Notably, prior to these reports, an attempt to produce cloned embryos by the similar technique shown by the Roslin’sgroup has been made by Prather et al. [16], who performed transfer of nuclei from two, four and eight-cell embryos to the enucleated oocytes and successfully produced one piglets that had been derived from the four-cell embryo’s nucleus. This approach is called “blastomere transplantation,” which is basically different from the somatic cell-based SCNT. Generally, fibroblasts used as SCNT donors can proliferate actively in vitro, and therefore are considered to be ideal cells for transgenesis or gene targeting. If these engineered fibroblasts are used for SCNT, the resulting cloned embryos and piglets should have the engineered traits in their genome. This possibility was first proven by the scientists at the Roslin Institute[17, 18] who successfully produced genetically engineered (GE) cloned sheep through SCNT using GE fetal fibroblasts. Since then, SCNT using GE cells as SCNT donors has been a common approach for production of knockout (KO) or Tg piglets [19, 20]. However, as mentioned below, the efficiency of producing cloned GE piglets is extremely low and the preparation of GE donor cells is laborious and time-consuming [21]. During the past two decades, production of only a few GE (KO) piglets has been reported by traditional approaches [22, 23, 24, 25, 26, 27]. Moreover, almost all resulting KO piglets were heterozygous with respect to the KO allele, thus requiring additional tasks such as breeding (likely one or two generations), sequence targeting, or in vitrocell cloning to obtain homozygous KO animals [28], which is also laborious, expensive, and time-consuming.

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) [29], 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 [30].

The ZFN technique uses the ZF protein (which binds to the target DNA) and the endonuclease FokI (which cleaves DNA) [31]. ZF protein has several protein motifs capable of recognizing specific sequences of three nucleotides and binding to them. Notably, Urnov et al. [32] first demonstrated that ZFN is effective to induce DNA editing at the endogenous target gene in mammalian cells. Its targeting efficiency was over 18% in the absence of drug selection, which is ~1000-fold higher than that achieved by traditional gene targeting.

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 FokI nuclease, as in ZFN, thus the term TALE nucleases (TALENs) [33, 34, 35]. Notably, the design and engineering of TALENs is simpler than that of ZFN, and thus can be done faster [35, 36].

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 prerequisitefor this system. This development dramatically reduced both the complexity and time required for the design and implementation of gene editing.


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.

MethodGenome editing tool (mode for gene modification)Method for gene modificationOutcomeTarget geneReferences
SCNTZFN (indels)Using adult porcine ear fibroblasts hemizygous for the eGFP transgeneSeven of nine embryos (Day 12) exhibited loss of fluorescenceeGFPtransgene[41]
SCNTZFN (indels)Using porcine fibroblasts transfected with ZFN plasmidOf 10 live piglets delivered, two carried the predicted ZFN-induced mutation; lower expression of both PPAR-γ1and PPAR-γ2was observed in those clonesPPARγ[42]
SCNTZFN (indels)Using porcine fetal fibroblasts transfected with ZFN plasmidOf six fetuses, all completely lacked α-Gal epitopesGGTA1[43]
MITALEN (indels)Cytoplasmic MI of TALEN mRNA toward IVF-derived zygotesCI 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 LDLRgeneLDLR[44]
MIZFN, TALEN (indels)Cytoplasmic MI of ZFN or TALEN mRNA toward in vivofertilized zygotesOf 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%)NF-kappaB subunit[45]
SCNTTALENs (indels)Using porcine fetal fibroblasts transfected with TALEN plasmidThree piglets with biallelic mutations of the GGTA1gene exhibited loss of α-Gal epitopes on the surface of cellsGGTA1[46]
SCNTTALENs (indels/KI)Using porcine fibroblasts transfected with TALEN mRNA + ssODNOf eight piglets born from DAZL-modified cells, three are still born; of the six piglets from APC-modified cells, only one aliveDAZL, APC[47]
SCNTZFNs (indels)Using porcine fetal fibroblasts transfected with ZFN mRNAThe resulting IL2RGKO pigs completely lacked a thymus and were deficient in T and NK cells, similar to human X-linked SCID patientsIL2RG[48]
SCNTZFN (indels)Using porcine adult liver-derived cells transfected with ZFN plasmid through the two-stepsFour viable and healthy cloned pigs obtained exhibited disruption of the GGTA1and the CMAHlociGGTA1, CMAH[49]
SCNTZFN (KI)Using porcine fibroblast cells transfected with ZFN plasmid and donor DNASuccessfully produced healthy monoallelic/biallelic CMAH KO pigsCMAH[50]
SCNT/MICRISPR/Cas9 (KI/indels)Using porcine fetal fibroblasts transfected with sgRNA-Cas9 plasmids + donor DNA/cytoplasmic MI of Cas9 mRNA/sgRNA toward IVF-derived zygotesOf the CD163recipients, five delivered healthy piglets by cesarean section; 12 of the 13 piglets contained either a biallelic or homozygous deletion of CD1DeGFP, CD163, CD1D[51]
MICRISPR/Cas9 (indels)Cytoplasmic MI of Cas9 mRNA/sgRNA toward in vivofertilized zygotesTen 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 diseasevWF[52]
SCNTCRISPR/Cas9 (indels)Using porcine fetal fibroblasts transfected with Cas9/sgRNA expression plasmidA total of three piglets were obtained; fibroblasts from all three animals were negative for class I SLA cell surface expressionclass I MHC[53]
SCNTTALENs (indels)Using pig fetal fibroblasts transfected with TALEN plasmidOf 27 live cloned piglets obtained, nine were targeted with biallelic mutations in RAG1, three were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2RAG1, RAG2[54]
SCNTZFN (indels)Using pig fetal fibroblasts transfected with ZFN plasmidThree GGTA1null piglets showing loss of α-Gal epitope expression were bornGGTA1[55]
SCNTCRISPR/Cas9 (indels)Using pig liver-derived cells transfected with two or three plasmids expressing Cas9 and sgRNA targeting to GGTA1, CMAH, or putative iGb3S genesOf 10 fetuses obtained, five had mutations in both the GGTA1and CMAHgenesGGTA1, CMAH, putative iGb3S[56]
SCNTZFNs (indels)Using pig fetal fibroblasts transfected with ZFN plasmidThe MSTN-mutant pigs grew normally, had increased muscle mass with decreased fat accumulationMSTN[57]
SCNTTALENs (indels)Using pig liver-derived cells transfected with TALEN plasmidLivers from ASGR1−/− pigs exhibit decreased human platelet uptakeASGR1[58]
SCNTZFN/TALEN (indels)Using pig fetal fibroblasts transfected with TALEN or ZFN mRNAOne of the cloned pigs generated GalT/CMAH-double homozygous KO pigsCMAH[59]
SCNTCRISPR/Cas9 (KI)Using pig fetal fibroblasts transfected with targeting donor vector and two expression vectors for sgRNA and Cas9Highly efficient KI (up to 54%) was achieved after drug selection; one cloned piglet obtained showed correct targetingH11[60]
SCNTCRISPR/Cas9Using pig fetal fibroblasts transfected with sgRNA, Cas9 expression plasmidsFour cloned double KO piglets showing loss of expression for both PARK2and PINK1were producedTYR, PARK2, PINK1[61]
MICRISPR/Cas9 (KI)Cytoplasmic MI of Cas9 mRNA + sgRNA + ssODN toward in vivofertilized zygotesTwo live-born piglets obtained showed the white coat-color phenotype over its entire bodyMITF[62]
MICRISPR/Cas9 (KI)Cytoplasmic MI of Cas9 mRNA + sgRNA + circular vector toward in vivofertilized zygotesAll 16 piglets born were healthy and carried the expected KI allele; the KI allele was successfully transmitted through germlineAlb[63]
MICRISPR/Cas9 (indels)Cytoplasmic MI of Cas9 mRNA and sgRNA toward in vivofertilized zygotesBi-allelic modifications of pig Npc1l1were achieved at the efficiency as high as 100%Npc1l1[64]
SCNTCRISPR/Cas9 (indels)Using pig fetal fibroblasts transfected with sgRNA-Cas9 encoding vectorOf eight marker-gene-free cloned pigs with biallelic mutations obtained, some showed phenotypes similar to DMMSTN[65]
SCNTCRISPR/Cas9 (indels)Using liver-derived cells transfected with sgRNA-Cas9 encoding vectorsOne triple knockout pig was obtained; Cells from this cloned pig exhibited reduced human IgM and IgG bindingGGTA1, CMAH, β4GalNT2[66]
SCNTCRISPR/Cas9 (indels)Using pig fetal fibroblasts transfected with sgRNA-Cas9 encoding vectorThree cloned piglets with biallelic mutation produced showed no antibody-producing B cellsIgM JH[67]
EPCRISPR/Cas9 (indels)Using Cas9 protein and sgRNA (RNP) toward IVF-derived zygotesThe use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and efficient production of Myostatinmutant pigsMSTN[68]
MICRISPR/Cas9 (indels)Cytoplasmic MI of Cas9 mRNA and sgRNA toward in vivofertilized zygotesOf two piglets obtained, one piglets exhibited DMDphenotype, as exemplified by degenerative and disordered skeletal and cardiac muscleDMD[69]
SCNTZFN (indels)Using pig fetal fibroblasts transfected with ZFN-encoding mRNAThe heterozygous FBN1mutant pigs obtained exhibited abnormal phenotype, which resembles MFS found in humansFBN1[70]
SCNTCRISPR/Cas9 (KI)Using pig fetal fibroblasts transfected with Cas9-sgRNA expression vector + donor DNA containing Cre/loxP systemTwo male live piglets with mono-allelic MSTNKO obtained exhibited enhanced myofiber quantity, but the myofiber size remained unalteredMSTN[71]
SCNTTALENs (indels)Using pig fetal fibroblasts transfected with TALEN plasmidsIn total, 12 live and two stillborn piglets were collected; all fetuses and piglets exhibited homozygous GGTA1-null mutationGGTA1[72]
SCNTTALENs (indels)Using porcine fetal fibroblasts co-transfected with TALEN and hDAF expression plasmidsSix live-born piglets and three stillborn piglets were obtained; the piglets showed eight base mono-allelic mutations of GGTA1and hDAFexpressionGGTA1[73]
SCNTCRISPR/Cas9 (indels)Using fetal fibroblast cells transfected with sgRNA-Cas9 encoding vectorFour live RUNX3KO piglets with monoallelic mutation showed the lack of RUNX3 protein in their internal organ systemRUNX3[74]
SCNTTALENs (indels)Using dermal fibroblasts transfected with TALEN plasmidsMSTNKO piglets exhibited a double-muscled phenotype, possessing a higher body weight and longissimus muscle mass measuring 170% that of wild-type piglets, with double the number of muscle fibersMSTN[75]
SCNTTALENs/Cas9 (KI)Using fetal fibroblast cells transfected with Cas9/sgRNA or TALEN vector + ssODNOf seven cloned piglets, some expressed human insulinINS[76]
SCNTCRISPR/Cas9 (KI)Using fetal fibroblast cells transfected with sgRNA-Cas9 encoding vector + ssODNOne cloned stillborn piglet harbored the orthologous p.C313Y mutation at the MSTNlocusAPP, LRRK2, MSTN[77]
SCNTTALENs (indels)Using ear fibroblasts transfected with TALEN vectorsThirty GGTA1biallelic KO piglets were successfully delivered and grew normally.GGTA1[78]
MICRISPR/Cas9 (KI)Cytoplasmic MI of Cas9 mRNA + sgRNA + ssODN toward in vivofertilized zygotesOf five piglets delivered alive, three exhibited pigmentary disorders with light-colored iris in eye, which was observed in patients harboring Sox10mutationsSox10[79]
MICRISPR/Cas9 (indels)Cytoplasmic MI of sgRNA-Cas9 encoding vector toward in vivofertilized zygotesOf six healthy fetuses recovered, four exhibited loss of α-Gal epitope expression, indicating a biallelic KO of GGTA1GGTA1[80]
MICRISPR/Cas9 (indels)Cytoplasmic MI of Cas9 mRNA + three types of sgRNAs toward in vivofertilized zygotesOf two live-born piglets delivered, one piglet showed biallelic modification of all three genes, and another showed biallelic modification of the DJ-1and PINK1genes and monoallelic mutations of parkingeneParkin, DJ-1, PINK1[81]
SCNT-MICRISPR/Cas9 (indels)Cytoplasmic MI of RNP toward SCNT embryosSix fetuses recovered revealed that all fetuses carried biallelic edits for the GRB10gene (6/6, 100%)GRB10[82]
SCNTCRISPR/Cas9 (indels)Using kidney fibroblasts transfected with ZFN vectorsTwo healthy normal females with GGTA1, CMAHdouble KO phenotypes are currently being raised for matingGGTA1, CMAH[83]
MICRISPR/Cas9 (indels)Cytoplasmic MI of Cas9 mRNA + sgRNA toward IVF-derived zygotesSeventeen live piglets and two stillborn were produced; all had mutations in both genes (no pigs with wild-type sequence)RAG2, IL2RG[84]
MICRISPR/Cas9 (indels)Cytoplasmic MI of Cas9 mRNA + sgRNA toward in vivofertilized zygotesEighteen piglets recovered showed either mono- or bi-allelic modifications and no wild-type animals; NANOS2KO pigs phenocopied KO mice with male specific germline ablationNANOS2[85]
SCNTCRISPR/Cas9 (indels)Using fetal fibroblasts transfected with sgRNA and Cas9 expression vectorsSix biallelic KO pigs with mutations in ApoEand LDLRgenes were obtained successfully in a single step.ApoE, LDLR[86]
SCNTTALENsUsing fetal fibroblasts transfected with TALEN plasmidAll six live piglets obtained carried biallelic mutations in the P53locusP53[87]
SCNTTALEN (indels)Using fetal fibroblasts transfected with TALEN plasmidA total of 18 live piglets were obtained; they showed hypermuscular characteristicsMSTN[88]
SCNTCRISPR/Cas9 (indels)Using fetal fibroblasts transfected with sgRNA-Cas9 encoding vectorsA total of 37 PERV-inactivated piglets were generated; 15 piglets remain alivePERV[89]
SCNTCRISPR/Cas9 (indels)Using fetal fibroblasts transfected with sgRNA-Cas9 encoding vectorsOf 26 female piglets delivered, 23 piglets carried mutations in the MSTNlocus; the bi-allelic KO pigs were viable and exhibited partial double-muscled phenotypeMSTN[90]
SCNTCRISPR/Cas9 (KI)Using fetal fibroblasts transfected with Cas9-gRNA plasmid and targeting vectorTwelve male piglets were born and expressed UCP1in a tissue-specific mannerUCP1[91]
MICRISPR/Cas9 (KI)Cytoplasmic MI of RNP toward in vivofertilized zygotesA total of 18 fetuses/born piglets were obtained; successful insertion of pseudo attPsites within the COL1Alocus was observedCOL1A[92]
MICRISPR/Cas9 (indels)Cytoplasmic MI of Cas9 mRNA + sgRNAs toward in vivo-derived zygotesIndels in 92–100% of the embryos analyzed; all resulting 12 piglets had biallelic edits of TMRPSS2TMPRSS2[93]
MICRISPR/Cas9 (indels)Direct pronuclear microinjection of Cas9-gRNA plasmidOf seven born piglets, one exhibited biallelic KO phenotype and one did monoallelic KO oneGGTA1[94]
MICRISPR/Cas9 (indels)Cytoplasmic MI of Cas9 mRNA and dual sgRNAs toward in vivofertilized zygotesOf nine fetuses examined, three exhibited bi-allelic mutations at the PDX1locus; in those fetuses pancreatic primordium was highly disorganizedPDX1[95]
SCNT (Handmade cloning)CRISPR/Cas9 (indels)Using fetal fibroblasts transfected with sgRNA and Cas9 expression vectorsEleven live bi-allelic GGTA1/CMAHdouble KO piglets were obtained with the identical phenotypeGGTA1, CMAH[96]
EPCRISPR/Cas9Using Cas9 protein and sgRNA (RNP) toward IVF-derived zygotesOf 11 piglets born, nine survived; six of nine carried mutations in TP53; three of the genome-edited pigs (50%) exhibited various tumor phenotypesTP53[97]
SCNTCRISPR/Cas9 (KI)Using fetal fibroblasts transfected with sgRNA- Cas9 plasmid + donor DNAThree piglets born grew and developed normally; all these piglets had fat-1KI at the Rosa26 locusRosa26[98]
SCNTCRISPR/Cas9 (KI)Using fetal fibroblasts transfected with sgRNA- Cas9 plasmid + donor DNAOf seven naturally delivered piglets, six showed successful KI; the KI allele was successfully transmitted through germlineHTT[99]
SCNTCRISPR/Cas9 (indels)Using fetal fibroblasts transfected with sgRNA- Cas9 plasmidOf a total of 17 piglets obtained, 12 appeared healthy; all had mutations at the target locusFBXO40[100]

Table 1.

Summary of production of genome-edited pigs.

Abbreviations: APC, adenomatous polyposis coli; Alb, albumin; GGTA1, α-1,3-galactosyltransferase gene; APP, amyloid precursor protein; ApoE, apolipoprotein E; ASGR1, asialoglycoprotein receptor; β4GalNT2, β1,4-N-acetylgalactosaminyl transferase; class I MHC, classI major histocompatibility complex; CRISPR/Cas9, clustered regulated interspaced short palindromic repeat and CRISPR-associated Cas; COL1A, collagen type I alpha 1 chain; CMAH, cytidine monophosphate-N-acetylneuraminic acid hydroxylase; DAZL, deleted in azoospermia-like; DM, double muscling; DMD, Duchenne muscular dystrophy; PARKIN, E3 ubiquitin ligase PARK2; EP, electroporation; eGFP, enhanced green fluorescent protein; FBXO40, F-box protein 40; FBN1, fibrillin-1; GM, gene-modified; GRB10, growth-hormone receptor binding protein-10; H11, Hipp11; hDAF, human decay-accelerating factor; HTT, huntingtin; indels, insertion or deletion of nucleotides; INS, insulin; IL2RG, interleukin-2 receptor gamma; IVF, in vitrofertilized; iGb3S, isogloboside 3 synthase; IgM JH, JH region of the pig IgM heavy chain; KI, knock-in; KO, knockout; LRRK2, leucine-rich repeat kinase 2; LDLR, low density lipoprotein receptor; MFS, Marfan syndrome; MI, microinjection; SCNT-MI, microinjection following somatic cell nuclear transfer; MITF, Microphthalmia-associated transcription factor; MSTN, myostatin; NANOS2, nanosC2HC-type zinc finger 2; fat-1, n-3 fatty acid desaturase; Npc1l1, Niemann-Pick C1-Like 1; PDX-1, pancreas duodenum homeobox 1; DJ-1, PARK7; PARK2, parkin; PPARγ, peroxisome proliferator-activated receptor-gamma; PERV, porcine endogenous retrovirus; PINK1, PTEN-induced putative kinase 1; RNP, ribonucleoprotein; RUNX3, Runt-related transcription factor 3; sgRNA, single guide RNA; ssODN, single-stranded DNA oligonucleotides; SCNT, somatic cell nuclear transfer; Sox10, SRY (sex determining region Y)-box 10; TALENs, transcription activator-like effector nucleases; TMPRSS2, transmembrane protease, serine S1, member 2; TYR, tyrosinase; UCP1, uncoupling protein 1; vWF, von Willebrand factor; ZFNs, zinc finger nucleases.

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 (EGFP) gene in a hemizygous manner. Whyte et al. [41] demonstrated that a ZFN pair efficiently inactivated the expression of EGFPthat was integrated into the chromosomes of porcine fibroblasts via the NHEJ pathway with an efficiency of ~5%. From this experiment, it was found that the endogenous NHEJ pathway is effective for inducing mutation in a porcine target gene. Furthermore, SCNT using GE cells with the mutated allele as a SCNT donor demonstrated that seven of the nine resulting cloned fetuses (at Day 12) stopped expressing EGFP. Yang et al. [42] co-transfected porcine fibroblasts with ZFN and pcDNA3.1 plasmids (providing neo) by electroporation (EP), performed SCNT using these GE cells, and finally obtained peroxisome proliferator-activated receptor γ (PPARγ) mono-allelic KO pigs, which are expected to generate a porcine cardiomyopathy model. Hauschild et al. [43] attempted to destroy GGTA1, an endogenous gene encoding an enzyme required for production of a xenogeneic antigen called α-Gal epitope, using ZFNs in porcine fetal cells. They found that α-Gal epitope-negative cells with the bi-allelic KO phenotype can be efficiently isolated by FACS, and the resultant GE cells have the potential to make cloned piglets. Importantly, this experiment suggests that it is possible to produce individuals with a bi-allelic KO phenotype with this technology. In other words, bi-allelic KO piglets can be directly created without breeding or subcloning, which contrasts with past instances where only heterozygous KO piglets have been produced through traditional gene targeting. Hauschild et al. [43] also showed that neither off-target cleavage nor integration of the ZFN-coding plasmid occurred.

The successful production of genome-edited piglets with bi-allelic KO genotype obtained after cytoplasmic MI of in vivo-derived porcine zygotes using either ZFN or TALEN mRNA was first reported by Lillico et al. [45]. This MI-based production of GE animals was also shown to be successful in mice [101, 102, 103, 104].

Hai et al. [52] first demonstrated that GE pigs can be produced using the CRISPR/Cas9 system. They performed cytoplasmic MI with Cas9 mRNA and sgRNA targeted to von Willebrand factorgene (vWF) to produce a pig modelfor type 1 von Willebrand disease. In this study, 10 of 16 resulting piglets had indels with an efficiency of 63%, and most pigs contained more than two different alleles, suggesting mono-allelic mutants.

Successful knock-in (KI) of a GOI into the target locus was first reported in pigs by Ruan et al. [60] and Peng et al. [63]. Zhou et al. [61] demonstrated the production of SCNT-treated piglets with mutations in multiple genes after a single transfection.

Fischer et al. [83] 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 in vitroEP in the presence of RNP [68]. Sheets et al. [82] produced GE fetuses by cytoplasmic MI of RNP into oocytes reconstituted with intact cells using the SCNT technology. By this treatment, they reported highly efficient (100%) generation of bi-allelic modification in the resultant cloned fetuses. The significance of this approach is be that researchers can obtain GE pigs with a defined genetic background, even though the starting oocytes are derived from ovaries obtained from a slaughterhouse.


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 in vitroEP of zygotes in the presence of genome editing reagents (Figure 1C); the fourth is MI of genome editing reagents into SCNT-treated embryos reconstituted using a normal cell (Figure 1D). Furthermore, we will provide a new approach based on in vitroEP of the SCNT-treated embryos reconstituted using a normal cell as the fifth group of methods for possible production of GE pigs (Figure 2A). In the following sections, each of these methods are described.

Figure 1.

Several methods to create genome-edited pigs. (A) Microinjection (MI)-based method using zygotes. (B) Somatic cell nuclear transfer (SCNT)-based method using gene-engineered (GE) cells as aSCNTdonor. (C) Electroporation (EP)-based method using zygotes. (D)MI-based method using the SCNT-treated embryos.

Figure 2.

A new method for production of genome-edited pigs. (A)EP-based method using the SCNT-treated embryos, which is termed “GENTEP.” (B) Experimental outline for checking the validity of GENTEP.

4.1 MI

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 [62]. Yu et al. [69] 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. [52], the in vitrodevelopmental efficiencies of embryos injected with Cas9 mRNA/sgRNA (~79%) and embryos injected with water (~77%) were both very high and comparable with each other, suggesting that the MI and the Cas9 mRNA/sgRNA had little effect on early embryonic development. On the contrary, Whitworth et al. [51] reported that a higher concentration of sgRNA induces toxicity in porcine embryos. According to Whitworth et al. [51], 10 ng/μL of sgRNA and Cas9 mRNA are recommended.

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 “in vivo-derived zygotes,” and the other is acquisition of zygotes by in vitrofertilization (IVF) between in vitromatured oocytes (derived from the ovaries obtained from the slaughterhouse) and sperm. Generally, it is believed that the in vivo-derived zygotes exhibit superior development performance comparing to the IVF-derived zygotes [51, 62]. Indeed, the number of laboratories using IVF-derived zygotes for production of GE pigs is low (~30% (5/17); see Table 1). However, acquisition of viable in vivo-derived zygotes is laborious and often associated with the sacrifice of pregnant females, which appears to be one of the major goals for improvement in genetic engineering technology using pigs.

The frequent generation of individuals with mosaic genotypes is also a serious problem associated with MI-based GE pig production. Sato et al. [105] 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. [44] suggested that 100% of bovine embryos exhibited fluorescence expression after cytoplasmic MI of EGFPmRNA, but only ~40% of porcine embryos did. It is probable that an endogenous system for translation to protein from mRNA may not be sufficiently established in those porcine embryos, especially at the stage immediately after fertilization or zygotic activation. Indeed, Sato et al. [106] demonstrated that this mosaicism can be partially improved when cytoplasmic MI is performed with oocytes 12 h after activation. In contrast, other researchers reported that only 10–20% of MI-treated embryos exhibited mosaicism [51, 84]. Notably, Whitworth et al. [51] performed cytoplasmic MI with Cas9 mRNA + sgRNA toward fertilized oocytes at 14 h post-fertilization. In this context, the use of sgRNA and an RNP instead of Cas9 mRNA may be the key to solving this issue of mosaicism, as the Cas9 protein is more rapidly translated, folded, and complexed with sgRNAs prior to editing, unlike the Cas9 mRNA [107, 108, 109]. For example, in mice, delivering RNPs into zygotes causes rapid genome editing in the target locus, which also maximizes efficiency while minimizing mosaicism [110, 111, 112]. Indeed, Sheets et al. [82] demonstrated that after MI with RNP, 100% of piglets produced had the bi-allelic KO genotype.

Interestingly, Petersen et al. [80] demonstrated that cytoplasmic MI of DNA vectors coding for CRISPR/Cas9 targeting the porcine GGTA1gene enabled biallelic knockout of GGTA1in 7/12 fetuses and piglets (58.3%). As mentioned previously, it is difficult to visualize porcine pronuclei at zygote stage under normal conditions due to high lipid content in the cytoplasm. Researchers therefore must centrifuge them briefly prior to MI. The fact that cytoplasmic MI of DNA vectors can induce genome editing at a target locus may be beneficial for researchers, because preparation of plasmid DNA is easier than that of mRNA, and it is generally more resistant against degradation than mRNA. According to Petersen et al. [80], it currently remains unknown how the circular DNA plasmid translocates from the cytoplasm to the nucleus. They speculate that the SV40 nuclear translocation signal of the CRISPR/Cas9 plasmid could play an important role by facilitating nuclear translocation via association with ubiquitous transcription factors.

4.2 SCNT

SCNT using GE cells as an SCNT donor is another way to produce GE pigs. The merit of this approach is the use of in vitrocultivated cells such as fetal fibroblasts to which various genetic engineering techniques (i.e., introduction of multiple KO, KI, and transgenes) can be applied easily. After gene transfer, these cells are subjected to cell selection through drug selection or fluorescence activated cell sorting (FACS) to enrich GE cells as a pure population. Thus, it is highly probable that the resulting SCNT-derived GE founder pigs have a predictable genotype and low rates of mosaicism. Unfortunately, as mentioned previously, the efficiency of SCNT to produce cloned piglets is still very low. Much effort has been focused on improving the low efficiency associated with the SCNT, which includes improvement of the oocyte/zygote culture system and application of chemical reagents to alter the epigenetic status of transferred nuclei. For improving the culture method, researchers have used vitamin C [113], α-tocopherol [114], melatonin [115] or alanyl-glutamine dipeptide (instead of glutamine) [116]. For altering the epigenetic status, researchers have used histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) [117, 118], valproic acid (VPA) [119, 120, 121], scriptaid [122, 123, 124], LBH589 (panobinostat) [125], oxamflatin [126], PXD101 (belinostat) [127], quisinostat [128], MGCD0103 [129], or histone methyltransferase inhibitors such as MM-102 [130]. Lin et al. [131] employed tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum (ER) stress, and demonstrated that TUDCA can enhance the developmental potential of porcine SCNT embryos by attenuating ER stress and reducing apoptosis. Wang et al. [132] demonstrated that administration of siRNA or microRNA-148a, both of which can suppress the function of DNA methyltransferase 1 (DNMT1) at a transcriptional level, is effective for enhancing the developmental potential of SCNT embryos. Furthermore, Matoba et al. [133] succeeded in drastically increasing SCNT efficiency by cytoplasmic MI of mRNA coding for histone demethylase (Kdm4d) in mice.

4.3 EP

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. [134]. Since then, many researchers have successfully induced gene edits by using this technology in mice [135, 136], bovines [137] and pigs [68]. 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. [68] 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. [82] 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 in vitroculture and screening. In this study, all (6/6) of the resultant clone fetuses exhibited 100% bi-allelic modification. Unfortunately, they failed to describe successful production of live birth piglets, but it seems that this approach is a powerful tool for GE pig production.

4.5 EP after SCNT

Similar to the approach shown by Sheets et al. [82], we tried to obtain cloned GE piglets through in vitroEP in the SCNT-treated embryos, which is called Genome Editing via Nuclear Transfer and subsequent Electroporation or GENTEP (Figure 2A). Some results obtained from GENTEP-related experiments are presented below.

SCNT-derived embryos were obtained by inserting fetal fibroblasts derived from microminiature pigs (MMP) [138] 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. [119] (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 in vitroEP in the presence of RNP targeted to the pig low density lipoprotein receptor (LDLR) gene (Figure 2B). Parthenotes (~6 h after electric activation) were also used for in vitroEP using tetramethylrhodamine-labeled dextran 3 kDa (used as an indicator for successful gene delivery) (as shown in Figure 3A) or as controls for immunocytochemistry using anti-LDLR antibody (as shown in Figure 3B).

Figure 3.

Validity check of GENTEP. (A) EP of parthenotes (6 h after activation) in the presence of tetramethylrhodamine-labelled dextran 3 kDa. Porcine parthenotes were subjected toin vitroEP, and then cultured for 7 days up to blastocysts. Note that almost all of the EP-treated blastocysts are fluorescent (arrows in a and b), while intact parthenotes do not fluoresce (c and d). Bar = 100 μm. (B) Staining with anti-LDLR antibody. The intact parthenote (hatched blastocysts) exhibits the reactivity to the antibody (arrowheads in a–c), but not to the second antibody alone (arrowheads in d–f). No reactivity to the antibody was also seen in the GENTEP-derived blastocyst (arrowheads in g–i). Nuclear staining withDAPIwas performed after staining with the second antibody. Note that porcine zona pellucida was slightly stained with the second antibody, since it was found to be reactive with the second antibody alone (see d–f). Bar = 100 μm.

First, we examined whether the in vitroEP we used here is effective for successful gene delivery to porcine embryos and does not cause any deleterious effects on their embryonic development, using porcine parthenotes (6 h after activation). The EP procedure was based on the method described by Hashimoto and Takemoto [135]. An electroporation chamber (#LF610P4-4_470; BEX Co. Ltd.), in which two platinum block electrodes were situated with a 1-mm gap between them (Figure 1C), was placed under a stereoscopic microscope and connected to an electric pulse generator (CUY21EDITII Genome Editor™, BEX Co. Ltd.). About 20 parthenotes were placed into a 5-μL drop containing 2 μg/μL tetramethylrhodamine-dextran 3 kDa (#D3307; Thermo Fisher Scientific Inc., Waltham, MA, USA) in Opti-MEM (Invitrogen, Carlsbad, CA, USA) between the electrodes (Figure 1C). EP was performed under these conditions: 30 V, square pulses, 1.0 ms in duration at 99 ms intervals, repeated seven times. The EP-treated parthenotes were then cultured for 7 days up to blastocysts to evaluate the in vitrodevelopmental rate and uptake of fluorescent dye into the embryos. Approximately 40% of the EP-treated parthenotes developed to the blastocyst stage and ~80% of them exhibited bright tetramethylrhodamine-derived fluorescence [arrows in Figure 3A(a,b)]. This result suggests that our EP condition is useful for effective delivery of a foreign substance into porcine embryos and not harmful for their development.

Second, we performed CRISPR/Cas9-based genome editing (targeted to the endogenous LDLRgene) with porcine SCNT-treated embryos. We designed sgRNA capable of recognizing a 20 bp sequence spanning the translation initiation codon (ATG) upstream of the protospacer adjacent motif (PAM) sequence (CGG) on the first exon of porcine LDLR(left panel of Figure 4A). The sgRNA was synthesized by Integrated DNA Technologies, Inc. (IDT; Coralville, Iowa, USA) as Alt-R™ CRISPR crRNA product. The crRNA and tracrRNA (purchased from IDT) were combined for annealing and then mixed with recombinant Cas9 protein (TaKaRa Shuzo Co. Ltd., Shiga, Japan) to form RNP, according to the method of Ohtsuka et al. [139]. The final concentrations of the components in RNP were 30 μM/mL (for crRNA/tracrRNA) and 1 mg/mL (for Cas9 protein). The SCNT-treated embryos 6 or 12 h after activation were transferred to a 5-μL drop (containing RNP in Opti-MEM) and immediately subjected to in vitroEP under these conditions: 30 V, square pulses, 1.0 ms in duration at 99 ms intervals (or 0.5 ms in duration at 99.5 ms intervals), both repeated seven times. After EP, the embryos were promptly cultivated in normal medium for 7 days up to blastocysts and then subjected to analysis of molecular biology (possible mutations in the first exon of LDLR) and immunocytochemistry (possible loss of LDLR protein synthesis) parameters, as described in Figure 2B. In each group, 8–17% of the EP-treated embryos developed to blastocysts (Table 2). These rates appear to be comparable to the yield (24.2%) in experiments performed using intact MMP fetal fibroblasts as SCNT donors [119]. All of the blastocysts obtained were then fixed with 4% paraformaldehyde, and a section of these embryos was subjected to immunocytochemical staining using anti-LDLR antibody (Figure 2B). The EP-treated cloned blastocyst (termed GENTEP-1) was unreactive to anti-LDLR (arrows in Figure 3B(g–i)). In contrast, a parthenote (blastocyst) exhibited positive reactivity to anti-LDLR (arrow in Figure 3B(a–c)). Staining with the second antibody alone failed to react with the antibody (arrow in Figure 3B(d–f)). Furthermore, each of these fixed blastocysts was subjected to genomic DNA isolation to examine possible mutations at the individual embryo level (Figure 2B). Next, GenomiPhi-based whole genome amplification (WGA) was performed using the isolated genomic DNA as a template, as described previously [140]. PCR was then performed using the WGA products as a PCR template. The primer sets used are LDLR-S (5′-AAACCTCACATTGAAATGCTG-3′)/LDLR-RV (5′-CCTAAACTCTCGCGCCCCCCT-3′) for the first round of PCR and LDLR-2S (5′-CTGCAAATGACTGGGGCCCCG-3′)/LDLR-2RV (5′-CTCCAACCACGTAAGAATGAC-3′) for nested PCR (left panel of Figure 4A). Nested PCR using the LDLR-2S/LDLR-2RV primer set yields 355-bp products (left panel of Figure 4A). The typical example when the nested PCR products (lanes 1–9) are loaded onto a 2% agarose gel is shown in the right panel of Figure 4A. Almost all of the samples tested exhibited 355-bp products, except for lane 3 showing bands of reduced size, suggesting occurrence of a large deletion (probably over 100 bp) around the LDLRsequence recognized by sgRNA. In Figure 4B, an example of the results obtained from direct sequencing of nested PCR products using LDLR-2S primer is shown. The sample GENTEP-1, which has been shown to exhibit loss of the reactivity to anti-LDLR (see Figure 3B-g-i), had a large deletion including a sequence spanning ATG and PAM. Notably, there was no appreciable overlapping in ideograms of the sample, suggesting a homozygous bi-allelic KO phenotype (Figure 4C). Subcloning of the PCR products derived from the sample GENTEP-1 into TA cloning vector and subsequent sequencing demonstrated that all six clones obtained exhibited the same sequence as the parental product (data not shown). When the remaining PCR products were sequenced it was found that almost all (82%, 9/11) of the samples exhibited the homozygous bi-allelic KO genotype (Figure 4C).

Figure 4.

Molecular biological analysis of the GENTEP-treated embryos (termed GENTEP-1 to -11) at a single embryo level. (A) Structure of porcineLDLRgene and a target sequence recognized by sgRNA (left panel), and the results of nested PCR (right panel). The target sequence (shown in blue) spanning ATG (shown in red) is located on the first exon ofLDLR. PAM, protospacer adjacent motif. Primers used for first PCR and nested PCR are shown above theLDRL. In the right panel, a part of the nested PCR products (lanes 1–9) loaded onto 2% agarose gel is shown. Arrow indicates the PCR products of 355 bp in size. M, 100-bp ladder markers. (B) Ideogram pattern in the GENTEP-1 and -2 samples obtained after direct sequencing of the nested PCR products using LDLR-2S primer. (C) Various indels found in each GENTEP-treated embryo. ATG is shown in red. Sequence recognized by sgRNA is shown in blue.

Stage at EP after activation of SCNT-treated embryosEP condition2Total number of SCNT-treated embryos examinedNo. of embryos cleaved to the two-cell stage (%)No. of embryos developed to blastocysts (%)
6 h0.5128 (66.7)1 (8.3)
1.01211 (91.7)2 (16.7)
12 h0.53321 (63.6)5 (15.2)
1.03523 (65.7)3 (8.6)

Table 2.

Summary of the properties of blastocysts derived from EP1 toward the SCNT-treated porcine embryos.

EP in the presence of RNP [10 μM of crRNA/tracrRNA mixture (targeted to LDLRgene) + 0.3 μg/μL of Cas9 protein] is performed on SCNT-treated embryos 6 or 12 h after activation. The EP-treated embryos were then cultured for 7 days to the blastocyst stage for the presence of mutations in the target gene at molecular biological and immunocytochemical levels.

EP was performed under the electric condition of 30 V in voltage, 0.5 ms in length of square pulse with 99.5-ms intervals (0.5) or 1.0 ms in length of square pulse with 99-ms intervals (1.0), and seven times of pulse stimulation using an electroporation chamber (#LF610P4-4_470; BEX Co. Ltd.) connected to an electric pulse generator (CUY21EDITII. Genome Editor™, BEX Co. Ltd.).


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 in vivo. Unlike small animals such as mice and rats, large animals have long gestation periods and it is costly to prepare large animal recipients. Therefore, this testing in vivoappears to be difficult in larger animals such as pigs. To overcome this issue, a single embryo (blastocyst) assay to evaluate the operability of the genome editing reagents prepared was provided by Wang et al. [62] who later re-validated those sets using porcine parthenotes. To our knowledge, this assay was first developed using mice by Sakurai et al. [141] who reported that it is useful for confirming the fidelity of sgRNAs used.

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 [142] and others [44]. 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 [144]; 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 FokI nuclease domain (fCas9) to improve the DNA cleavage specificity [146]. Recently, it was reported that Cpf1, a putative Class 2 CRISPR effector, mediates target DNA editing differently from Cas9 [147]. It generates a 5-nucleotide staggered cut with a 5′ overhang, which is particularly advantageous in facilitating an NHEJ-based KI into a genome. Several unique enzymes that can decrease the probability of off-target cleavage have also been produced. For example, two engineered enzymes produced from SpCas9 from Streptococcus pyogeneswith the goal of enhancing specificity, called eSpCas9 [148] and SpCas9-HF [149], are reported to reduce the probability of mismatched DNA binding. A hybrid enzyme combining the Cas9-nickase and PmCDA1, an activation-induced cytidine deaminase (AID) ortholog, could perform targeted nucleotide substitution [150]. Furthermore, a CRISPR system using a new Cas-related enzyme called Cas13a that targets RNA has also been recently developed [151].

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. [154] 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. [61] demonstrated successful generation of PARK2 (parkin) and PTEN-induced putative kinase 1 (PINK1) double-KO pigs through SCNT with GE fetal fibroblasts after co-transfection of Cas9, PARK2-sgRNA, and PINK1-sgRNA-expressing vectors by electroporation. The percentage of PARK2−/−/PINK1−/− double-KO cells was up to 38.1%. SCNT using these double-KO cells resulted in the birth of 20 cloned piglets. Of these, four piglets developed normally, and both parkin and PINK1 in those individuals were depleted at the protein level. Estrada et al. [66] also succeeded in obtaining one triple-KO cloned piglet with mutations in GGTA1(coding for α-1,3-galactosyltransferase), CMAH(coding for cytidine monophosphate-N-acetylneuraminic acid hydroxylase) and β4GalNT2(coding for β1,4-N-acetylgalactosaminyl transferase) after SCNT. Wang et al. [81] generated PARK7 (DJ-1)/parkin/PINK1triple-gene modified pigs using the CRISPR/Cas9 system in one step through direct zygote injection of Cas9 mRNA and three types of sgRNAs. According to Wang et al. [81], of two live-born piglets delivered, one piglet showed biallelic modification of all three genes, and another showed biallelic modification of the DJ-1and PINK1genes and monoallelic mutation of the parkingene.

5.4 KI

As shown in Table 1, in 2015 successful KI in pigs was reported by several groups. For example, Wang et al. [62] performed MI with in vivofertilized zygotes (derived from colored pigs) using Cas9 mRNA + sgRNA + single-stranded DNA oligonucleotides (ssODN), targeting microphthalmia-associated transcription factor (MITF), a master regulator gene of melanocyte development, and obtained two live-born piglets showing the white coat color phenotype over its entire body. Peng et al. [63] tried to create KI piglets with a MI approach using a circular vector as donor DNA. They designed an sgRNA targeting the starting codon region (including the adjacent 5′ and ATG) and generated a targeted fragment (donor for HR) with the insert flanked by 1-kb HA on both sides. They performed cytoplasmic MI of Cas9 mRNA + in vitrosynthesized sgRNA + circular vector containing the targeting fragment, and finally obtained 16 live piglets, all of which were found to carry the expected KI allele. Notably, they confirmed expression of human albumin (Alb) protein generated from the KI allele in the plasma of these cloned pigs. This means that expression of a transgene (human Alb as GOI) is possible under the control of an endogenous promoter system (in this case, Alb promoter).

Ruan et al. [60] demonstrated production of GE pigs with successful KI of GOI into the target Hipp11(H11) locus, which is considered as “safe harbor” genomic locus that allows gene expression without disrupting internal gene function, like the Rosa26locus. They utilized a positive and negative selection method to insert GFPinto the pH 11locus in pig fetal fibroblast cells by electroporation. The targeting donor vector (4.2 kb in size) contains a reporter cassette with neoand GFP genes which are flanked by a 0.8-kb HA to the H11locus on each side with the diphtheria toxin A (DTA) gene at the 3′ end. Cells were transfected with the linearized donor vector and two expression vectors for sgRNA (targeted to the H11locus) and Cas9. After drug selection, they obtained GE cells with successful KI at the H11locus with efficiencies up to 54%. Next, they performed SCNT using these correctly targeted clones, and obtained one cloned piglet which was later confirmed to show correct targeting.

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% [155]. 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 [156]. Chu et al. [157] 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 [158] and porcine fetal fibroblasts [159]. On the contrary, Li et al. [160] 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 in vivogenome editing experiments, because successful delivery of the expression vectors of sgRNAs alone or RNA itself into selected tissues caused generation of genome-edited tissues. For example, Platt et al. [161] demonstrated that in vivoviral administration of Kirsten rat sarcoma viral oncogene homolog (Kras), transformation related protein 53 (Trp53), and serine/threonine-protein kinase 11 (Stk11)-gRNAs to the Cas9-expressing line caused lung carcinomas within a short period. This suggests that if a Cas9-expressing pig is produced, it will provide an easy and efficient way to produce genetic modifications, which should substantially facilitate studying gene functions, modeling human diseases, and promoting agricultural productivity. Based on this concept, Wang et al. [163] first produced Cre-dependent Cas9-expressing pigs to enable efficient in vivogenome editing. They first transfected the linear-targeting donor containing Cre-dependent Cas9-expression cassette and TALEN plasmids directed to Rosa26locus into porcine fetal fibroblasts and finally selected clones carrying KI cassette. These clones were then used for SCNT to produce cloned GE piglets. They showed that cells isolated from several organs of GE pigs exhibited Cre-induced activation of Cas9 expression. This Cas9 pig line will be used for various studies as indicated above.


6. Conclusion

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.


Author contributions

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.


  1. 1. Cooper DKC, Gollackner B, Sachs DH. Will the pig solve the transplantation backlog? Annual Review of Medicine. 2002;53:133-147. DOI: 10.1146/
  2. 2. Fan N, Lai L. Genetically modified pig models for human diseases. Journal of Genetics and Genomics. 2013;40:67-73. DOI: 10.1016/j.jgg.2012.07.014
  3. 3. Yang H, Wu Z. Genome editing of pigs for agriculture and biomedicine. Frontiers in Genetics. 2018;9:360. DOI: 10.3389/fgene.2018.00360
  4. 4. Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA, Ruddle FH. Genetic transformation of mouse embryos by microinjection of purified DNA. Proceedings of the National Academy of Sciences of the United States of America. 1980;77:7380-7384. DOI: 10.1073/pnas.77.12.7380
  5. 5. Hammer RE, Pursel VG, Rexroad CE Jr, Wall RJ, Bolt DJ, et al. Production of transgenic rabbits, sheep and pigs by microinjection. Nature. 1985;315:680-683. DOI: 10.1038/315680a0
  6. 6. Bestor TH, Chandler VL, Feinberg AP. Epigenetic effects in eukaryotic gene expression. Developmental Genetics. 1994;15:458-462. DOI: 10.1002/dvg.1020150603
  7. 7. Doetschman T, Gregg RG, Maeda N, Hooper ML, Melton DW, et al. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature. 1987;330:576-578. DOI: 10.1038/330576a0
  8. 8. Bouabe H, Okkenhaug K. Gene targeting in mice: A review. Methods in Molecular Biology. 2013;1064:315-336. DOI: 10.1007/978-1-62703-601-6_23
  9. 9. Notarianni E, Galli C, Laurie S, Moor RM, Evans MJ. Derivation of pluripotent, embryonic cell lines from the pig and sheep. Journal of Reproduction and Fertility. Supplement. 1991;43:255-260
  10. 10. Chen LR, Shiue YL, Bertolini L, Medrano JF, BonDurant RH, et al. Establishment of pluripotent cell lines from porcine preimplantation embryos. Theriogenology. 1999;52:195-212. DOI: 10.1016/S0093-691X(99)00122-3
  11. 11. Ezashi T, Telugu BP, Alexenko AP, Sachdev S, Sinha S, et al. Derivation of induced pluripotent stem cells from pig somatic cells. Proceedings of the National Academy of Sciences of the United States of America. 2009;106:10993-10998. DOI: 10.1073/pnas.0905284106
  12. 12. Brevini TA, Pennarossa G, Gandolfi F. No shortcuts to pig embryonic stem cells. Theriogenology. 2010;74:544-550. DOI: 10.1016/j.theriogenology.2010.04.020
  13. 13. Hou DR, Jin Y, Nie XW, Zhang ML, Ta N, et al. Derivation of porcine embryonic stem-like cells fromin vitro-produced blastocyst-stage embryos. Scientific Reports. 2016;6:25838. DOI: 10.1038/srep25838
  14. 14. Campbell KH, McWhir J, Ritchie WA, Wilmut I. Sheep cloned by nuclear transfer from a cultured cell line. Nature. 1996;380:64-66. DOI: 10.1038/380064a0
  15. 15. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH. Viable offspring derived from fetal and adult mammalian cells. Nature. 1997;385:810-813. DOI: 10.1038/385810a0
  16. 16. Prather RS, Sims MM, First NL. Nuclear transplantation in early pig embryos. Biology of Reproduction. 1989;41:414-418. DOI: 10.1095/biolreprod41.3.414
  17. 17. Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, et al. Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts. Science. 1997;278:2130-2133. DOI: 10.1126/science.278.5346.2130
  18. 18. Denning C, Burl S, Ainslie A, Bracken J, Dinnyes A, et al. Deletion of the alpha(1,3)galactosyl transferase (GGTA1) gene and the prion protein (PrP) gene in sheep. Nature Biotechnology. 2001;19:559-562. DOI: 10.1038/89313
  19. 19. Polejaeva IA, Chen SH, Vaught TD, Page RL, Mullins J, et al. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature. 2000;407:86-90. DOI: 10.1038/35024082
  20. 20. Sachs DH, Galli C. Genetic manipulation in pigs. Current Opinion in Organ Transplantation. 2009;14:148-153. DOI: 10.1097/MOT.0b013e3283292549
  21. 21. Wells KD, Prather RS. Genome-editing technologies to improve research, reproduction, and production in pigs. Molecular Reproduction and Development. 2017;84:1012-1017. DOI: 10.1002/mrd.22812
  22. 22. Park KW, Cheong HT, Lai L, Im GS, Kühholzer B, et al. Production of nuclear transfer-derived swine that express the enhanced green fluorescent protein. Animal Biotechnology. 2001;12:173-181. DOI: 10.1081/ABIO-100108344
  23. 23. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, et al. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Science. 2002;295:1089-1092. DOI: 10.1126/science.1068228
  24. 24. Dai Y, Vaught TD, Boone J, Chen SH, Phelps CJ, et al. Targeted disruption of the alpha1,3-galactosyltransferase gene in cloned pigs. Nature Biotechnology. 2002;20:251-255. DOI: 10.1038/nbt0302-251
  25. 25. Lai LX, Kang JX, Li RF, Wang JD, Witt WT, et al. Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nature Biotechnology. 2006;24:435-436. DOI: 10.1038/nbt1198
  26. 26. Rogers CS, Stoltz DA, Meyerholz DK, Ostedgaard LS, Rokhlina T, et al. Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science. 2008;321:1837-1841. DOI: 10.1126/science.1163600
  27. 27. Suzuki S, Iwamoto M, Saito Y, Fuchimoto D, Sembon S, et al. Il2rg gene-targeted severe combined immunodeficiency pigs. Cell Stem Cell. 2012;10:753-758. DOI: 10.1016/j.stem.2012.04.021
  28. 28. Tan W, Proudfoot C, Lillico SG, Whitelaw CB. Gene targeting, genome editing: From Dolly to editors. Transgenic Research. 2016;25:273-287. DOI: 10.1007/s11248-016-9932-x
  29. 29. Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157:1262-1278. DOI: 10.1016/j.cell.2014.05.010
  30. 30. Sonoda E, Hochegger H, Saberi A, Taniguchi Y, Takeda S. Differential usage of non-homologous end-joining and homologous recombination in double strand break repair. DNA Repair. 2006;5:1021-1029. DOI: 10.1016/j.dnarep.2006.05.022
  31. 31. Kim YG, Cha J, Chandrasegaran S. Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domain. Proceedings of the National Academy of Sciences of the United States of America. 1996;93:1156-1160. DOI: 10.1073/pnas.93.3.1156
  32. 32. Urnov FD, Miller JC, Lee YL, Beausejour CM, Rock JM, et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature. 2005;435:646-651. DOI: 10.1038/nature03556
  33. 33. Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics. 2010;186:757-761. DOI: 10.1534/genetics.110.120717
  34. 34. Miller JC, Tan S, Qiao G, Barlow KA, Wang J, et al. A TALE nuclease architecture for efficient genome editing. Nature Biotechnology. 2011;29:143-148. DOI: 10.1038/nbt.1755
  35. 35. Reyon D, Tsai SQ , Khayter C, Foden JA, Sander JD, et al. FLASH assembly of TALENs for high-throughput genome editing. Nature Biotechnology. 2012;30:460-465. DOI: 10.1038/nbt.2170
  36. 36. Cermak T, Doyle EL, Christian M, Wang L, Zhang Y, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Research. 2011;39:e82. DOI: 10.1093/nar/gkr218
  37. 37. Horvath P, Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010;327:167-170. DOI: 10.1126/science.1179555
  38. 38. Bhaya D, Davison M, Barrangou R. CRISPR-Cas systems in bacteria and archaea: Versatile small RNAs for adaptive defense and regulation. Annual Review of Genetics. 2011;45:273-297. DOI: 10.1146/annurev-genet-110410-132430
  39. 39. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337:816-821. DOI: 10.1126/science.1225829
  40. 40. Gaj T, Gersbach CA, Barbas CFIII. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology. 2013;31:397-405. DOI: 10.1016/j.tibtech.2013.04.004
  41. 41. Whyte JJ, Zhao J, Wells KD, Samuel MS, Whitworth KM, et al. Gene targeting with zinc finger nucleases to produce cloned eGFP knockout pigs. Molecular Reproduction and Development. 2011;78:2. DOI: 10.1002/mrd.21271
  42. 42. Yang D, Yang H, Li W, Zhao B, Ouyang Z, et al. Generation of PPARgamma mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning. Cell Research. 2011;21:979-982. DOI: 10.1038/cr.2011.70
  43. 43. Hauschild J, Petersen B, Santiago Y, Queisser AL, Carnwath JW, et al. Efficient generation of a biallelic knockout in pigs using zinc-finger nucleases. Proceedings of the National Academy of Sciences of the United States of America. 2011;108:12013-12017. DOI: 10.1073/pnas.1106422108
  44. 44. Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, et al. Efficient TALEN-mediated gene knockout in livestock. Proceedings of the National Academy of Sciences of the United States of America. 2012;109:17382-17387. DOI: 10.1073/pnas.1211446109
  45. 45. Lillico SG, Proudfoot C, Carlson DF, Stverakova D, Neil C, et al. Live pigs produced from genome edited zygotes. Scientific Reports. 2013;3:2847. DOI: 10.1038/srep02847
  46. 46. Xin J, Yang H, Fan N, Zhao B, Ouyang Z, et al. Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs. PLoS ONE. 2013;8:e84250. DOI: 10.1371/journal.pone.0084250
  47. 47. Tan W, Carlson DF, Lancto CA, Garbe JR, Webster DA, et al. Efficient nonmeiotic allele introgression in livestock using custom endonucleases. Proceedings of the National Academy of Sciences of the United States of America. 2013;110:16526-16531. DOI: 10.1073/pnas.1310478110
  48. 48. Watanabe M, Nakano K, Matsunari H, Matsuda T, Maehara M, et al. Generation of interleukin-2 receptor gamma gene knockout pigs from somatic cells genetically modified by zinc finger nuclease-encoding mRNA. PLoS ONE. 2013;8:e76478. DOI: 10.1371/journal.pone.0076478
  49. 49. Lutz AJ, Li P, Estrada JL, Sidner RA, Chihara RK, et al. Double knockout pigs deficient in N-glycolylneuraminic acid and galactose alpha-1,3-galactose reduce the humoral barrier to xenotransplantation. Xenotransplantation. 2013;20:27-35. DOI: 10.1111/xen.12019
  50. 50. Kwon DN, Lee K, Kang MJ, Choi YJ, Park C, et al. Production of biallelic CMP-Neu5Ac hydroxylase knock-out pigs. Scientific Reports. 2013;3:1981. DOI: 10.1038/srep01981
  51. 51. Whitworth KM, Lee K, Benne JA, Beaton BP, Spate LD, et al. CRISPR/Cas9 system to produce genetically engineered pigs fromin vitro-derived oocytes and embryos. Biology of Reproduction. 2014;91:78. DOI: 10.1095/biolreprod.114.121723
  52. 52. Hai T, Teng F, Guo R, Li W, Zhou Q. One-step generation of knockout pigs by zygote injection of CRISPR/Cas system. Cell Research. 2014;24:372-375. DOI: 10.1038/cr.2014.11
  53. 53. Reyes LM, Estrada JL, Wang ZY, Blosser RJ, Smith RF, et al. Creating class I MHC-null pigs using guide RNA and the Cas9 endonuclease. Journal of Immunology. 2014;193:5751-5757. DOI: 10.4049/jimmunol.1402059
  54. 54. Huang J, Guo X, Fan N, Song J, Zhao B, et al. RAG1/2 knockout pigs with severe combined immunodeficiency. Journal of Immunology. 2014;193:1496-1503. DOI: 10.4049/jimmunol.1400915
  55. 55. Bao L, Chen H, Jong U, Rim C, Li W, et al. Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transfer. Science China. Life Sciences. 2014;57:263-268. DOI: 10.1007/s11427-013-4601-2
  56. 56. Li P, Estrada JL, Burlak C, Montgomery J, Butler JR, et al. Efficient generation of genetically distinct pigs in a single pregnancy using multiplexed single-guide RNA and carbohydrate selection. Xenotransplantation. 2015;22:20-31. DOI: 10.1111/xen.12131
  57. 57. Qian L, Tang M, Yang J, Wang Q , Cai C, et al. Targeted mutations in myostatin by zinc-finger nucleases result in double-muscled phenotype in Meishan pigs. Scientific Reports. 2015;5:14435. DOI: 10.1038/srep14435
  58. 58. Paris LL, Estrada JL, Li P, Blankenship RL, Sidner RA, et al. Reduced human platelet uptake by pig livers deficient in the asialoglycoprotein receptor 1 protein. Xenotransplantation. 2015;22:203-210. DOI: 10.1111/xen.12164
  59. 59. Miyagawa S, Matsunari H, Watanabe M, Nakano K, Umeyama K, et al. Generation of alpha1,3-galactosyltransferase and cytidine monophospho-N-acetylneuraminic acid hydroxylase gene double-knockout pigs. Journal of Reproduction and Development. 2015;61:449-457. DOI: 10.1262/jrd.2015-058
  60. 60. Ruan J, Li H, Xu K, Wu T, Wei J, et al. Highly efficient CRISPR/Cas9-mediated transgene knockin at the H11 locus in pigs. Scientific Reports. 2015;5:14253. DOI: 10.1038/srep14253
  61. 61. Zhou X, Xin J, Fan N, Zou Q , Huang J, et al. Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cellular and Molecular Life Sciences. 2015;72:1175-1184. DOI: 10.1007/s00018-014-1744-7
  62. 62. Wang X, Zhou J, Cao C, Huang J, Hai T, et al. Efficient CRISPR/Cas9-mediated biallelic gene disruption and site-specific knockin after rapid selection of highly active sgRNAs in pigs. Scientific Reports. 2015;5:13348. DOI: 10.1038/srep13348
  63. 63. Peng J, Wang Y, Jiang J, Zhou X, Song L, et al. Production of human albumin in pigs through CRISPR/Cas9-mediated knockin of human cDNA into swine albumin locus in the zygotes. Scientific Reports. 2015;5:16705. DOI: 10.1038/srep16705
  64. 64. Wang Y, Du Y, Shen B, Zhou X, Li J, et al. Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA. Scientific Reports. 2015;5:8256. DOI: 10.1038/srep08256
  65. 65. Wang K, Ouyang H, Xie Z, Yao C, Guo N, et al. Efficient generation of myostatin mutations in pigs using the CRISPR/Cas9 system. Scientific Reports. 2015;5:16623. DOI: 10.1038/srep16623
  66. 66. Estrada JL, Martens G, Li P, Adams A, Newell KA, et al. Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes. Xenotransplantation. 2015;22:194-202. DOI: 10.1111/xen.12161
  67. 67. Chen F, Wang Y, Yuan Y, Zhang W, Ren Z, et al. Generation of B cell-deficient pigs by highly efficient CRISPR/Cas9-mediated gene targeting. Journal of Genetics and Genomics. 2015;42:437-444. DOI: 10.1016/j.jgg.2015.05.002
  68. 68. Tanihara F, Takemoto T, Kitagawa E, Rao S, Do LT, et al. Somatic cell reprogramming-free generation of genetically modified pigs. Science Advances. 2016;2:e1600803. DOI: 10.1126/sciadv.1600803
  69. 69. Yu HH, Zhao H, Qing YB, Pan WR, Jia BY, et al. Porcine zygote injection with Cas9/sgRNA results in DMD-modified pig with muscle dystrophy. International Journal of Molecular Sciences. 2016;17:E1668. DOI: 10.3390/ijms17101668
  70. 70. Umeyama K, Watanabe K, Watanabe M, Horiuchi K, Nakano K, et al. Generation of heterozygous fibrillin-1 mutant cloned pigs from genome-edited foetal fibroblasts. Scientific Reports. 2016;6:24413. DOI: 10.1038/srep24413
  71. 71. Bi Y, Hua Z, Liu X, Hua W, Ren H, et al. Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP. Scientific Reports. 2016;6:31729. DOI: 10.1038/srep31729
  72. 72. Cheng W, Zhao H, Yu H, Xin J, Wang J, et al. Efficient generation of GGTA1-null Diannan miniature pigs using TALENs combined with somatic cell nuclear transfer. Reproductive Biology and Endocrinology. 2016;14:77. DOI: 10.1186/s12958-016-0212-7
  73. 73. Kang JT, Kwon DK, Park AR, Lee EJ, Yun YJ, et al. Production of alpha1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology. Journal of Veterinary Science. 2016;17:89-96. DOI: 10.4142/jvs.2016.17.1.89
  74. 74. Kang JT, Ryu J, Cho B, Lee EJ, Yun YJ, et al. Generation of RUNX3 knockout pigs using CRISPR/Cas9-mediated gene targeting. Reproduction in Domestic Animals. 2016;51:970-978. DOI: 10.1111/rda.12775
  75. 75. Rao S, Fujimura T, Matsunari H, Sakuma T, Nakano K, et al. Efficient modification of the myostatin gene in porcine somatic cells and generation of knockout piglets. Molecular Reproduction and Development. 2016;83:61-70. DOI: 10.1002/mrd.22591
  76. 76. Yang Y, Wang K, Wu H, Jin Q , Ruan D, et al. Genetically humanized pigs exclusively expressing human insulin are generated through custom endonuclease-mediated seamless engineering. Journal of Molecular Cell Biology. 2016;8:174-177. DOI: 10.1093/jmcb/mjw008
  77. 77. Wang K, Tang X, Liu Y, Xie Z, Zou X, et al. Efficient generation of orthologous point mutations in pigs via CRISPR-assisted ssODN-mediated homology-directed repair. Molecular Therapy—Nucleic Acids. 2016;5:e396. DOI: 10.1038/mtna.2016.101
  78. 78. Feng C, Li X, Cui H, Long C, Liu X, et al. Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN mRNA and magnetic beads with somatic cell nuclear transfer. Journal of Integrative Agriculture. 2016;15:1540-1549. DOI: 10.1016/S2095-3119(16)61347-3
  79. 79. Zhou X, Wang L, Du Y, Xie F, Li L, et al. Efficient generation of gene-modified pigs harboring precise orthologous human mutation via CRISPR/Cas9-induced homology-directed repair in zygotes. Human Mutation. 2016;37:110-118. DOI: 10.1002/humu.22913
  80. 80. Petersen B, Frenzel A, Lucas-Hahn A, Herrmann D, Hassel P, et al. Efficient production of biallelic GGTA1 knockout pigs by cytoplasmic microinjection of CRISPR/Cas9 into zygotes. Xenotransplantation. 2016;23:338-346. DOI: 10.1111/xen.12258
  81. 81. Wang X, Cao C, Huang J, Yao J, Hai T, et al. One-step generation of triple gene-targeted pigs using CRISPR/Cas9 system. Scientific Reports. 2016;6:20620. DOI: 10.1038/srep20620
  82. 82. Sheets TP, Park CH, Park KE, Powell A, Donovan DM, et al. Somatic cell nuclear transfer followed by CRIPSR/Cas9 microinjection results in highly efficient genome editing in cloned pigs. International Journal of Molecular Sciences. 2016;17:2031. DOI: 10.3390/ijms17122031
  83. 83. Fischer K, Kraner-Scheiber S, Petersen B, Rieblinger B, Buermann A, et al. Efficient production of multi-modified pigs for xenotransplantation by ‘combineering’, gene stacking and gene editing. Scientific Reports. 2016;6:29081. DOI: 10.1038/srep29081
  84. 84. Lei S, Ryu J, Wen K, Twitchell E, Bui T, et al. Increased and prolonged human norovirus infection in RAG2/IL2RG deficient gnotobiotic pigs with severe combined immunodeficiency. Scientific Reports. 2016;6:25222. DOI: 10.1038/srep25222
  85. 85. Park KE, Kaucher AV, Powell A, Waqas MS, Sandmaier SE, et al. Generation of germline ablated male pigs by CRISPR/Cas9 editing of the NANOS2 gene. Scientific Reports. 2017;7:40176. DOI: 10.1038/srep40176
  86. 86. Huang L, Hua Z, Xiao H, Cheng Y, Xu K, et al. CRISPR/Cas9-mediated ApoE−/− and LDLR−/− double gene knockout in pigs elevates serum LDL-C and TC levels. Oncotarget. 2017;8:37751-37760. DOI: 10.18632/oncotarget.1715
  87. 87. Shen Y, Xu K, Yuan Z, Guo J, Zhao H, et al. Efficient generation of P53 biallelic knockout Diannan miniature pigs via TALENs and somatic cell nuclear transfer. Journal of Translational Medicine. 2017;15:224. DOI: 10.1186/s12967-017-1327-0
  88. 88. Kang J, Kim S, Zhu H, Jin L, Guo Q , et al. Generation of cloned adult muscular pigs with myostatin gene mutation by genetic engineering. RSC Advances. 2017;7:12541-12549. DOI: 10.1039/C6RA28579A
  89. 89. Niu D, Wei HJ, Lin L, George H, Wang T, et al. Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9. Science. 2017;357:1303-1307. DOI: 10.1126/science.aan4187
  90. 90. Wang K, Tang X, Xie Z, Zou X, Li M, et al. CRISPR/Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs. Transgenic Research. 2017;26:799-805. DOI: 10.1007/s11248-017-0044-z
  91. 91. Zheng Q , Lin J, Huang J, Zhang H, Zhang R, et al. Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity. Proceedings of the National Academy of Sciences of the United States of America. 2017;114:E9474-E9482. DOI: 10.1073/pnas.1707853114
  92. 92. Park KE, Powell A, Sandmaier SE, Kim CM, Mileham A, et al. Targeted gene knock-in by CRISPR/Cas ribonucleoproteins in porcine zygotes. Scientific Reports. 2017;7:42458. DOI: 10.1038/srep42458
  93. 93. Whitworth KM, Benne JA, Spate LD, Murphy SL, Samuel MS, et al. Zygote injection of CRISPR/Cas9 RNA successfully modifies the target gene without delaying blastocyst development or altering the sex ratio in pigs. Transgenic Research. 2017;26:97-107. DOI: 10.1007/s11248-016-9989-6
  94. 94. Chuang CK, Chen CH, Huang CL, Su YH, Peng SH, et al. Generation of GGTA1 mutant pigs by direct pronuclear microinjection of CRISPR/Cas9 plasmid vectors. Animal Biotechnology. 2017;28:174-181. DOI: 10.1080/10495398.2016.1246453
  95. 95. Wu J, Vilarino M, Suzuki K, Okamura D, Bogliotti YS, et al. CRISPR-Cas9 mediated one-step disabling of pancreatogenesis in pigs. Scientific Reports. 2017;7:10487. DOI: 10.1038/s41598-017-08596-5
  96. 96. Gao H, Zhao C, Xiang X, Li Y, Zhao Y, et al. Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning. Journal of Reproduction and Development. 2017;63:17-26. DOI: 10.1262/jrd.2016-079
  97. 97. Tanihara F, Hirata M, Nguyen NT, Le QA, Hirano T, et al. Generation of a TP53-modified porcine cancer model by CRISPR/Cas9-mediated gene modification in porcine zygotes via electroporation. PLoS ONE. 2018;13:e0206360. DOI: 10.1371/journal.pone.0206360
  98. 98. Li M, Ouyang H, Yuan H, Li J, Xie Z, et al. Site-specific Fat-1 knock-in enables significant decrease of n-6PUFAs/n-3PUFAs ratio in pigs. G3: Genes, Genomes, Genetics. 2018;8:1747-1754. DOI: 10.1534/g3.118.200114
  99. 99. Yan S, Tu Z, Liu Z, Fan N, Yang H, et al. A huntingtin knockin pig model recapitulates features of selective neurodegeneration in huntington’s disease. Cell. 2018;173:989-1002.e13. DOI: 10.1016/j.cell.2018.03.005
  100. 100. Zou Y, Li Z, Zou Y, Hao H, Li N, et al. An FBXO40 knockout generated by CRISPR/Cas9 causes muscle hypertrophy in pigs without detectable pathological effects. Biochemical and Biophysical Research Communications. 2018;498:940-945. DOI: 10.1016/j.bbrc.2018.03.085
  101. 101. Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, et al. One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 2013;154:1370-1379. DOI: 10.1016/j.cell.2013.08.022
  102. 102. Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153:910-918. DOI: 10.1016/j.cell.2013.04.025
  103. 103. Fujii W, Kawasaki K, Sugiura K, Naito K. Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease. Nucleic Acids Research. 2013;41:e187. DOI: 10.1093/nar/gkt772
  104. 104. Shen B, Zhang J, Wu H, Wang J, Ma K, et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Research. 2013;23:720-723. DOI: 10.1038/cr.2013.46
  105. 105. Sato M, Koriyama M, Watanabe S, Ohtsuka M, Sakurai T, et al. Direct injection of CRISPR/Cas9-related mRNA into cytoplasm of parthenogenetically activated porcine oocytes causes frequent mosaicism for indel mutations. International Journal of Molecular Sciences. 2015;16:17838-17856. DOI: 10.3390/ijms160817838
  106. 106. Sato M, Kosuke M, Koriyama M, Inada E, Saitoh I, et al. Timing of CRISPR/Cas9-related mRNA microinjection after activation as an important factor affecting genome editing efficiency in porcine oocytes. Theriogenology. 2018;108:29-38. DOI: 10.1016/j.theriogenology.2017.11.030
  107. 107. Sung YH, Kim JM, Kim HT, Lee J, Jeon J, et al. Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases. Genome Research. 2014;24:125-131. DOI: 10.1101/gr.163394.113
  108. 108. Liang X, Potter J, Kumar S, Zou Y, Quintanilla R, et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. Journal of Biotechnology. 2015;208:44-53. DOI: 10.1016/j.jbiotec.2015.04.024
  109. 109. Jung CJ, Zhang J, Trenchard E, Lloyd KC, West DB, et al. Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein. Transgenic Research. 2017;26:263-277. DOI: 10.1007/s11248-016-9998-5
  110. 110. Aida T, Chiyo K, Usami T, Ishikubo H, Imahashi R, et al. Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice. Genome Biology. 2015;16:87. DOI: 10.1186/s13059-015-0653-x
  111. 111. Hashimoto M, Yamashita Y, Takemoto T. Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse. Developmental Biology. 2016;418:1-9. DOI: 10.1016/j.ydbio.2016.07.017
  112. 112. Tu Z, Yang W, Yan S, Yin A, Gao J, et al. Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos. Scientific Reports. 2017;7:42081. DOI: 10.1038/srep42081
  113. 113. Huang Y, Tang X, Xie W, Zhou Y, Li D, et al. Vitamin C enhancesin vitroandin vivodevelopment of porcine somatic cell nuclear transfer embryos. Biochemical and Biophysical Research Communications. 2011;411:397-401. DOI: 10.1016/j.bbrc.2011.06.160
  114. 114. Jeong YW, Park SW, Hossein MS. Anti-apoptotic and embryotrophic effects of a-tocopherol and L-ascorbic acid on porcine embryos derived fromin vitrofertilisation and somatic cell nuclear transfer. Theriogenology. 2006;66:2104-2112. DOI: 10.1016/j.theriogenology.2006.06.007
  115. 115. Liang S, Jin YX, Yuan B, Zhang JB, Kim NH. Melatonin enhances the developmental competence of porcine somatic cell nuclear transfer embryos by preventing DNA damage induced by oxidative stress. Scientific Reports. 2017;7:11114. DOI: 10.1038/s41598-017-11161-9
  116. 116. Wang XX, Jin YZ, Zuo ZR, Liu ZG. Replacement of glutamine with alanyl-glutamine dipeptide enhances the developmental competence of porcine (sus scrofa) embryos produced by somatic cell nuclear transfer. Journal of Agricultural Biotechnology. 2017;25:110-115
  117. 117. Himaki T, Yokomine TA, Sato M, Takao S, Miyoshi K, et al. Effects of trichostatin A onin vitrodevelopment and transgene function in somatic cell nuclear transfer embryos derived from transgenic Clawn miniature pig cells. Animal Science Journal. 2010;81:558-563. DOI: 10.1111/j.1740-0929.2010.00772.x
  118. 118. Cong P, Zhu K, Ji Q , Zhao H, Chen Y. Effects of trichostatin A on histone acetylation and methylation characteristics in early porcine embryos after somatic cell nuclear transfer. Journal of Animal Science. 2013;84:639-649. DOI: 10.1111/asj.12059
  119. 119. Miyoshi K, Mori H, Mizobe Y, Akasaka E, Ozawa A, et al. Valproic acid enhancesin vitrodevelopment and Oct-3/4 expression of miniature pig somatic cell nuclear transfer embryos. Cellular Reprogramming. 2010;12:67-74. DOI: 10.1089/cell.2009.0032
  120. 120. Huang Y, Tang X, Xie W, Zhou Y, Li D, et al. Histone deacetylase inhibitor significantly improved the cloning efficiency of porcine somatic cell nuclear transfer embryos. Cellular Reprogramming. 2011;13:513-520. DOI: 10.1089/cell.2011.0032
  121. 121. Miyoshi K, Maeda K, Akioka K, Sato M, Kawaguchi H, et al. Birth of cloned microminipigs derived from somatic cell nuclear transfer embryos that have been transiently treated with valproic acid. Cellular Reprogramming. 2016;18:390-400. DOI: 10.1089/cell.2016.0025
  122. 122. Zhao J, Ross JW, Hao Y, Spate LD, Walters EM, et al. Significant improvement in cloning efficiency of an inbred miniature pig by histone deacetylase inhibitor treatment after somatic cell nuclear transfer. Biology of Reproduction. 2009;81:525-530. DOI: 10.1095/biolreprod.109.077016
  123. 123. Zhao J, Hao Y, Ross JW, Spate LD, Walters EM, et al. Histone deacetylase inhibitors improvein vitroandin vivodevelopmental competence of somatic cell nuclear transfer porcine embryos. Cellular Reprogramming. 2010;12:75-83. DOI: 10.1089/cell.2009.0038
  124. 124. Zhang L, Huang Y, Wu Y, Si J, Huang Y, et al. Scriptaid upregulates expression of development-related genes, inhibits apoptosis, and improves the development of somatic cell nuclear transfer mini-pig embryos. Cellular Reprogramming. 2017;19:19-26. DOI: 10.1089/cell.2016.0033
  125. 125. Jin JX, Li S, Gao QS, Hong Y, Jin L, et al. Significant improvement of pig cloning efficiency by treatment with LBH589 after somatic cell nuclear transfer. Theriogenology. 2013;80:630-635. DOI: 10.1016/j.theriogenology.2013.06.006
  126. 126. Hou L, Ma F, Yang J, Riaz H, Wang Y, et al. Effects of histone deacetylase inhibitor oxamflatin onin vitroporcine somatic cell nuclear transfer embryos. Cellular Reprogramming. 2014;16:253-265. DOI: 10.1089/cell.2013.0058
  127. 127. Jin JX, Kang JD, Li S, Jin L, Zhu HY, et al. PXD101 significantly improves nuclear reprogramming and thein vitrodevelopmental competence of porcine SCNT embryos. Biochemical and Biophysical Research Communications. 2015;456:156-161. DOI: 10.1016/j.bbrc.2014.11.051
  128. 128. Jin L, Guo Q , Zhu HY, Xing XX, Zhang GL, et al. Quisinostat treatment improves histone acetylation and developmental competence of porcine somatic cell nuclear transfer embryos. Molecular Reproduction and Development. 2017;84:340-346. DOI: 10.1002/mrd.22787
  129. 129. Jin L, Zhu HY, Guo Q , Li XC, Zhang YC, et al. Effect of histone acetylation modification with MGCD0103, a histone deacetylase inhibitor, on nuclear reprogramming and the developmental competence of porcine somatic cell nuclear transfer embryos. Theriogenology. 2017;87:298-305. DOI: 10.1016/j.theriogenology.2016.09.011
  130. 130. Zhang Z, Zhai Y, Ma X, Zhang S, An X, et al. Down-regulation of H3K4me3 by MM-102 facilitates epigenetic reprogramming of porcine somatic cell nuclear transfer embryos. Cellular Physiology and Biochemistry. 2018;45:1529-1540. DOI: 10.1159/000487579
  131. 131. Lin T, Lee JE, Oqani RK, Kim SY, Cho ES, et al. Tauroursodeoxycholic acid improves pre-implantation development of porcine SCNT embryo by endoplasmic reticulum stress inhibition. Reproductive Biology. 2016;16:269-278. DOI: 10.1016/j.repbio.2016.10.003
  132. 132. Wang P, Li X, Cao L, Huang S, Li H, et al. MicroRNA-148a overexpression improves the early development of porcine somatic cell nuclear transfer embryos. PLoS ONE. 2017;12:e0180535. DOI: 10.1371/journal.pone.0180535
  133. 133. Matoba S, Liu Y, Lu F, Iwabuchi KA, Shen L, et al. Embryonic development following somatic cell nuclear transfer impeded by persisting histone methylation. Cell. 2014;159:884-895. DOI: 10.1016/j.cell.2014.09.055
  134. 134. Kaneko T, Sakuma T, Yamamoto T, Mashimo T. Simple knockout by electroporation of engineered endonucleases into intact rat embryos. Scientific Reports. 2014;4:6382. DOI: 10.1038/srep06382
  135. 135. Hashimoto M, Takemoto T. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing. Scientific Reports. 2015;5:11315. DOI: 10.1038/srep11315
  136. 136. Qin W, Dion SL, Kutny PM, Zhang Y, Cheng AW, et al. Efficient CRISPR/Cas9-mediated genome editing in mice by zygote electroporation of nuclease. Genetics. 2015;200:423-430. DOI: 10.1534/genetics.115.176594
  137. 137. Wei J, Gaynor P, Cole S, Brophy B, Oback B, et al. Developing the laboratory conditions for bovine zygote-mediated genome editing by electroporation. In: Proceedings of the World Congress on Genetics Applied to Livestock Production. Volume Technologies—Gene Editing; 2018. p. 1118
  138. 138. Miyoshi N, Horiuchi M, Inokuchi Y, Miyamoto Y, Miura N, et al. Novel microminipig model of atherosclerosis by fat and high cholesterol diet, established in Japan. In Vivo. 2010;24:671-680
  139. 139. Ohtsuka M, Sato M, Miura H, Takabayashi S, Matsuyama M, et al. i-GONAD: A robust method for in situ germline genome engineering using CRISPR nucleases. Genome Biology. 2018;19:25. DOI: 10.1186/s13059-018-1400-x
  140. 140. Sato M, Miyoshi M, Nakamura S, Ohtsuka M, Sakurai T, et al. Efficient generation of somatic cell nuclear transfer-competent porcine cells with mutated alleles at multiple target loci by using CRISPR/Cas9 combined with targeted toxin-based selection system. International Journal of Molecular Sciences. 2017;18:2610. DOI: 10.3390/ijms18122610
  141. 141. Sakurai T, Watanabe S, Kamiyoshi A, Sato M, Shindo T. A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice. BMC Biotechnology. 2014;14:69. DOI: 10.1186/1472-6750-14-69
  142. 142. Akasaka E, Ozawa A, Mori H, Mizobe Y, Yoshida M, et al. Whole-genome amplification-based GenomiPhi for multiple genomic analysis of individual early porcine embryos. Theriogenology. 2011;75:1543-1549. DOI: 10.1016/j.theriogenology.2010.12.018
  143. 143. Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, et al. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology. 2013;31:822-826. DOI: 10.1038/nbt.2623
  144. 144. Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell. 2013;154:1380-1389. DOI: 10.1016/j.cell.2013.08.021
  145. 145. Kim S, Kim D, Cho SW, Kim J, Kim JS. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Research. 2014;24:1012-1019. DOI: 10.1101/gr.171322.113
  146. 146. Guilinger JP, Thompson DB, Liu DR. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nature Biotechnology. 2014;32:577-582. DOI: 10.1038/nbt.2909
  147. 147. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015;163:759-771. DOI: 10.1016/j.cell.2015.09.038
  148. 148. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, et al. Rationally engineered Cas9 nucleases with improved specificity. Science. 2016;351:84-88. DOI: 10.1126/science.aad5227
  149. 149. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ , Nguyen NT, et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature. 2016;529:490-495. DOI: 10.1038/nature16526
  150. 150. Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, et al. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science. 2016;353:pii: aaf8729. DOI:10.1126/science.aaf8729
  151. 151. Abudayyeh OO, Gootenberg JS, Essletzbichler P, Han S, Joung J, et al. RNA targeting with CRISPR-Cas13. Nature. 2017;550:280-284. DOI: 10.1038/nature24049
  152. 152. Cong L, Ran FA, Cox D, Lin S, Barretto R, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819-823. DOI: 10.1126/science.1231143
  153. 153. Mali P, Yang L, Esvelt KM, Aach J, Guell M, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339:823-826. DOI: 10.1126/science.1232033
  154. 154. Sakurai T, Kamiyoshi A, Kawate H, Mori C, Watanabe S, et al. A non-inheritable maternal Cas9-based multiple-gene editing system in mice. Scientific Reports. 2016;6:20011. DOI: 10.1038/srep20011
  155. 155. Mao Z, Bozzella M, Seluanov A, Gorbunova V. Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair (Amst). 2008;7:1765-1771. DOI: 10.1016/j.dnarep.2008.06.018
  156. 156. Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, et al. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nature Biotechnology. 2015;33:538-542. DOI: 10.1038/nbt.3190
  157. 157. Chu VT, Weber T, Wefers B, Wurst W, Sander S, et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nature Biotechnology. 2015;33:543-548. DOI: 10.1038/nbt.3198
  158. 158. Song J, Yang D, Xu J, Zhu T, Chen YE, et al. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nature Communications. 2016;7:10548. DOI: 10.1038/ncomms10548
  159. 159. Gerlach M, Kraft T, Brenner B, Petersen B, Niemann H, et al. Efficient knock-in of a point mutation in porcine fibroblasts using the CRISPR/Cas9-GMNN fusion gene. Genes. 2018;9:296. DOI: 10.3390/genes9060296
  160. 160. Li G, Zhang X, Zhong C, Mo J, Quan R, et al. Small molecules enhance CRISPR/Cas9-mediated homology-directed genome editing in primary cells. Scientific Reports. 2017;7:8943. DOI: 10.1038/s41598-017-09306-x
  161. 161. Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014;159:440-455. DOI: 10.1016/j.cell.2014.09.014
  162. 162. Cebrian-Serrano A, Zha S, Hanssen L, Biggs D, Preece C, et al. Maternal supply of Cas9 to zygotes facilitates the efficient generation of site-specific mutant mouse models. PLoS ONE. 2017;12:e0169887. DOI: 10.1371/journal.pone.0169887
  163. 163. Wang K, Jin Q , Ruan D, Yang Y, Liu Q , et al. Cre-dependent Cas9-expressing pigs enable efficientin vivogenome editing. Genome Research. 2017;27:2061-2071. DOI: 10.1101/gr.222521.117

Written By

Masahiro Sato, Kazuchika Miyoshi, Hiroaki Kawaguchi, Emi Inada, Issei Saitoh and Akihide Tanimoto

Submitted: January 21st, 2019 Reviewed: June 13th, 2019 Published: July 22nd, 2019