Recent Advance in Genome Editing-Based Gene Modification in Pigs

Abstract

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 transferderived embryos that have been reconstituted with normal nuclei. 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. Background of second-generation genome editingAs 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 Fok I (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 (a...

Pork Production with Entire Males: Directions for Control of Boar Taint

“…The application of genome editing in livestock species [103] and specifically in pig [104] has been recently reviewed. The most efficient genome editing tool is the CRISPR/Cas9 system that originated as a defense mechanism of bacteria against foreign viral DNA.…”

In vitro Electroporation in the Presence of CRISPR/Cas9 Reagents as a Safe and Effective Method for Producing Biallelic Knock-Out Porcine Embryos

“…For instance, when TP53 was targeted, all the tested blastocysts (15) exhibited mutations in the TP53 target region, while approximately 45% of the blastocysts carried bi-allelic KO mutations [15]. Notably, in our previous study concerning the in vitro EP of SCNT-treated embryos in the presence of the RNP targeted toward the low-density lipoprotein receptor (LDLR) gene, almost all (82%, 9/11) of the resulting embryos (blastocysts) exhibited the bi-allelic KO genotype [14]. Taken together, these findings suggest that the in vitro EP-based genome-editing technology used in the present study is safe and efficient for the production of porcine embryos with bi-allelic mutated phenotypes.…”

“…Unfortunately, the in vitro EP-based technology for obtaining genome-edited piglets continues to be applied scarcely, with only a few laboratories worldwide reporting success in producing genome-edited porcine embryos and piglets using this technology [13][14][15][16][17][18][19][20]56]. Tanihara and Otoi successfully knocked-out the genes encoding myostatin (MSTN) [13,20], porcine endogenous retroviruses (PERV) polymerase (pol) [18], TP53 (which encodes p53) [15], cluster of differentiation 163 (CD163) [16], and pancreas duodenum homeobox 1 (PDX-1) [17] by using in vitro EP in porcine embryos.…”

“…Currently, several methods are available for the production of GM embryos and piglets, such as 1) microinjection of nucleic acids (NAs) into the pronuclei of zygotes [8]; 2) somatic cell nuclear transfer (SCNT) from a GM donor cell into the enucleated porcine oocytes [9][10][11]; 3) microinjection of nucleic acids (NAs) into SCNT-treated embryos [12]; 3) in vitro electroporation (EP) of porcine zygotes in the presence of NAs [13]; and 5) in vitro EP of the SCNT-treated embryos in the presence of NAs [14]. According to Sato et al [14], in the case of the 61 genome-edited piglets produced since 2013, most of these experiments were focused either on using SCNT-based production of GM piglets or on zygote microinjection [14], while the in vitro EP-based production of GM piglets was used by only a few laboratories [13,[15][16][17][18][19][20]. This is in contrast to the case of genome-edited mice.…”

In vitro Electroporation in the Presence of CRISPR/Cas9 Reagents as a Safe and Effective Method for Producing Biallelic Knock-Out Porcine Embryos

“…Currently, several methods are available for the production of GM embryos and piglets, such as 1) microinjection of nucleic acids (NAs) into the pronuclei of zygotes [8]; 2) somatic cell nuclear transfer (SCNT) from a GM donor cell into the enucleated porcine oocytes [9][10][11]; 3) microinjection of nucleic acids (NAs) into SCNT-treated embryos [12]; 3) in vitro electroporation (EP) of porcine zygotes in the presence of NAs [13]; and 5) in vitro EP of the SCNT-treated embryos in the presence of NAs [14]. According to Sato et al [14], in the case of the 61 genome-edited piglets produced since 2013, most of these experiments were focused either on using SCNT-based production of GM piglets or on zygote microinjection [14], while the in vitro EP-based production of GM piglets was used by only a few laboratories [13,[15][16][17][18][19][20]. This is in contrast to the case of genome-edited mice.…”