Cytoplasmic Injection Research Articles

Bone morphogenetic protein 15 gene disruption affects the in vitro maturation of porcine oocytes by impairing spindle assembly and organelle function

Bone morphogenetic protein 15 (BMP15) plays a crucial role in the porcine follicular development. However, its exact functions in the in vitro maturation (IVM) of porcine oocytes remain largely unknown. Here, through cytoplasmic injection of a preassembled crRNA–tracrRNA–Cas9 ribonucleoprotein complex, we achieved BMP15 disruption in approximately 54 % of the cultured porcine oocytes. Editing BMP15 impaired the IVM of porcine oocytes, as indicated by the significantly increased abnormal spindle assembly and reduced first polar body (PB1) extrusion. The editing also impaired cytoplasmic maturation of porcine oocytes, as reflected by reduced abundant of Golgi apparatus and impaired functions of mitochondria. The impaired IVM of porcine oocytes by editing BMP15 possibly was associated with the attenuated SMAD1/5 and EGFR-ERK1/2 signaling in the cumulus granulosa cells (CGCs) and the inhibited MOS/ERK1/2 signaling in oocytes. The attenuated MOS/ERK1/2 signaling may contribute to the inactivation of maturation promoting factor (MPF) and the increased abnormal spindle assembly, leading to reduced PB1 extrusion. It also may contribute to reduced Golgi apparatus formation, and impaired functions of mitochondria. These findings expand our understanding of the regulatory role of BMP15 in the IVM of porcine oocytes and provide a basis for manipulation of porcine reproductive performance.

CRISPR-Cas9 Genome Editing of Rat Embryos using Adeno-Associated Virus (AAV) and 2-Cell Embryo Electroporation.

Genome editing technology is widely used to produce genetically modified animals, including rats. Cytoplasmic or pronuclear injection of DNA repair templates and CRISPR-Cas reagents is the most common delivery method into embryos. However, this type of micromanipulation necessitates access to specialized equipment, is laborious, and requires a certain level of technical skill. Moreover, microinjection techniques often result in lower embryo survival due to the mechanical stress on the embryo. In this protocol, we developed an optimized method to deliver large DNA repair templates to work in conjunction with CRISPR-Cas9 genome editing without the need for microinjection. This protocol combines AAV-mediated DNA delivery of single-stranded DNA donor templates along with the delivery of CRISPR-Cas9 ribonucleoprotein (RNP) by electroporation to modify 2-cell embryos. Using this novel strategy, we have successfully produced targeted knock-in rat models carrying insertion of DNA sequences from 1.2 to 3.0 kb in size with efficiencies between 42% and 90%.

Generation of Fel d 1 chain 2 genome-edited cats by CRISPR-Cas9 system

Allergens from domestic cats (Felis catus) cause allergy-related health problems worldwide. Fel d 1 is a major allergen that causes severe allergic reactions in humans, including rhinitis, conjunctivitis, and life-threatening asthma. Therefore, patients with cat allergies anticipate hypoallergenic cats. We successfully generated Fel d 1 chain 2 (CH2) genome-edited cats using the CRISPR-Cas9 system in this study. T7 endonuclease 1 assay and Sanger sequencing were used to confirm the mutation in CH2 genome-edited cats. Fel d 1 level in CH2 genome-edited cats were assessed by enzyme-linked immunosorbent assay (ELISA). Remarkably, ELISA showed that the level of Fel d 1 in the CH2 homozygous genome-edited cat (Name: Alsik) was extremely low compared with that in wild type domestic cats and could be hypoallergenic cats. Additionally, we successfully cloned the CH2 homozygous genome-edited cat using cytoplasm injection clone technology. The cloned CH2 homozygous genome-edited cat was verified using microsatellite analysis. Creating hypoallergenic cats using the CRISPR-Cas9 system is a significant step forward because these cats can safely approach allergic patients.

Effect of additional cytoplasm injection on the cloned bovine embryo organelle distribution and stress mitigation

Although somatic cell nuclear transfer (SCNT) is a critical component of animal cloning, this approach has several issues. We previously introduced the cytoplasm injection cloning technology (CICT), which significantly improves the quality and quantity of cloned embryos. This study examined the residual status of fused cytoplasmic organelles, such as the endoplasmic reticulum (ER) and lysosomes, in the CICT group during early embryo development. We found that extra-cytoplasmic organelles stained using the ER-Tracker™ Green dye and LysoTracker™ Deep Red probe were fused and dispersed throughout the recipient oocyte and were still visible in day 8 blastocysts. We screened for ER stress, autophagy, and apoptosis-related genes to elucidate the association between the added organelles and improved embryo quality in CICT-cloned embryos. We found that CHOP, ATF4, ATG5, ATG7, and LC3 genes showed non-significantly up- or downregulated expression between CICT- and in vitro fertilization (IVF)-derived embryos but showed significantly (p < 0.05) upregulated expression in SCNT-cloned embryos. Surprisingly, a non-significant difference in the expression of some genes, such as ATF6 and caspase-3, was observed between the CICT- and SCNT-cloned embryos. Our findings imply that compared to conventional SCNT cloning, CICT-derived cloned embryos with additional cytoplasm have much higher organelle activity, lower autophagy, lower rates of apoptosis, and higher embryo development rates.

Cytoplasmic contractile injection systems mediate cell death in Streptomyces

Contractile injection systems (CIS) are bacteriophage tail-like structures that mediate bacterial cell–cell interactions. While CIS are highly abundant across diverse bacterial phyla, representative gene clusters in Gram-positive organisms remain poorly studied. Here we characterize a CIS in the Gram-positive multicellular model organism Streptomyces coelicolor and show that, in contrast to most other CIS, S. coelicolor CIS (CISSc) mediate cell death in response to stress and impact cellular development. CISSc are expressed in the cytoplasm of vegetative hyphae and are not released into the medium. Our cryo-electron microscopy structure enabled the engineering of non-contractile and fluorescently tagged CISSc assemblies. Cryo-electron tomography showed that CISSc contraction is linked to reduced cellular integrity. Fluorescence light microscopy furthermore revealed that functional CISSc mediate cell death upon encountering different types of stress. The absence of functional CISSc had an impact on hyphal differentiation and secondary metabolite production. Finally, we identified three putative effector proteins, which when absent, phenocopied other CISSc mutants. Our results provide new functional insights into CIS in Gram-positive organisms and a framework for studying novel intracellular roles, including regulated cell death and life-cycle progression in multicellular bacteria.

Genome Editing of Pig.

Pigs have anatomical and physiological characteristics similar to humans; therefore, genetically modified pigs have the potential to become a valuable bioresource in biomedical research. In fact, considering the increasing need for translational research, pigs are useful for studying intractable diseases, organ transplantation, and regenerative medicine as large-scale experimental animals with excellent potential for extrapolation to humans. With the advent of zinc finger nucleases (ZFNs), breakthroughs in genome editing tools such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) have facilitated the efficient generation of genetically modified pigs. Genome editing has been used in pigs for more than 10 years; now, along with knockout pigs, knock-in pigs are also gaining increasing importance. In this chapter, we describe the establishment of gene-modified cells (nuclear donor cells), which are necessary for gene knockout and production of knock-in pigs via somatic cell nuclear transplantation, as well as the production of gene knockout pigs using a simple cytoplasmic injection method.

Generation of Genome-Edited Mice by Cytoplasmic Injection of CRISPR-Cas9 RNA.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR-Cas9) gene editing enables rapid production of genome-edited animals. The Cas9/guide RNA (gRNA) component can be introduced into zygotes in several ways. Here, we provide an instructional guide for the generation of knockout mice using cytoplasmic injection of in vitro transcribed Cas9 RNA and gRNA.

Cytoplasmic Microinjection of &lt;i&gt;piggyBac&lt;/i&gt; Transposase mRNA and Transposon Vectors for Efficient &lt;i&gt;In Vitro&lt;/i&gt; Production of Transgenic Porcine Parthenotes

The efficient production of transgenic (Tg) piglets has remained a challenge in the field of domestic animal studies. Unlike mice, the pronuclei of pig zygotes cannot be easily studied because of the abundance of lipid droplets. Therefore, the zygotes must be briefly centrifuged before pronuclear injection (PNI) to move the lipid droplets to the periphery of the zygote for PNI-mediated production of Tg piglets. However, this procedure is temporal because lipid droplets return to the original space during PNI, hampering the consecutive PNI. Cytoplasmic injection (CPI) of nucleic acids is comparatively simple than PNI because CPI does not require such pre-centrifugation. Unfortunately, CPI using purified DNA fragments is inadequate for creating Tg piglets because it is challenging to integrate nucleic acids into the host genome. &lt;em&gt;PiggyBac&lt;/em&gt; (PB), one of the transposons, is a valuable tool enabling efficient chromosomal integration of a transgene. The PB-mediated gene transfer requires two components, namely, transposase and transposons harboring the gene of interest (GOI) flanked by PB acceptor sites. We speculate that the CPI of transposase mRNA and transposons could accelerate the chromosomal integration of the GOI in pig zygotes. To prove this hypothesis, we performed CPI using transposase mRNA (super PB transposase mRNA) + transposon DNA carrying the enhanced green fluorescent protein (&lt;em&gt;EGFP&lt;/em&gt;) cDNA (referred to as “pT-EGFP”), transposase expression plasmid DNA (referred to as “pTrans”) + pT-EGFP, pT-EGFP alone, or non-transposon EGFP expression plasmid DNA using porcine parthenotes. Consequently, 50% (2/4 tested) of green-fluorescent embryos exhibited chromosomal integration of GOI. In contrast, green-fluorescent embryos derived from CPI with pTrans + pT-EGFP or pT-EGFP alone did not show chromosomal integration. We used mRNA for super PB transposase, which is an engineered hyperactive version of the wild-type PB transposase. To conclude, the PB system, based on the CPI of super PB transposase mRNA + transposon DNA, could be useful for producing Tg porcine parthenotes.

Characterization of SHARPIN knockout Syrian hamsters developed using CRISPR/Cas9 system.

SHARPIN (SHANK-associated RH domain interactor) is a component of the linear ubiquitination complex that regulates the NF-κB signaling pathway. To better understand the function of SHARPIN, we sought to establish a novel genetically engineered Syrian hamster with SHARPIN disruption using the CRISPR/Cas9 system. A single-guide ribonucleic acid targeting exon 1 of SHARPIN gene was designed and constructed. The zygotes generated by cytoplasmic injection of the Cas9/gRNA ribonucleoprotein were transferred into pseudopregnant hamsters. Neonatal mutants were identified by genotyping. SHARPIN protein expression was detected using Western blotting assay. Splenic, mesenteric lymph nodes (MLNs), and thymic weights were measured, and organ coefficients were calculated. Histopathological examination of the spleen, liver, lung, small intestine, and esophagus was performed independently by a pathologist. The expression of lymphocytic markers and cytokines was evaluated using reverse transcriptase-quantitative polymerase chain reaction. All the offspring harbored germline-transmitted SHARPIN mutations. Compared with wild-type hamsters, SHARPIN protein was undetectable in SHARPIN-/- hamsters. Spleen enlargement and splenic coefficient elevation were spotted in SHARPIN-/- hamsters, with the descent of MLNs and thymuses. Further, eosinophil infiltration and structural alteration in spleens, livers, lungs, small intestines, and esophagi were obvious after the deletion of SHARPIN. Notably, the expression of CD94 and CD22 was downregulated in the spleens of knockout (KO) animals. Nonetheless, the expression of CCR3, CCL11, Il4, and Il13 was upregulated in the esophagi. The expression of NF-κB and phosphorylation of NF-κB and IκB protein significantly diminished in SHARPIN-/- animals. A novel SHARPIN KO hamster was successfully established using the CRISPR/Cas9 system. Abnormal development of secondary lymphoid organs and eosinophil infiltration in multiple organs reveal its potential in delineating SHARPIN function and chronic inflammation.

PKR and TLR3 trigger distinct signals that coordinate the induction of antiviral apoptosis

RIG-I-like receptors (RLRs), protein kinase R (PKR), and endosomal Toll-like receptor 3 (TLR3) sense viral non-self RNA and are involved in cell fate determination. However, the mechanisms by which intracellular RNA induces apoptosis, particularly the role of each RNA sensor, remain unclear. We performed cytoplasmic injections of different types of RNA and elucidated the molecular mechanisms underlying viral dsRNA-induced apoptosis. The results obtained revealed that short 5′-triphosphate dsRNA, the sole ligand of RIG-I, induced slow apoptosis in a fraction of cells depending on IRF-3 transcriptional activity and IFN-I production. However, intracellular long dsRNA was sensed by PKR and TLR3, which activate distinct signals, and synergistically induced rapid apoptosis. PKR essentially induced translational arrest, resulting in reduced levels of cellular FLICE-like inhibitory protein and functioned in the TLR3/TRIF-dependent activation of caspase 8. The present results demonstrated that PKR and TLR3 were both essential for inducing the viral RNA-mediated apoptosis of infected cells and the arrest of viral production.

Cytoplasmic Injection of Zygotes to Genome Edit Naturally Occurring Sequence Variants Into Bovine Embryos.

Genome editing provides opportunities to improve current cattle breeding strategies through targeted introduction of natural sequence variants, accelerating genetic gain. This can be achieved by harnessing homology-directed repair mechanisms following editor-induced cleavage of the genome in the presence of a repair template. Introducing the genome editors into zygotes and editing in embryos has the advantage of uncompromised development into live animals and alignment with contemporary embryo-based improvement practices. In our study, we investigated the potential to introduce sequence variants, known from the pre-melanosomal protein 17 (PMEL) and prolactin receptor (PRLR) genes, and produce non-mosaic, edited embryos, completely converted into the precision genotype. Injection of gRNA/Cas9 editors into bovine zygotes to introduce a 3 bp deletion variant into the PMEL gene produced up to 11% fully converted embryos. The conversion rate was increased to up to 48% with the use of TALEN but only when delivered by plasmid. Testing three gRNA/Cas9 editors in the context of several known PRLR sequence variants, different repair template designs and delivery as DNA, RNA or ribonucleoprotein achieved full conversion rates up to 8%. Furthermore, we developed a biopsy-based screening strategy for non-mosaic embryos which has the potential for exclusively producing non-mosaic animals with intended precision edits.

Sperm DNA Fragmentation Index: A Comparison Study of Success Rates among Natural, Intrauterine Insemination (IUI) and In Vitro Fertilization (IVF)-Intra Cytoplasmic Sperm Injection (ICSI) Pregnancy Programs

Background: Some studies have reported a relationship between the sperm DNA fragmentation index (DFI) and the rate of fertilization and pregnancy. This study was designed to assess the mean sperm DNA fragmentation in pregnancies that occur in infertile couples, whether in natural pregnancy, intrauterine insemination (IUI), and in vitro fertilization (IVF) – intra cytoplasmic injection (ICSI).&#x0D; Methods: This research is an observational analytic study with a cross-sectional design. The sample taken in this study were infertile patients that underwent natural pregnancy or IUI or IVF-ICSI at Yasmin Infertility Clinic of Dr. Ciptomangunkusumo Hospital period 2018-2020 with a consecutive sampling technique. The research data was processed and analyzed by the Mann-Whitney test using the SPSS application.&#x0D; Results: The mean DFI of sperm in men with infertile couples who successfully conceived naturally was 10.7% (mild), while IUI was 20.4% (moderate), and IVF-ICSI was 30.5% (poor). The mean DFI in semen samples of men from infertile couples who underwent a natural program was significantly lower in those who successfully conceived compared to those who did not. Similar results were also shown in the IUI and IVF-ICSI programs, which showed a significantly lower DFI compared to non-pregnant women.&#x0D; Conclusion: DFI can be applied as a marker for selecting the type of pregnancy program in infertility management.

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice.

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (>1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (>1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

A Simplified Protocol to Incorporate the Fluorescent Unnatural Amino Acid ANAP into Xenopus laevis Oocyte-Expressed P2X7 Receptors.

The long intracellular P2X7 C-terminus accounts for diverse downstream effects of P2X7 activation. Although the recent determination of the cryo-EM structure of the full-length P2X7 receptor finally revealed the structure and several unexpected features of the large cytoplasmic domain, its molecular function remains enigmatic. Incorporation of unnatural amino acids (UAA) via an amber Stop codon has been a powerful tool for structure-function analysis of proteins. Voltage clamp fluorometry (VCF) with the fluorescent unnatural amino acid L-3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (ANAP) provides a means to study intracellular domain movements of ion channel receptors. In the Xenopus laevis oocyte expression system, site-specific introduction of this environment-sensitive fluorophore can be achieved by the nuclear injection of cDNA encoding an orthogonal amber suppressor tRNA/aminoacyl-tRNA synthetase pair and subsequent cytoplasmic injection of ANAP together with the respective cRNA containing the amber Stop codon. Here, we describe this protocol for expression of ANAP-labeled P2X7. In addition, we provide a simplified alternative protocol, in which we coinject cRNAs encoding the tRNA synthetase and mutant P2X7 together with the synthesized amber suppressor tRNA and ANAP in one step into the cytosol. We found that the new protocol yielded more reproducible results and was less harmful for the oocytes. By selective fluorescence labeling of the ANAP-labeled P2X7 protein in the oocyte plasma membrane and VCF recordings, we show that this method results in comparable levels of functional ANAP-labeled P2X7 protein.

Highly Efficient Synthetic CRISPR RNA/Cas9-Based Mutagenesis for Rapid Cardiovascular Phenotypic Screening in F0 Zebrafish

The zebrafish is a valuable vertebrate model to study cardiovascular formation and function due to the facile visualization and rapid development of the circulatory system in its externally growing embryos. Despite having distinct advantages, zebrafish have paralogs of many important genes, making reverse genetics approaches inefficient since generating animals bearing multiple gene mutations requires substantial efforts. Here, we present a simple and robust synthetic CRISPR RNA/Cas9-based mutagenesis approach for generating biallelic F0 zebrafish knockouts. Using a dual-guide synthetic CRISPR RNA/Cas9 ribonucleoprotein (dgRNP) system, we compared the efficiency of biallelic gene disruptions following the injections of one, two, and three dgRNPs per gene into the cytoplasm or yolk. We show that simultaneous cytoplasmic injections of three distinct dgRNPs per gene into one-cell stage embryos resulted in the most efficient and consistent biallelic gene disruptions. Importantly, this triple dgRNP approach enables efficient inactivation of cell autonomous and cell non-autonomous gene function, likely due to the low mosaicism of biallelic disruptions. In support of this finding, we provide evidence that the F0 animals generated by this method fully phenocopied the endothelial and peri-vascular defects observed in corresponding stable mutant homozygotes. Moreover, this approach faithfully recapitulated the trunk vessel phenotypes resulting from the genetic interaction between two vegfr2 zebrafish paralogs. Mechanistically, investigation of genome editing and mRNA decay indicates that the combined mutagenic actions of three dgRNPs per gene lead to an increased probability of frameshift mutations, enabling efficient biallelic gene disruptions. Therefore, our approach offers a highly robust genetic platform to quickly assess novel and redundant gene function in F0 zebrafish.

Effects of Donor Cell Types on the Development of Bovine Embryos Using Cytoplasm Injection Cloning Technology

Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic cells (ECs) were used as donor cells to clone bovine embryos using CICT. We initially used AF cells to develop cloned embryos and then cultured the cloned day-8 blastocysts for 10 days to obtain ECs as donor cells for second embryo cloning. We found that the bovine blastocysts cloned using AF cells had significantly reduced developmental rates, embryo quality, and ratios of inner cell mass (ICM) to the total number of cells compared to those using ECs as donor cells. Furthermore, there were significant differences in the DNA methyltransferase-, histone deacetylation-, apoptosis-, and development-related genes at the blastocyst stage in embryos cloned from AFs compared to those in embryos cloned from ECs. Our results suggest that using ECs as donor cells for nuclear transfer enhances the quantity and quality of cloned embryos. However, further investigation is required in terms of determining pregnancy rates and developing cloned embryos from different donor cell types.

Electroporation-Mediated Genome Editing of Livestock Zygotes.

The introduction of genome editing reagents into mammalian zygotes has traditionally been accomplished by cytoplasmic or pronuclear microinjection. This time-consuming procedure requires expensive equipment and a high level of skill. Electroporation of zygotes offers a simplified and more streamlined approach to transfect mammalian zygotes. There are a number of studies examining the parameters used in electroporation of mouse and rat zygotes. Here, we review the electroporation conditions, timing, and success rates that have been reported for mice and rats, in addition to the few reports about livestock zygotes, specifically pigs and cattle. The introduction of editing reagents at, or soon after, fertilization can help reduce the rate of mosaicism, the presence of two of more genotypes in the cells of an individual; as can the introduction of nuclease proteins rather than mRNA encoding nucleases. Mosaicism is particularly problematic in large livestock species with long generation intervals as it can take years to obtain non-mosaic, homozygous offspring through breeding. Gene knockouts accomplished via the non-homologous end joining pathway have been more widely reported and successfully accomplished using electroporation than have gene knock-ins. Delivering large DNA plasmids into the zygote is hindered by the zona pellucida (ZP), and the majority of gene knock-ins accomplished by electroporation have been using short single stranded DNA (ssDNA) repair templates, typically less than 1 kb. The most promising approach to deliver larger donor repair templates of up to 4.9 kb along with genome editing reagents into zygotes, without using cytoplasmic injection, is to use recombinant adeno-associated viruses (rAAVs) in combination with electroporation. However, similar to other methods used to deliver clustered regularly interspaced palindromic repeat (CRISPR) genome-editing reagents, this approach is also associated with high levels of mosaicism. Recent developments complementing germline ablated individuals with edited germline-competent cells offer an approach to avoid mosaicism in the germline of genome edited founder lines. Even with electroporation-mediated delivery of genome editing reagents to mammalian zygotes, there remain additional chokepoints in the genome editing pipeline that currently hinder the scalable production of non-mosaic genome edited livestock.

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

The production of genetically modified (GM) pigs is considered valuable in biomedical research for the development of model animals for various diseases and pigs with resistance against viral infection. The porcine genome may be modified using several methods, such as somatic cell nuclear transfer (SCNT) using GM cells as the SCNT donor, direct injection of the transgene or the genome editing components (GEC) into fertilized eggs referred to as zygotes, the in vitro electroporation (EP) of the zygotes in the presence of GECs, viral infection using retroviruses, injection of the GECs into the SCNT-treated embryos, and the in vitro EP of the SCNT-treated embryos in the presence of GECs. In our previous study, we administered a cytoplasmic injection of CRISPR/Cas9-based GEC into parthenogenetically-activated porcine oocytes (referred to as parthenotes) and observed that these oocytes comprised a mixture of genome-edited and genome-unedited cells, referred to as the “mosaic”. In contrast, when in vitro EP of the SCNT-treated embryos in the presence of GEC was performed, bi-allelic knock out (KO) of the target gene was detected in most oocytes (82%; 9/11). The production of bi-allelic KO piglets is particularly beneficial for investigating GM domestic animals as it does not require further breeding trials to obtain bi-allelic KO individuals, which would otherwise be a time-consuming and laborious task. In this context, the present study was aimed to confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos without multiple breeding trials, for which parthenotes were subjected to EP in the presence of a ribonucleoprotein containing Cas9 protein and single-guide RNA (targeted toward GGTA1). The treated embryos were cultured until they transformed into blastocysts. The genomic DNA isolated from these blastocysts was used for molecular biology analysis to detect the possible insertion and deletion of sequences (indels) at the GGTA1 locus. Among the 32 blastocysts obtained, 21 (66%) were observed to be the bi-allelic KO ones. The remaining embryos either had a normal phenotype (25%; 8/32) or mosaic mutations (9%; 3/32). These findings confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos.

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

The production of genetically modified (GM) pigs is considered valuable in biomedical research for the development of model animals for various diseases and pigs with resistance against viral infection. The porcine genome may be modified using several methods, such as somatic cell nuclear transfer (SCNT) using GM cells as the SCNT donor, direct injection of the transgene or the genome editing components (GEC) into fertilized eggs referred to as zygotes, the in vitro electroporation (EP) of the zygotes in the presence of GECs, viral infection using retroviruses, injection of the GECs into the SCNT-treated embryos, and the in vitro EP of the SCNT-treated embryos in the presence of GECs. In our previous study, we administered a cytoplasmic injection of CRISPR/Cas9-based GEC into parthenogenetically-activated porcine oocytes (referred to as parthenotes) and observed that these oocytes comprised a mixture of genome-edited and genome-unedited cells, referred to as the “mosaic”. In contrast, when in vitro EP of the SCNT-treated embryos in the presence of GEC was performed, bi-allelic knock out (KO) of the target gene was detected in most oocytes (82%; 9/11). The production of bi-allelic KO piglets is particularly beneficial for investigating GM domestic animals as it does not require further breeding trials to obtain bi-allelic KO individuals, which would otherwise be a time-consuming and laborious task. In this context, the present study was aimed to confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos without multiple breeding trials, for which parthenotes were subjected to EP in the presence of a ribonucleoprotein containing Cas9 protein and single-guide RNA (targeted toward GGTA1). The treated embryos were cultured until they transformed into blastocysts. The genomic DNA isolated from these blastocysts was used for molecular biology analysis to detect the possible insertion and deletion of sequences (indels) at the GGTA1 locus. Among the 32 blastocysts obtained, 21 (66%) were observed to be the bi-allelic KO ones. The remaining embryos either had a normal phenotype (25%; 8/32) or mosaic mutations (9%; 3/32). These findings confirm the efficiency of in vitro EP in producing bi-allelic KO porcine embryos.