RESUMEN
Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation. Centrifugation at 2000 × g did not adversely affect blastocyst formation rates in zygotes electroporated with gRNA targeting the GGTA1 gene; instead, it led to increased total and monoallelic mutation rates compared with control zygotes without centrifugation. However, the groups had no significant differences in biallelic mutation rates. In zygotes electroporated with gRNA targeting the CMAH gene, centrifugation treatments exceeding 1000 × g significantly increased both biallelic mutation rates and mutation efficiency. The combination of centrifugation and zona pellucida removal did not have a detrimental effect on blastocyst formation rates. It led to a higher rate of double biallelic mutations in embryos targeting both GGTA1 and CMAH compared to embryos without centrifugation treatment. In summary, our results demonstrate that pre-electroporation treatments, including centrifugation and zona pellucida removal, positively influenced the reduction of mosaic mutations, with the effectiveness of centrifugation depending on the specific gRNA used.
Asunto(s)
Sistemas CRISPR-Cas , Centrifugación , Electroporación , Edición Génica , Animales , Edición Génica/métodos , Porcinos , Electroporación/métodos , Sistemas CRISPR-Cas/genética , Embrión de Mamíferos/metabolismo , Zona Pelúcida/metabolismo , Cigoto/metabolismo , Blastocisto/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , Mutación/genética , GalactosiltransferasasRESUMEN
We evaluated the quality and fertilizing ability of frozen-thawed porcine sperm that were selected using a commercially available device (MIGLIS, Menicon Life Science) consisting of three parts: an outer lid, an inner lid, and a tube. Firstly, to determine an adequate concentration of caffeine for separation, frozen-thawed sperm were incubated with different concentrations of caffeine (0, 1, 2.5, 5, and 10 mM) in a MIGLIS device. To determine the appropriate incubation time for separating sperm in the MIGLIS device, frozen-thawed sperm were incubated with 2.5 mM caffeine for 5, 10, 15, or 20 min. To evaluate the fertilization and embryo development of oocytes fertilized with frozen-thawed sperm separated into two regions (outer and inner) in the MIGLIS device, the separated sperm from the three boars was used to fertilize in vitro-matured oocytes and cultured in vitro for 7 days. Sperm quality parameters of sperm collected from the inner tube after incubation with 2.5 mM caffeine were superior to sperm incubated without caffeine. Moreover, sperm collected from the inner tube after incubation for 10 min had a higher progressive motility. The rate of blastocyst produced from spermatozoa collected from the inner tube after incubation with 2.5 mM caffeine for 10 min significantly increased compared to that produced from spermatozoa from the outer tube, regardless of the boar. In conclusion, sperm sorting using the MIGLIS device may be useful for separating high-quality sperm after incubation with 2.5 mM caffeine for 10 min to improve blastocyst formation.
Asunto(s)
Cafeína , Criopreservación , Fertilización In Vitro , Preservación de Semen , Motilidad Espermática , Espermatozoides , Animales , Masculino , Cafeína/farmacología , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Fertilización In Vitro/veterinaria , Criopreservación/veterinaria , Criopreservación/métodos , Preservación de Semen/veterinaria , Preservación de Semen/métodos , Femenino , Motilidad Espermática/efectos de los fármacos , Porcinos , Desarrollo Embrionario/efectos de los fármacos , Oocitos/efectos de los fármacos , Oocitos/fisiología , Blastocisto/efectos de los fármacos , Blastocisto/fisiologíaRESUMEN
The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates. Therefore, this study elucidated the effects of culture methods and reagent combinations on the CRISPR/Cas9 gene editing systems by using three lipofection reagents: Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (CM), Lipofectamine™ 2000 Transfection Reagent (LP), and jetCRISPR™ RNP Transfection Reagent (Jet). Porcine zona pellucida-free zygotes were incubated for 5 h with Cas9, a guide RNA targeting CD163, and the above lipofection reagents. When examining the effect of culture methods using 4-well (multiple embryo culture) and 25-well plates (single embryo culture) on the efficiency of CM-mediated zygote transfection, the culture of embryos in 25-well plates significantly increased the blastocyst formation rate; however, there was no difference in mutation rates between the 4-well and 25-well plates. When assessing the effects of individual or combined reagents on the efficiency of zygote transfection, the mutation rate was significantly lower for individual LP compared to individual CM- and Jet-mediated transfections. Moreover, combinations of lipofection transfection reagents did not significantly increase the mutation rate or mutation efficiency.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Transfección , Cigoto , Animales , Sistemas CRISPR-Cas/genética , Edición Génica/métodos , Cigoto/metabolismo , Porcinos , Transfección/métodos , ARN Guía de Sistemas CRISPR-Cas/genética , Técnicas de Cultivo de Embriones/métodos , Indicadores y Reactivos , Mutación , LípidosRESUMEN
Programmed cell death-1 (PD-1) is an immunoinhibitory receptor required to suppress inappropriate immune responses such as autoimmunity. Immune checkpoint antibodies that augment the PD-1 pathway lead to immune-related adverse events (irAEs), organ non-specific side effects due to autoimmune activation in humans. In this study, we generated a PD-1 mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes to evaluate the PD-1 gene deficiency phenotype. We optimized the efficient guide RNAs (gRNAs) targeting PD-1 in zygotes and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. One recipient gilt became pregnant and gave birth to two piglets. Sequencing analysis revealed that both piglets were biallelic mutants. At 18 mo of age, one pig showed non-purulent arthritis of the left elbow/knee joint and oligozoospermia, presumably related to PD-1 modification. Although this study has a limitation because of the small number of cases, our phenotypic analysis of PD-1 modification in pigs will provide significant insight into human medicine and PD-1-deficient pigs can be beneficial models for studying human irAEs.
Asunto(s)
Sistemas CRISPR-Cas , Electroporación , Fertilización In Vitro , Edición Génica , Receptor de Muerte Celular Programada 1 , Cigoto , Animales , Edición Génica/métodos , Electroporación/métodos , Cigoto/metabolismo , Fertilización In Vitro/métodos , Porcinos , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/metabolismo , Sistemas CRISPR-Cas/genética , Animales Modificados Genéticamente , ARN Guía de Sistemas CRISPR-Cas/genética , Femenino , Secuencia de Bases , Mutación/genética , FenotipoRESUMEN
The generation of genetically engineered pig models that develop pancreas-specific tumors has the potential to advance studies and our understanding of pancreatic cancer in humans. TP53 mutation causes organ-nonspecific cancers, and PDX1-knockout results in the loss of pancreas development. The aim of the present study was to generate a PDX1-knockout pig chimera carrying pancreas complemented by TP53 mutant cells via phytohemagglutinin (PHA)-mediated blastomere aggregation using PDX1 and TP53 mutant blastomeres, as a pig model for developing tumors in the pancreas with high frequency. First, the concentration and exposure time to PHA to achieve efficient blastomere aggregation were optimized. The results showed that using 300 µg/mL PHA for 10 min yielded the highest rates of chimeric blastocyst formation. Genotyping analysis of chimeric blastocysts derived from aggregated embryos using PDX1- and TP53-edited blastomere indicated that approximately 28.6% carried mutations in both target regions, while 14.3-21.4% carried mutations in one target. After the transfer of the chimeric blastocysts into one recipient, the recipient became pregnant with three fetuses. Deep sequencing analysis of the PDX1 and TP53 regions using ear and pancreas samples showed that one fetus carried mutations in both target genes, suggesting that the fetus was a chimera derived from embryo-aggregated PDX1 and TP53 mutant blastomeres. Two out of three fetuses carried only the PDX1 mutation, indicating that the fetuses developed from embryos not carrying TP53-edited blastomeres. The results of the present study could facilitate the further improvement and design of high-frequency developing pancreatic tumor models in pigs.
Asunto(s)
Blastómeros , Proteínas de Homeodominio , Mutación , Fitohemaglutininas , Transactivadores , Proteína p53 Supresora de Tumor , Animales , Blastómeros/metabolismo , Blastómeros/citología , Transactivadores/genética , Transactivadores/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Mutación/genética , Porcinos , Fitohemaglutininas/farmacología , Quimera/genética , Blastocisto/metabolismo , FemeninoRESUMEN
This study examined the effects of ergothioneine (EGT) supplementation as an antioxidant on the quality of boar spermatozoa when using liquid and frozen preservation methods. In the first experiment, boar semen was preserved in an extender supplemented with 0, 50, 100 and 200 µM EGT, at 15 °C, part of the samples for one and another part for three weeks. In comparison with the control (without EGT), EGT supplementation at 100 µM significantly increased the percentage of total motility of spermatozoa that were preserved as a liquid both for one and three weeks (P < 0.05). EGT supplementation did not affect the quality of preserved spermatozoa, irrespective of the EGT concentration. In the second experiment, semen was frozen and thawed in the freezing extender supplemented with 0, 50, 100 and 200 µM EGT. In comparison with the control, the 100 µM EGT supplementation significantly increased the percentages of total and progressive motility of frozen-thawed spermatozoa (P < 0.05). EGT (100 µM) supplementation did not affect the viability, the plasma membrane integrity, or the acrosomal integrity of frozen-thawed spermatozoa. These findings indicate that supplementing extenders with 100 µM EGT may improve the motility of boar sperm in both liquid and freezing preservation methods.
Asunto(s)
Ergotioneína , Masculino , Porcinos , Animales , Ergotioneína/farmacología , Semen , Suplementos Dietéticos , Antioxidantes/farmacología , EspermatozoidesRESUMEN
Sterilization of the culture medium using ultraviolet (UV)-C reduces the potential adverse effects of microorganisms and allows for long-term use. In the present study, we investigated the effects of a medium directly irradiated with UV-C prior to in vitro culture on the development and quality of porcine in vitro-fertilized embryos and the free amino acid composition of the culture media. The culture media (porcine zygote medium [PZM-5] and porcine blastocyst medium [PBM]) were irradiated with UV-C at 228 and 260 nm for 1 and 3 days, respectively. Next, the culture media were irradiated with UV-C at 228 nm for 3, 7, or 14 days. After in vitro fertilization, the embryos were cultured in the UV-C-irradiated media for 7 days. Free amino acid levels in culture media irradiated with 228 and 260 nm UV-C for 3 days were analysed. The blastocyst formation rate of embryos cultured in media irradiated with 260 nm UV-C for 3 days was significantly lower than that of embryos cultured in non-irradiated control media. However, 228 nm UV-C irradiation for up to 14 days did not affect blastocyst formation rates and quality in the resulting blastocysts. Moreover, 260 nm UV-C irradiation significantly increased the taurine concentration in both culture media and decreased methionine concentration in the PBM. In conclusion, UV-C irradiation at 228 nm before in vitro culture had no detrimental effects on embryonic development. However, 260 nm UV-C irradiation decreased embryo development and altered the composition of free amino acids in the medium.
Asunto(s)
Aminoácidos , Desarrollo Embrionario , Animales , Femenino , Embarazo , Porcinos , Cigoto , Fertilización In Vitro/veterinaria , Medios de CultivoRESUMEN
This study aimed to assess the potential of the centrifuge-free commercial device (MIGLIS®) in selecting functional frozen-thawed bovine sperm by migration-sedimentation, its effect on embryo development, and compare the potential with that of centrifugation-based techniques, including washing and Percoll density gradient centrifugation (DGC). In experiment 1, different dilutions (1.5×, 2×, and 3×) of frozen-thawed spermatozoa were assessed to identify the adequate one for the MIGLIS method. In experiment 2, the recovery rates, quality, and reactive oxygen species (ROS) concentrations of the spermatozoa selected using MIGLIS, washing, and Percoll DGC were compared. In experiment 3, the resultant in vitro fertilised embryos from spermatozoa selected using the three methods were evaluated for blastocyst formation rates and intracellular ROS concentrations at the 2-4 cell stage. The intracellular ROS concentrations were investigated using 2', 7'-dichlorodihydrofluorescein diacetate staining. Using the MIGLIS device, significantly more spermatozoa were recovered at 2× dilution compared with the other dilution ratio, but the motility was not affected by the dilution ratio. On the selection of spermatozoa using the three methods, employing MIGLIS decreased the recovery rates. However, the MIGLIS method increased motility, viability, and acrosome integrity rates compared to those in spermatozoa from the other methods. The ROS concentration of spermatozoa in the MIGLIS method was significantly lower than that in the washing method. Nevertheless, blastocyst formation rates were similar among the three methods, but the ROS concentration of early-stage embryos produced using MIGLIS was significantly lower than those produced using Percoll DGC. In conclusion, the MIGLIS method has the potential to select functional, high-quality frozen-thawed bovine spermatozoa.
Asunto(s)
Preservación de Semen , Semen , Masculino , Animales , Bovinos , Especies Reactivas de Oxígeno , Criopreservación/veterinaria , Criopreservación/métodos , Motilidad Espermática , Espermatozoides , Preservación de Semen/veterinaria , Preservación de Semen/métodos , Centrifugación/veterinariaRESUMEN
The transfection efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas ribonucleoprotein complexes was compared using three nonviral vector transfection reagents: nonliposomal polymeric (TransIT-X2), lipid nanoparticle delivery (CRISPRMAX), and peptide (ProteoCarry) systems. Porcine zona pellucida-free zygotes and embryos were incubated for 5 h with CRISPR-associated protein 9 (Cas9), guide RNA (gRNA) targeting GGTA1, and one of the reagents. In Experiment 1, optimization of Cas9 protein to gRNA molar ratios of 1:2, 2:2, and 4:2, along with single or double doses of reagents, was performed on zygotes at 10 h post-in vitro fertilization. In Experiment 2, optimization of timing was performed at 10 or 29 h post-in vitro fertilization, using optimal molar ratios and reagent doses. Blastocyst formation, mutation rates, and mutation efficiency were measured in each experiment. For each reagent, a 4:2 Cas9:gRNA molar ratio and addition of a double reagent dose exhibited a higher mutation rate; however, blastocyst rate tended to decrease compared with that of control. Moreover, the optimal transfection time varied depending on the reagent, and the proportions of blastocysts carrying mutations were <34%. In conclusion, the above three transfectants allowed gene editing of porcine zygotes and embryos; however, this newly established chemistry-based technology needs further improvement, especially regarding editing efficiency and embryo development.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Porcinos/genética , Animales , Edición Génica/veterinaria , Proteína 9 Asociada a CRISPR/genética , Cigoto , Desarrollo EmbrionarioRESUMEN
Bilirubin is excreted into the bile from hepatocytes, mainly as monoglucuronosyl and bisglucuronosyl conjugates, reflecting bilirubin glucuronidation activity. However, there is limited information on the in vitro evaluation of liver cell lines or primary hepatocytes. This study aimed to investigate variations in the bilirubin metabolic function of canine and human hepatocyte spheroids formed in a three-dimensional (3D) culture system indicated by the formation of bilirubin glucuronides when protease inhibitors such as atazanavir, indinavir, ritonavir, and nelfinavir were treated with bilirubin. The culture supernatant was collected for bilirubin glucuronidation assessment and the cells were used to evaluate viability. On day 8 of culture, both canine and human hepatocyte spheroids showed high albumin secretion and distinct spheroid formation, and their bilirubin glucuronidation activities were evaluated considering cell viability. Treatment with atazanavir and ritonavir remarkably inhibited bilirubin glucuronide formation, wherein atazanavir showed the highest inhibition, particularly in human hepatocyte spheroids. These results may reflect the effects on cellular uptake of bilirubin and its intracellular metabolic function. Thus, primary hepatocytes cultured in a 3D culture system may be a useful in vitro system for the comprehensive evaluation of bilirubin metabolic function and risk assessment in bilirubin metabolic disorders for drug development.
Asunto(s)
Hepatocitos , Inhibidores de Proteasas , Humanos , Animales , Perros , Sulfato de Atazanavir/metabolismo , Sulfato de Atazanavir/farmacología , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Bilirrubina/metabolismo , Bilirrubina/farmacología , Hígado/metabolismo , Ritonavir/farmacología , Ritonavir/metabolismo , Esferoides Celulares/metabolismoRESUMEN
Pigs rarely develop cancer; however, tumour protein p53 (TP53)-modified pigs may have an increased incidence of cancer. In this study, two pigs with mosaic mutations induced by gene editing were compared to determine the role of the wild-type TP53 sequence in tumorigenesis and to speculate how amino acid changes in TP53 sequences are related to tumorigenesis. The pig without tumours had a wild-type TP53 sequence and a 1-bp deletion in the TP53 sequence that resulted in a premature stop codon. In contrast, the pig with nephroblastoma had 6- and 7-bp deletions in the TP53 sequence, resulting in the absence of two amino acids and a premature stop codon, respectively. Our results indicated that TP53 mutations with truncated amino acids may be related to tumour formation.
RESUMEN
Genetic mosaicism is considered one of the main limitations of the electroporation method used to transfer CRISPR-Cas9/guide RNA (gRNA) into porcine zygotes. We hypothesized that fertilization of oocytes with sperm from gene-deficient boars, in combination with electroporation (EP) to target the same region of the gene in subsequent zygotes, would increase the gene modification efficiency. As myostatin (MSTN) and α1,3-galactosyltransferase (GGTA1) have beneficial effects on agricultural production and xenotransplantation, respectively, we used these two genes to test our hypothesis. Spermatozoa from gene-knockout boars were used for oocyte fertilization in combination with EP to transfer gRNAs targeting the same gene region to zygotes. No significant differences in the rates of cleavage and blastocyst formation as well as in the mutation rates of blastocysts were observed between the wild-type and gene-deficient spermatozoa groups, irrespective of the targeted gene. In conclusion, the combination of fertilization with gene-deficient spermatozoa and gene editing of the same targeted gene region using EP had no beneficial effects on embryo genetic modification, indicating that EP alone is a sufficient tool for genome modification.
Asunto(s)
Edición Génica , Cigoto , Masculino , Animales , Porcinos , Edición Génica/veterinaria , Sistemas CRISPR-Cas , Semen , Electroporación/veterinaria , ARN Guía de Sistemas CRISPR-CasRESUMEN
It is important to prevent microbial contamination during liquid preservation of semen in pigs. We examined the effects of curcumin supplementation on the quality of porcine spermatozoa irradiated with ultraviolet-C (UV-C) at 228 nm. UV-C is used to disinfect gases and solid surfaces. In the first experiment, porcine semen was preserved with 0, 10, 25, 50 or 100 µM curcumin under UV-C irradiation at 228 nm for 7 days at 15°C. The irradiation did not affect the motility and viability of preserved spermatozoa but decreased the percentage of plasma membrane integrity of spermatozoa. Curcumin supplementation at 25 µM significantly improved the plasma membrane and acrosome integrity of irradiated spermatozoa compared with spermatozoa preserved without curcumin (p < .05). In the second experiment, semen was preserved with or without 25 µM curcumin with UV-C irradiation at 228 or 260 nm for 3 days at 15°C. Curcumin supplementation increased the percentages of total motility, sperm viability and plasma membrane integrity of preserved spermatozoa at both irradiation wavelengths (p < .05). All quality parameters of 260 nm irradiated spermatozoa decreased compared to those of the other groups, irrespective of curcumin supplementation. The collective findings indicate that porcine spermatozoa can retain their viability even after continuous long-duration irradiation with 228 nm UV-C. Curcumin supplementation improves the quality of UV-C irradiated spermatozoa during semen preservation.
Asunto(s)
Curcumina , Preservación de Semen , Porcinos , Masculino , Animales , Semen , Curcumina/farmacología , Espermatozoides , Acrosoma , Análisis de Semen/veterinaria , Preservación de Semen/veterinaria , Suplementos Dietéticos , Motilidad EspermáticaRESUMEN
BACKGROUND: Pigs are excellent large animal models with several similarities to humans. They provide valuable insights into biomedical research that are otherwise difficult to obtain from rodent models. However, even if miniature pig strains are used, their large stature compared with other experimental animals requires a specific maintenance facility which greatly limits their usage as animal models. Deficiency of growth hormone receptor (GHR) function causes small stature phenotypes. The establishment of miniature pig strains via GHR modification will enhance their usage as animal models. Microminipig is an incredibly small miniature pig strain developed in Japan. In this study, we generated a GHR mutant pig using electroporation-mediated introduction of the CRISPR/Cas9 system into porcine zygotes derived from domestic porcine oocytes and microminipig spermatozoa. METHODS AND RESULTS: First, we optimized the efficiency of five guide RNAs (gRNAs) designed to target GHR in zygotes. Embryos that had been electroporated with the optimized gRNAs and Cas9 were then transferred into recipient gilts. After embryo transfer, 10 piglets were delivered, and one carried a biallelic mutation in the GHR target region. The GHR biallelic mutant showed a remarkable growth-retardation phenotype. Furthermore, we obtained F1 pigs derived from the mating of GHR biallelic mutant with wild-type microminipig, and GHR biallelic mutant F2 pigs through sib-mating of F1 pigs. CONCLUSIONS: We have successfully demonstrated the generation of biallelic GHR-mutant small-stature pigs. Backcrossing of GHR-deficient pig with microminipig will establish the smallest pig strain which can contribute significantly to the field of biomedical research.
Asunto(s)
Sistemas CRISPR-Cas , Cigoto , Masculino , Humanos , Porcinos/genética , Animales , Femenino , Sistemas CRISPR-Cas/genética , Receptores de Somatotropina/genética , Porcinos Enanos , OocitosRESUMEN
It is important to prevent contamination inside the incubator as a method of preventing microbial infections during the embryo culture. In the present study, we examined the effects of ultraviolet-C (UV-C) irradiation, used for microorganism inactivation, on embryo development and the growth of bacteria, including Escherichia coli and Staphylococcus aureus, and the fungus Cladosporium cladosporioides. In the embryo irradiation experiment, we examined the effects of the plastic lid of the culture dish, irradiation distances (10, 20, and 25 cm), and different irradiation wavelengths (228 and 260 nm) during embryo culture for 7 days on the development and quality of porcine in vitro-fertilized embryos. None of the embryos cultured in dishes without plastic lids developed into blastocysts after irradiation with 228 nm UV-C. When porcine embryos were cultured in a culture dish with lids, the 228 nm UV-C irradiation decreased blastocyst formation rates of the embryos but not their quality, irrespective of the UV-C irradiation distance. Moreover, irradiation with 260 nm UV-C, even with plastic lids, had more detrimental effects on embryo development than irradiation with 228 nm UV-C. Investigation of the inactivating effects of UV-C irradiation at 228 nm and 260 nm on the growth of the bacteria and fungus showed that 260 nm UV-C reduced the viability to a greater extent than 228 nm UV-C. Moreover, the disinfection efficacy for the bacteria increased when the irradiation duration increased and the distance decreased. In conclusion, porcine embryos can develop into blastocysts without loss of quality even after continuous long-duration irradiation (7 days) with 228 nm UV-C, which can inactivate the growth of bacteria and the tested fungus; however, the development rate of the embryo is reduced.
Asunto(s)
Blastocisto , Desarrollo Embrionario , Animales , Porcinos , Desarrollo Embrionario/fisiología , Blastocisto/fisiología , Embrión de Mamíferos , Fertilización In Vitro/veterinaria , Escherichia coli , Bacterias , Rayos UltravioletaRESUMEN
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) technology is growing rapidly and has been greatly influencing the efficiency and effectiveness of genetic modifications in different applications. One aspect of research gaining importance in the development of the CRISPR/Cas9 system is the introduction of CRISPR materials into target organisms. Although we previously demonstrated the efficacy of electroporation- and lipofection-mediated CRISPR/Cas9 gene disruption in porcine zygotes, we still believe that the efficiency of this system could be improved by combining these two methods. The present study was thus conducted to clarify the effects of a combination of electroporation and lipofection for delivering CRISPR/Cas9 components into zona pellucida (ZP)-intact and -free zygotes. The results revealed that electroporation alone significantly increased the biallelic mutation rates in the resulting blastocysts compared to lipofection alone, irrespective of the presence of ZP. None of ZP-intact zygotes treated by lipofectamine alone had any mutations, suggesting that removal of the ZP is necessary for enabling CRISPR/Cas9-based genome editing via lipofection treatment in the zygotes. Additional lipofectamine treatment after electroporation did not improve the rates of total and biallelic mutations in the resulting blastocysts derived from either ZP-intact or -free zygotes.