Towards a better future for DNA barcoding: Evaluating monophyly- and distance-based species identification using COI gene fragments of Dacini fruit flies.

The utility of a universal DNA 'barcode' fragment (658 base pairs of the Cytochrome C Oxidase I [COI] gene) has been established as a useful tool for species identification, and widely criticized as one for understanding the evolutionary history of a group. Large amounts of COI sequence data have been produced that hold promise for rapid species identification, for example, for biosecurity. The fruit fly tribe Dacini holds about a thousand species, of which 80 are pests of economic concern. We generated a COI reference library for 265 species of Dacini containing 5601 sequences that span most of the COI gene using circular consensus sequencing. We compared distance metrics versus monophyly assessments for species identification and although we found a 'soft' barcode gap around 2% pairwise distance, the exceptions to this rule dictate that a monophyly assessment is the only reliable method for species identification. We found that all fragments regularly used for Dacini fruit fly identification >450 base pairs long provide similar resolution. 11.3% of the species in our dataset were non-monophyletic in a COI tree, which is mostly due to species complexes. We conclude with recommendations for the future generation and use of COI libraries. We revise the generic assignment of Dacus transversus stat. rev. Hardy 1982, and Dacus perpusillus stat. rev. Drew 1971 and we establish Dacus maculipterus White 1998 syn. nov. as a junior synonym of Dacus satanas Liang et al. 1993.

Factors Shaping Public Support for More Carbon Capture and Storage Projects in the United States.

This study contributes to the literature examining public acceptance of carbon capture and storage (CCS) projects in the US. The examination of factors that shape public support for CCS projects provides policymakers with insights to address public concerns, balance CCS development with public sentiments, and make informed decisions about optimal locations and timing. Based on a nationally representative survey on 1850 respondents, the study finds that in the US, there is very low familiarity (6.4%) regarding CCS technology and some limited opposition (11.5%) to increased CCS development. Regression results suggest that support for increased CCS projects in the US is influenced by perceptions of technical and social risks (leakage and community danger, respectively) but not cost of living risks, perceptions of environmental and economic benefits, familiarity with the technology, confidence in government regulations, and a desire for the US to lead in CCS. We fail to find the 'Not-in-My-Backyard' effect, and individuals supporting the development of more CCS in their states also support it at a national level. Understanding these factors helps policymakers anticipate challenges in implementing CCS initiatives and allows for the development of strategies to address concerns.

Fatty Acids and Their Lipogenic Enzymes in Anorexia Nervosa Clinical Subtypes.

Disordered eating behavior differs between the restricting subtype (AN-R) and the binging and purging subtype (AN-BP) of anorexia nervosa (AN). Yet, little is known about how these differences impact fatty acid (FA) dysregulation in AN. To address this question, we analyzed 26 FAs and 7 FA lipogenic enzymes (4 desaturases and 3 elongases) in 96 women: 25 AN-R, 25 AN-BP, and 46 healthy control women. Our goal was to assess subtype-specific patterns. Lauric acid was significantly higher in AN-BP than in AN-R at the fasting timepoint (p = 0.038) and displayed significantly different postprandial changes 2 h after eating. AN-R displayed significantly higher levels of n-3 alpha-linolenic acid, stearidonic acid, eicosapentaenoic acid (EPA), docosapentaenoic acid, and n-6 linoleic acid and gamma-linolenic acid compared to controls. AN-BP showed elevated EPA and saturated lauric acid compared to controls. Higher EPA was associated with elevated anxiety in AN-R (p = 0.035) but was linked to lower anxiety in AN-BP (p = 0.043). These findings suggest distinct disordered eating behaviors in AN subtypes contribute to lipid dysregulation and eating disorder comorbidities. A personalized dietary intervention may improve lipid dysregulation and enhance treatment effectiveness for AN.

Use of a Smartphone-Based Medication Adherence Platform to Improve Outcomes in Uncontrolled Type 2 Diabetes Among Veterans: Prospective Case-Crossover Study.

BACKGROUND: Medication nonadherence is a problem that impacts both the patient and the health system. OBJECTIVE: The objective of this study was to evaluate the impact of a novel smartphone app with patient-response-directed clinical intervention on medication adherence and blood glucose control in noninsulin-dependent patients with type 2 diabetes mellitus (T2DM). METHODS: We enrolled 50 participants with T2DM not on insulin with smartphones from a rural health care center in Northern Nevada for participation in this case-crossover study. Participants underwent a standard of care arm and an intervention arm. Each study arm was 3 months long, for a total of 6 months of follow-up. Participants had a hemoglobin A1c (HbA1c) lab draw at enrollment, 3 months, and 6 months. Participants had monthly "medication adherence scores" (MAS) and "Self-Efficacy for Appropriate Medication Use Scale" (SEAMS) questionnaires completed at baseline and monthly for the duration of the study. Our primary outcomes of interest were the changes in HbA1c between study arms. Secondary outcomes included the evaluation of the difference in the proportion of participants achieving a clinically meaningful reduction in HbA1c and the difference in the number of participants requiring diabetes therapy escalation between study arms. Exploratory outcomes included the analysis of the variation in medication possession ratio (MPR), MAS, and SEAMS during each study arm. RESULTS: A total of 30 participants completed both study arms and were included in the analysis. Dropouts were higher in participants enrolled in the standard of care arm first (9/25, 36% vs 4/25, 16%). Participants had a median HbA1c of 9.1%, had been living with T2DM for 6 years, had a median age of 66 years, and had a median of 8.5 medications. HbA1c reduction was 0.69% in the intervention arm versus 0.35% in the standard of care arm (P=.30). A total of 70% (21/30) of participants achieved a clinically meaningful reduction in HbA1c of 0.5% in the app intervention arm versus 40% (12/30) in the standard of care arm (odds ratio 2.29, 95% CI 0.94-5.6; P=.09). Participants had higher odds of a therapy escalation while in the standard of care arm (18/30, 60% vs 5/30, 16.7%, odds ratio 4.3, 95% CI 1.2-15.2; P=.02). The median MPR prior to enrollment was 109%, 112% during the study's intervention arm, and 102% during the standard of care arm. The median real-time MAS was 93.2%. The change in MAS (1 vs -0.1; P=.02) and SEAMS (1.9 vs -0.2; P<.001) from baseline to month 3 was higher in the intervention arm compared to standard of care. CONCLUSIONS: A novel smartphone app with patient-response-directed provider intervention holds promise in the ability to improve blood glucose control in complex non-insulin-dependent T2DM and is worthy of additional study.

Macrophage-Hitchhiked Orally Administered ß-Glucans-Functionalized Nanoparticles as "Precision-Guided Stealth Missiles" for Targeted Pancreatic Cancer Therapy.

The prognosis in cases of pancreatic ductal adenocarcinoma (PDAC) with current treatment modalities is poor owing to the highly desmoplastic tumor microenvironment (TME). Herein, a ß-glucans-functionalized zinc-doxorubicin nanoparticle system (ßGlus-ZnD NPs) that can be orally administered, is developed for targeted PDAC therapy. Following oral administration in PDAC-bearing mice, ßGlus-ZnD NPs actively target/transpass microfold cells, overcome the intestinal epithelial barrier, and then undergo subsequent phagocytosis by endogenous macrophages (ßGlus-ZnD@Mϕ). As hitchhiking cellular vehicles, ßGlus-ZnD@Mϕ transits through the intestinal lymphatic system and enters systemic circulation, ultimately accumulating in the tumor tissue as a result of the tumor-homing and "stealth" properties that are conferred by endogenous Mϕ. Meanwhile, the Mϕ that hitchhikes ßGlus-ZnD NPs is activated to produce matrix metalloproteinases, destroying the desmoplastic stromal barrier, and differentiates toward the M1 -like phenotype, modulating the TME and recruiting effector T cells, ultimately inducing apoptosis of the tumor cells. The combination of ßGlus-ZnD@Mϕ and immune checkpoint blockade effectively inhibits the growth of the primary tumor and suppresses the development of metastasis. It thus represents an appealing approach to targeted PDAC therapy.

Cargo-free particles divert neutrophil-platelet aggregates to reduce thromboinflammation.

The combination of inflammation and thrombosis is a hallmark of many cardiovascular diseases. Under such conditions, platelets are recruited to an area of inflammation by forming platelet-leukocyte aggregates via interaction of PSGL-1 on leukocytes and P-selectin on activated platelets, which can bind to the endothelium. While particulate drug carriers have been utilized to passively redirect leukocytes from areas of inflammation, the downstream impact of these carriers on platelet accumulation in thromboinflammatory conditions has yet to be studied. Here, we explore the ability of polymeric particles to divert platelets away from inflamed blood vessels both in vitro and in vivo. We find that untargeted and targeted micron-sized polymeric particles can successfully reduce platelet adhesion to an inflamed endothelial monolayer in vitro in blood flow systems and in vivo in a lipopolysaccharide-induced, systemic inflammation murine model. Our data represent initial work in developing cargo-free, anti-platelet therapeutics specifically for conditions of thromboinflammation.

Skin penetration of caffeine from commercial eye creams and eye creams designed and optimized based on Hansen solubility parameters.

Computer-aided formulation design can streamline and speed up product development. In this study, ingredient screening and optimizing software, Formulating for Efficacy® (FFE), was used to design and optimize creams for the topical delivery of caffeine. FFE was set up to optimize lipophilic active ingredients, therefore, this study challenged the program's capabilities. The effect of two chemical penetration enhancers, including dimethyl isosorbide (DMI) and ethoxydiglycol (EDG), were studied based on their favorable Hansen Solubility Parameter physicochemical input parameters for the skin delivery of caffeine in the FFE® software application. Four oil-in-water emulsions containing 2% caffeine were formulated, one without a chemical penetration enhancer, one with five percent of DMI, one with five percent of EDG, and one with 2.5% of DMI and EDG each (DMI + EDG). Additionally, three commercial products were used as reference products. The cumulative amount of caffeine released and permeated, and the flux across Strat-M® membranes were determined using Franz diffusion cells. The eye creams had skin-compatible pH, excellent spreadability for the application area, were opaque emulsions with 14-17 µm droplet size, and were stable at 25 °C for 6 months. All four eye creams formulated released over 85% of caffeine in 24 h, outperforming the commercial products. DMI + EDG cream provided the highest permeation in vitro in 24 h, which was significantly higher than the commercial products (p < 0.05). FFE proved to be a valuable and quick tool to aid in the topical delivery of caffeine.

Abnormal Wall Shear Stress Area is Correlated to Coronary Artery Bypass Graft Remodeling 1 Year After Surgery.

Coronary artery bypass graft surgery is a common intervention for coronary artery disease; however, it suffers from graft failure, and the underlying mechanisms are not fully understood. To better understand the relation between graft hemodynamics and surgical outcomes, we performed computational fluid dynamics simulations with deformable vessel walls in 10 study participants (24 bypass grafts) based on CT and 4D flow MRI one month after surgery to quantify lumen diameter, wall shear stress (WSS), and related hemodynamic measures. A second CT acquisition was performed one year after surgery to quantify lumen remodeling. Compared to venous grafts, left internal mammary artery grafts experienced lower abnormal WSS (< 1 Pa) area one month after surgery (13.8 vs. 70.1%, p = 0.001) and less inward lumen remodeling one year after surgery (- 2.4% vs. - 16.1%, p = 0.027). Abnormal WSS area one month post surgery correlated with percent change in graft lumen diameter one year post surgery (p = 0.030). This study shows for the first time prospectively a correlation between abnormal WSS area one month post surgery and graft lumen remodeling 1 year post surgery, suggesting that shear-related mechanisms may play a role in post-operative graft remodeling and might help explain differences in failure rates between arterial and venous grafts.

Improving Agrobacterium tumefaciens-Mediated Genetic Transformation for Gene Function Studies and Mutagenesis in Cucumber (Cucumis sativus L.).

In the post-genomics era, Agrobacterium tumefaciens-mediated genetic transformation is becoming an increasingly indispensable tool for characterization of gene functions and crop improvement in cucumber (Cucumis sativus L.). However, cucumber transformation efficiency is still low. In this study, we evaluated the effects of several key factors affecting the shoot-regeneration rate and overall transformation efficiency in cucumber including genotypes, the age and sources of explants, Agrobacterium strains, infection/co-cultivation conditions, and selective agents. We showed that in general, North China cucumbers exhibited higher shoot-regeneration rate than US pickling or slicing cucumbers. The subapical ground meristematic regions from cotyledons or the hypocotyl had a similar shoot-regeneration efficiency that was also affected by the age of the explants. Transformation with the Agrobacterium strain AGL1 yielded a higher frequency of positive transformants than with GV3101. The antibiotic kanamycin was effective in selection against non-transformants or chimeras. Optimization of various factors was exemplified with the development of transgenic plants overexpressing the LittleLeaf (LL) gene or RNAi of the APRR2 gene in three cucumber lines. The streamlined protocol was also tested in transgenic studies in three additional genes. The overall transformation efficiency defined by the number of verified transgenic plants out of the number of seeds across multiple experiments was 0.2-1.7%. Screening among T1 OE transgenic plants identified novel, inheritable mutants for leaf or fruit color or size/shape, suggesting T-DNA insertion as a potential source of mutagenesis. The Agrobacterium-mediated transformation protocol from this study could be used as the baseline for further improvements in cucumber transformation.

Students' Perceived Well-Being and Online Preference: Evidence from Two Universities in Vietnam during COVID-19.

University education is still being impacted two years after the COVID-19 outbreak. We performed a rapid survey in February 2022 at two public universities in Vietnam to examine the effects of the pandemic on well-being and the factors that may associate with online class preference among university students as well as to investigate the need for support to improve resilience. A web-based survey included 1589 undergraduate students in total. Both quantitative and qualitative data analysis was carried out. Overall, approximately a quarter of respondents said that they perceived an influence on their health, 42.9% expressed stress, and more than 70% reported worrying about the future. In total, 61.9% of the respondents reported having satisfaction with online classes, while over half of them preferred a program of 50% online classes. Students who live in an urban area, are female, have had pre-COVID-19 campus life experience, have decreased income, and/or experience low online satisfaction and over-information may be in need of more support. The results show implications for universities to consider policies addressing well-being and post-pandemic online education. Providing support to university students to improve their resilience against the impact on their studying, campus life, health, and well-being should be prioritized during and post-pandemic.

Soluble Epoxide Hydrolase Is Associated with Postprandial Anxiety Decrease in Healthy Adult Women.

The metabolism of bioactive oxylipins by soluble epoxide hydrolase (sEH) plays an important role in inflammation, and sEH may be a risk modifier in various human diseases and disorders. The relationships that sEH has with the risk factors of these diseases remain elusive. Herein, sEH protein expression and activity in white blood cells were characterized before and after a high-fat meal in healthy women (HW) and women with anorexia nervosa (AN). sEH expression and sEH activity were significantly correlated and increased in both groups two hours after consumption of the study meal. Fasting sEH expression and activity were positively associated with body mass index (BMI) in both groups, while an inverse association with age was found in AN only (p value < 0.05). sEH was not associated with anxiety or depression in either group at the fasting timepoint. While the anxiety score decreased after eating in both groups, a higher fasting sEH was associated with a lower postprandial anxiety decrease in HW (p value < 0.05). sEH characterization using direct measurements verified the relationship between the protein expression and in vivo activity of this important oxylipin modulator, while a well-controlled food challenge study design using HW and a clinical control group of women with disordered eating elucidated sEH's role in the health of adult women.

Wall Shear Stress Differences Between Arterial and Venous Coronary Artery Bypass Grafts One Month After Surgery.

Although coronary artery bypass graft (CABG) surgery is a well-established intervention, graft failure can occur, and the underlying mechanisms remain incompletely understood. The purpose of this prospective study is to utilize computational fluid dynamics (CFD) to investigate how graft hemodynamics one month post surgery may vary among graft types, which have different long-term patency rates. Twenty-four grafts from 10 participants (64.6 ± 8.5 years, 9 men) were scanned with coronary CT angiography and 4D flow MRI one month after CABG surgery. Grafts included 10 left internal mammary arteries (LIMA), 3 radial arteries (RA), and 11 saphenous vein grafts (SVG). Image-guided CFD was used to quantify blood flow rate and wall area exposed to abnormal wall shear stress (WSS). Arterial grafts had a lower abnormal WSS area than venous grafts (17.9% vs. 70.1%; p = 0.001), and a similar trend was observed for LIMA vs. SVG (13.8% vs. 70.1%; p = 0.001). Abnormal WSS area correlated positively to lumen diameter (p < 0.001) and negatively to flow rate (p = 0.001). This CFD study is the first of its kind to prospectively reveal differences in abnormal WSS area 1 month post surgery among CABG types, suggesting that WSS may influence the differential long-term graft failure rates observed among these groups.

COVID-19 in Europe: Dataset at a sub-national level.

The COVID-19 pandemic has hit humanity, straining health care systems, economies, and governments worldwide. In one of the responses to the pandemic, a big global effort has been mounted to collect, analyze, and make data publicly available. However, many of the existing COVID-19 public datasets are (i) aggregated at country level, and (ii) tend not to bring the COVID-19-specific data coupled with socio-demographic, economic, public policy, health, pollution and environmental factors, all of which may be key elements to study the transmission of the SARS-CoV-2 and its severity. To aid the evaluation of the determinants and impact of the COVID-19 pandemic at a large scale, we present here a new dataset with socio-demographic, economic, public policy, health, pollution and environmental factors for the European Union at the small regions level (NUTS3). The database is freely accessible at http://dx.doi.org/10.17632/2ghxnrkr9p.4. This dataset can help to monitor the COVID-19 mortality and infections at the sub-national level and enable analysis that may inform future policymaking.

One-Step Generation of Multiple Gene-Edited Pigs by Electroporation of the CRISPR/Cas9 System into Zygotes to Reduce Xenoantigen Biosynthesis.

Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

Introduction of a point mutation in the KRAS gene of in vitro fertilized porcine zygotes via electroporation of the CRISPR/Cas9 system with single-stranded oligodeoxynucleotides.

This study aimed to investigate the efficiency of KRAS gene editing via CRISPR/Cas9 delivery by electroporation and analyzed the effects of the non-homologous end-joining pathway inhibitor Scr7 and single-stranded oligodeoxynucleotide (ssODN) homology arm length on introducing a point mutation in KRAS. Various concentrations (0-2 µM) of Scr7 were evaluated; all concentrations of Scr7 including 0 µM resulted in the generation of blastocysts with a point mutation and the wild-type sequence or indels. No significant differences in the blastocyst formation rates of electroporated zygotes were observed among ssODN homology arm lengths, irrespective of the gRNA (gRNA1 and gRNA2). The proportion of blastocysts carrying a point mutation with or without the wild-type sequence and indels was significantly higher in the ssODN20 group (i.e., the group with a ssODN homology arm of 20 bp) than in the ssODN60 group (gRNA1: 25.7% vs. 5.4% and gRNA2: 45.5% vs. 5.9%, p < .05). In conclusion, the CRISPR/Cas9 delivery with ssODN via electroporation is feasible for the generation of point mutations in porcine embryos. Further studies are required to improve the efficiency and accuracy of the homology-directed repair.

Generation of CD163-edited pig via electroporation of the CRISPR/Cas9 system into porcine in vitro-fertilized zygotes.

CD163 is a putative fusion receptor for virus of porcine reproductive and respiratory syndrome (PRRS). In this study, we introduced a CRISPR/Cas9 system [guide RNAs (gRNAs) with Cas9 protein] targeting the CD163 gene into in vitro-fertilized porcine zygotes by electroporation to generate CD163-modified pigs. First, we designed four types of gRNAs that targeted distinct sites in exon 7 of the CD163 gene. Cas9 protein with different gRNAs was introduced into in vitro-fertilized zygotes by electroporation. When the electroporated zygotes were allowed to develop to blastocysts in vitro and the genome editing efficiency was evaluated using these blastocysts, three (gRNA1, 2, and 4) of the four gRNAs tested successfully edited the CD163 gene. To generate CD163-knockout pigs, a total of 200 electroporated zygotes using these three gRNAs were transferred into the oviducts of oestrous-synchronized surrogate and the surrogate gave birth to eight piglets. Subsequent sequence analysis revealed that one of the piglets carried no wild-type sequence in CD163 gene. The other seven piglets carried only wild-type sequence. Thus, we successfully generated a CD163-edited pig by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes, although further improvement is required to generate genetically modified pigs with high efficiency.

Conductive Materials for Healing Wounds: Their Incorporation in Electroactive Wound Dressings, Characterization, and Perspectives.

The use of conductive materials to promote the activity of electrically responsive cells is an effective means of accelerating wound healing. This article focuses on recent advancements in conductive materials, with emphasis on overviewing their incorporation with non-conducting polymers to fabricate electroactive wound dressings. The characteristics of these electroactive dressings are deliberated, and the mechanisms on how they accelerate the wound healing process are discussed. Potential directions for the future development of electroactive wound dressings and their potential in monitoring the course of wound healing in vivo concomitantly are also proposed.

Curcumin supplementation in the maturation medium improves the maturation, fertilisation and developmental competence of porcine oocytes.

This study was conducted to determine the effects of supplementing the maturation medium with the antioxidant curcumin on the in vitro maturation (IVM), fertilisation and development of porcine oocytes. Curcumin supplementation was performed at concentrations of 0, 5, 10, 20, and 40 µM. At concentrations of 5-20 µM, curcumin had significant positive effects (P < 0.05) on maturation and fertilisation rates compared to the non-treated group. Of the groups cultured with 5-20 µM curcumin, the number of oocytes with DNA-fragmented nuclei after IVM was significantly lower than in groups matured without curcumin. Moreover, curcumin supplementation at 10 µM also gave a significantly higher rate of blastocyst formation compared with oocytes matured without curcumin. Increasing the curcumin concentration to 40 µM yielded negative effects on fertilisation and embryonic development compared with the groups treated with lower concentrations of curcumin. Supplementation with 10 µM curcumin had beneficial effects on the oocyte maturation rate and DNA fragmentation index compared to the non-treated group both in the presence and absence of hydrogen peroxide. These results indicate that curcumin supplementation at a suitable concentration (10 µM) is potentially useful for porcine oocyte culture systems, in terms of protecting oocytes from various forms of oxidative stress.

Efficient generation of GGTA1-deficient pigs by electroporation of the CRISPR/Cas9 system into in vitro-fertilized zygotes.

BACKGROUND: Xenoantigens are a major source of concern with regard to the success of interspecific xenografts. GGTA1 encodes α1,3-galactosyltransferase, which is essential for the biosynthesis of galactosyl-alpha 1,3-galactose, the major xenoantigen causing hyperacute rejection. GGTA1-modified pigs, therefore, are promising donors for pig-to-human xenotransplantation. In this study, we developed a method for the introduction of the CRISPR/Cas9 system into in vitro-fertilized porcine zygotes via electroporation to generate GGTA1-modified pigs. RESULTS: We designed five guide RNAs (gRNAs) targeting distinct sites in GGTA1. After the introduction of the Cas9 protein with each gRNA via electroporation, the gene editing efficiency in blastocysts developed from zygotes was evaluated. The gRNA with the highest gene editing efficiency was used to generate GGTA1-edited pigs. Six piglets were delivered from two recipient gilts after the transfer of electroporated zygotes with the Cas9/gRNA complex. Deep sequencing analysis revealed that five out of six piglets carried a biallelic mutation in the targeted region of GGTA1, with no off-target events. Furthermore, staining with isolectin B4 confirmed deficient GGTA1 function in GGTA1 biallelic mutant piglets. CONCLUSIONS: We established GGTA1-modified pigs with high efficiency by introducing a CRISPR/Cas9 system into zygotes via electroporation. Multiple gene modifications, including knock-ins of human genes, in porcine zygotes via electroporation may further improve the application of the technique in pig-to-human xenotransplantation.