scholarly journals Comparison of the effects of introducing the CRISPR/Cas9 system by microinjection and electroporation into porcine embryos at different stages

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Quynh Anh Le ◽  
Fuminori Tanihara ◽  
Manita Wittayarat ◽  
Zhao Namula ◽  
Yoko Sato ◽  
...  
Keyword(s):  
Mutation Rate ◽  
Gene Editing ◽  
Genetically Modified ◽  
Embryonic Stage ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Guide Rnas ◽  
Cas9 Protein ◽  
Mutation Efficiency ◽  
Allelic Mutation

Abstract Objective Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are used for generating genetically modified pigs. However, these methods create mosaic mutations in embryos. In this study, we evaluated whether the gene editing method and embryonic stage for gene editing affect the gene editing efficiency of porcine embryos. Results First, we designed five guide RNAs (gRNAs) targeting the B4GALNT2 gene and evaluated mutation efficiency by introducing each gRNA with Cas9 protein into zygotes by electroporation. Next, the optimized gRNA with Cas9 protein was introduced into 1-cell and 2-cell stage embryos by either microinjection or electroporation. The sequence of gRNA affected the bi-allelic mutation rate and mutation efficiency of blastocysts derived from electroporated embryos. Microinjection significantly decreased the cleavage rates in each embryonic stage and blastocyst formation rates in 2-cell stage embryos compared with electroporation (p < 0.05). However, the bi-allelic mutation rate and mutation efficiency of blastocysts from the 1-cell stage embryos edited using microinjection were significantly higher (p < 0.05) than those of blastocysts from the 2-cell stage embryos edited by both methods. These results indicate that the gene editing method and embryonic stage for gene editing may affect the genotype and mutation efficiency of the resulting embryos.

scholarly journals Evaluation of mutation rates, mosaicism and off target mutations when injecting Cas9 mRNA or protein for genome editing of bovine embryos

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sadie L. Hennig ◽  
Joseph R. Owen ◽  
Jason C. Lin ◽  
Amy E. Young ◽  
Pablo J. Ross ◽  
...  
Keyword(s):  
Genome Editing ◽  
Bovine Genome ◽  
Single Step ◽  
Bovine Embryos ◽  
Guide Rnas ◽  
Livestock Breeding ◽  
Cas9 Protein ◽  
Mutation Efficiency ◽  
Generation Sequencing ◽  
Cas9 Mrna

AbstractThe CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrated that the use of gRNA/Cas9 ribonucleoprotein complex resulted in a high editing efficiency at three different loci in bovine embryos and decreased levels of mosaicism relative to Cas9 mRNA. Additional optimization will be required to further reduce mosaicism to levels that make single-step embryo editing in cattle commercially feasible.

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

2021 ◽  
Vol 22 (5) ◽  
pp. 2249
Author(s):  
Fuminori Tanihara ◽  
Maki Hirata ◽  
Nhien Thi Nguyen ◽  
Osamu Sawamoto ◽  
Takeshi Kikuchi ◽  
...  
Keyword(s):  
Gene Editing ◽  
Multiple Gene ◽  
Knock Out ◽  
Editing Efficiency ◽  
Guide Rnas ◽  
Cas9 Protein ◽  
One Step ◽  
Step Generation ◽  
The One

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.

scholarly journals Evaluation of Mosaicism and Off Target Mutations in CRISPR-Mediated Genome Edited Bovine Embryos

2020 ◽  
Author(s):  
Sadie L. Hennig ◽  
Joseph R. Owen ◽  
Jason C. Lin ◽  
Amy E. Young ◽  
Pablo J. Ross ◽  
...  
Keyword(s):  
Genome Editing ◽  
Bovine Genome ◽  
Single Step ◽  
Bovine Embryos ◽  
Guide Rnas ◽  
Livestock Breeding ◽  
Cas9 Protein ◽  
Mutation Efficiency ◽  
Generation Sequencing ◽  
Cas9 Mrna

ABSTRACTThe CRISPR/Cas9 genome editing tool has the potential to improve the livestock breeding industry by allowing for the introduction of desirable traits. Although an efficient and targeted tool, the CRISPR/Cas9 system can have some drawbacks, including off-target mutations and mosaicism, particularly when used in developing embryos. Here, we introduced genome editing reagents into single-cell bovine embryos to compare the effect of Cas9 mRNA and protein on the mutation efficiency, level of mosaicism, and evaluate potential off-target mutations utilizing next generation sequencing. We designed guide-RNAs targeting three loci (POLLED, H11, and ZFX) in the bovine genome and saw a significantly higher rate of mutation in embryos injected with Cas9 protein (84.2%) vs. Cas9 mRNA (68.5%). In addition, the level of mosaicism was higher in embryos injected with Cas9 mRNA (100%) compared to those injected with Cas9 protein (94.2%), with little to no unintended off-target mutations detected. This study demonstrates that the use of Cas9 protein, rather than Cas9 mRNA, results in a higher editing efficiency in bovine embryos while lowering the level of mosaicism. However, further optimization must be carried out for the CRISPR/Cas9 system to become feasible for single-step embryo editing in a commercial system.

scholarly journals Somatic cell reprogramming-free generation of genetically modified pigs

2016 ◽  
Vol 2 (9) ◽  
pp. e1600803 ◽  
Author(s):  
Fuminori Tanihara ◽  
Tatsuya Takemoto ◽  
Eri Kitagawa ◽  
Shengbin Rao ◽  
Lanh Thi Kim Do ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Gene Editing ◽  
Genetically Modified ◽  
Efficient Production ◽  
Simple Method ◽  
Guide Rna ◽  
Somatic Cell Reprogramming ◽  
Cas9 Protein ◽  
Somatic Cell Nucleus

Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.

scholarly journals Consumer Acceptance of Gene-Edited versus Genetically Modified Foods in Korea

2021 ◽  
Vol 18 (7) ◽  
pp. 3805
Author(s):  
Eunae Son ◽  
Song Soo Lim
Keyword(s):  
Gene Editing ◽  
Consumer Preferences ◽  
Genetically Modified ◽  
Relevant Information ◽  
Consumer Acceptance ◽  
Genetically Modified Foods ◽  
Breeding Methods ◽  
Novel Foods ◽  
Novel Technology ◽  
Potential Market

Food made with gene-editing has received considerable attention in recent years because it is claimed to be a little different from traditional genetically modified breeding methods concerning safety. However, consumer acceptance of these novel foods and their potential market uptake remains to be answered. This study aims to assess differences in the acceptance of gene-edited and genetically modified foods in Korea. The choice-based conjoint analysis is adopted to estimate part-worth functions for the soybean oil attributes with 200 surveyed samples. The estimated part-worth values reveal how much each attribute affects consumers’ decision-making. Estimated results suggest that consumers tend to accept gene-editing more than genetically modified foods. The acceptance of novel technology is shown to correspond closely to the degree of consumers’ scientific knowledge, highlighting the importance of revealing relevant information regarding the technology. Results also show that country of origin is a significant food-specific attitudinal factor in shaping consumer preferences.

scholarly journals Application of CRISPR/Cas9 technology in wild apple (Malus sieverii) for paired sites gene editing

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Yan Zhang ◽  
Ping Zhou ◽  
Tohir A. Bozorov ◽  
Daoyuan Zhang
Keyword(s):  
Abiotic Stress ◽  
Human Activities ◽  
Genetic Improvement ◽  
Gene Editing ◽  
New Technologies ◽  
Tianshan Mountains ◽  
Biotic And Abiotic Stress ◽  
Guide Rna ◽  
Cas9 Protein ◽  
Albino Phenotype

Abstract Background Xinjiang wild apple is an important tree of the Tianshan Mountains, and in recent years, it has undergone destruction by many biotic and abiotic stress and human activities. It is necessary to use new technologies to research its genomic function and molecular improvement. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system has been successfully applied to genetic improvement in many crops, but its editing capability varies depending on the different combinations of the synthetic guide RNA (sgRNA) and Cas9 protein expression devices. Results In this study, we used 2 systems of vectors with paired sgRNAs targeting to MsPDS. As expected, we successfully induced the albino phenotype of calli and buds in both systems. Conclusions We conclude that CRISPR/Cas9 is a powerful system for editing the wild apple genome and expands the range of plants available for gene editing.

Synthesis and phosphorylation of uvomorulin during mouse early development

Development ◽  
1992 ◽  
Vol 115 (1) ◽  
pp. 313-318 ◽  
Author(s):  
M. Sefton ◽  
M.H. Johnson ◽  
L. Clayton
Keyword(s):  
Cell Adhesion ◽  
Early Development ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Blastocyst Stage ◽  
Embryonic Stage ◽  
Cell Stage ◽  
Mouse Oocytes ◽  
Maternal Mrna ◽  
Embryonic Genome

The cell adhesion molecule, uvomorulin, is synthesised in both the 135 × 10(3) M(r) precursor and 120 × 10(3) M(r) mature forms on maternal mRNA templates in unfertilized and newly fertilized mouse oocytes. Synthesis on maternal message ceases during the 2-cell stage to resume later on mRNA encoded presumptively by the embryonic genome. Uvomorulin is detectable by immunoblotting at all stages upto the blastocyst stage, but shows variations in its total amount and processing with embryonic stage. Whilst only trace levels of phosphorylated uvomorulin are detectable in early and late 4-cell embryos, uvomorulin in 8-cell embryos is phosphorylated.

scholarly journals Inhibition of histone deacetylase 1 (HDAC1) and HDAC2 enhances CRISPR/Cas9 genome editing

2019 ◽  
Vol 48 (2) ◽  
pp. 517-532 ◽  
Author(s):  
Bin Liu ◽  
Siwei Chen ◽  
Anouk La Rose ◽  
Deng Chen ◽  
Fangyuan Cao ◽  
...  
Keyword(s):  
Gene Editing ◽  
Gene Knockout ◽  
Rapid Development ◽  
Open Chromatin ◽  
Chromatin Compaction ◽  
Histone Deacetylase 1 ◽  
Cas9 Protein ◽  
Target Dna ◽  
Histone Modifiers ◽  
Low Efficiency

Abstract Despite the rapid development of CRISPR/Cas9-mediated gene editing technology, the gene editing potential of CRISPR/Cas9 is hampered by low efficiency, especially for clinical applications. One of the major challenges is that chromatin compaction inevitably limits the Cas9 protein access to the target DNA. However, chromatin compaction is precisely regulated by histone acetylation and deacetylation. To overcome these challenges, we have comprehensively assessed the impacts of histone modifiers such as HDAC (1–9) inhibitors and HAT (p300/CBP, Tip60 and MOZ) inhibitors, on CRISPR/Cas9 mediated gene editing efficiency. Our findings demonstrate that attenuation of HDAC1, HDAC2 activity, but not other HDACs, enhances CRISPR/Cas9-mediated gene knockout frequencies by NHEJ as well as gene knock-in by HDR. Conversely, inhibition of HDAC3 decreases gene editing frequencies. Furthermore, our study showed that attenuation of HDAC1, HDAC2 activity leads to an open chromatin state, facilitates Cas9 access and binding to the targeted DNA and increases the gene editing frequencies. This approach can be applied to other nucleases, such as ZFN and TALEN.

Genome mutation after introduction of the gene editing by electroporation of Cas9 protein (GEEP) system in matured oocytes and putative zygotes

2019 ◽  
Vol 55 (4) ◽  
pp. 237-242 ◽  
Author(s):  
Maki Hirata ◽  
Fuminori Tanihara ◽  
Manita Wittayarat ◽  
Takayuki Hirano ◽  
Nhien Thi Nguyen ◽  
...  
Keyword(s):  
Gene Editing ◽  
Cas9 Protein

70 XBP1 DYSREGULATION BY CRISPR/Cas9-MEDIATED GENE EDITING DURING PORCINE EMBRYO EARLY DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 142
Author(s):  
K. Gutierrez ◽  
W. G. Glanzner ◽  
N. Dicks ◽  
R. C. Bohrer ◽  
L. G. Currin ◽  
...  
Keyword(s):  
Er Stress ◽  
Gene Editing ◽  
Early Embryo ◽  
Early Embryo Development ◽  
Unfolded Protein ◽  
Guide Rnas ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Er Homeostasis

Early developing embryos are very sensitive to their developmental milieu. For instance, variations in temperature, pH, or culture media composition can trigger endoplasmic reticulum (ER) stress. Endoplasmic reticulum stress has been shown to reduce early embryo development and embryo quality. In response to ER stress, embryos activate coping mechanisms, such as the unfolded protein response, to re-establish ER homeostasis. The X box binding protein (XBP1) is one of the main transducers of the unfolded protein response. Under ER stress, XBP1 mRNA is unconventionally spliced by IRE1α to yield its activated isoform (XBP1s), which allows expression of genes involved in protein folding, transport, and degradation. XBP1s has been detected in oocytes and early stage embryos of different species, including Drosophila, Xenopus, zebrafish, mice, and pigs, suggesting an important role during early embryo development. In this study, we used the CRISPR/Cas9 gene editing technology to investigate the effect of XBP1 dysregulation during development of porcine embryos in vitro. Pig zygotes were produced by intracytoplasmic sperm injection using in vitro-matured oocytes. Treatments consisted of (a) Cas9 mRNA (Cas9) + 1 single guide RNAs targeting XBP1 gene region 1 (sgRNA-1); (b) Cas9 + 1 single guide RNAs targeting XBP1 gene region 2 (sgRNA-2); (c) Cas9 + sgRNA-1 + sgRNA-2; (d) Cas9 alone; and (e) sgRNA-1 + sgRNA-2. After injection, embryos were cultured in vitro for 5 to 7 days to assess development and cell numbers. Experiments were repeated 5 or more times, and data were analysed by ANOVA and means compared using Student’s t-test or Tukey–Kramer Honestly Significant Difference test. Embryo cleavage was similar between the groups (a = 59.8 ± 4.9%, b = 58.8 ± 5.3%, c = 68.86 ± 2.2%, d = 66.4 ± 5.9%, and e = 70.10 ± 1.9%), but development to the blastocyst stage was substantially reduced (P < 0.05) in the groups injected with Cas9 + sgRNAs (a = 18 ± 4.5%, b = 16 ± 1.5%, and c = 5.3 ± 2.8%) compared with controls (d = 33.7 ± 6.2% and e = 31.4 ± 1.2%). Moreover, we observed that only 22.7% of the embryos treated with Cas9 + sgRNA-1 + sgRNA-2 were able to develop beyond 8-cell stage compared with 62.5% in the control group injected with Cas9 alone. These findings suggest that XBP1 activity is required for maintenance of ER homeostasis and development of porcine embryos beyond the main period of embryo genome activation.

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