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Gene Editing and Delivery in Animal Genetic Engineering

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Genetics and Genomics".

Deadline for manuscript submissions: closed (31 August 2022) | Viewed by 22482

Special Issue Editor

Department of Genome Medicine, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan
Interests: development of techniques for manipulation of mammalian genome through gene delivery
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

A gene delivery system is an important tool that facilitates decrypting the function of a gene of interest and creating animal models for diseases. In particular, with the advancements in the field of genome editing technologies, the need for more convenient and easy technologies to create genetically modified animals is escalating.

For this Special Issue focusing on the application of gene delivery technology, we are pleased to invite articles that are focused on the central theme of genetic manipulation (transgenesis, knock out or knock in) of early embryos (or fetuses) and germ cells (oocytes, primordial germ cells, and sperm) in mammals.

Prof. Dr. Sato Masahiro
Guest Editor

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Keywords

  • genome manipulation
  • gene delivery
  • genome editing
  • embryos
  • germ cells
  • genetically modified animals

Published Papers (9 papers)

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Research

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19 pages, 3687 KiB  
Article
Sperm Energy Restriction and Recovery (SER) Alters Epigenetic Marks during the First Cell Cycle of Development in Mice
by Darya A. Tourzani, Qiangzong Yin, Erica A. Jackson, Oliver J. Rando, Pablo E. Visconti and Maria G. Gervasi
Int. J. Mol. Sci. 2023, 24(1), 640; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms24010640 - 30 Dec 2022
Cited by 2 | Viewed by 2138
Abstract
The sperm energy restriction and recovery (SER) treatment developed in our laboratory was shown to improve fertilization and blastocyst development following in vitro fertilization (IVF) in mice. Here, we investigated the effects of SER on early embryogenesis. Developmental events observed during the first [...] Read more.
The sperm energy restriction and recovery (SER) treatment developed in our laboratory was shown to improve fertilization and blastocyst development following in vitro fertilization (IVF) in mice. Here, we investigated the effects of SER on early embryogenesis. Developmental events observed during the first cell cycle indicated that progression through the pronuclear stages of SER-generated embryos is advanced in comparison with control-generated embryos. These findings prompted further analysis of potential effects of SER on pronuclear chromatin dynamics, focusing on the key H3K4me3 and H3K27ac histone modifications. Nearly all the SER-generated embryos displayed H3K4me3 in the male pronuclei at 12 h post-insemination (HPI), while a subset of the control-generated embryos did not. Additionally, SER-generated embryos displayed a more homogenous intensity of H3K27ac at 8 and 12 HPI compared to control embryos. These changes in histone modifications during the first cell cycle were accompanied by differences in gene expression at the two-cell stage; both of these changes in early embryos could potentially play a role in the improved developmental outcomes of these embryos later in development. Our results indicate that sperm incubation conditions have an impact on early embryo development and can be useful for the improvement of assisted reproductive technology outcomes. Full article
(This article belongs to the Special Issue Gene Editing and Delivery in Animal Genetic Engineering)
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11 pages, 4694 KiB  
Article
Generation of Flag/DYKDDDDK Epitope Tag Knock-In Mice Using i-GONAD Enables Detection of Endogenous CaMKIIα and β Proteins
by Kazushi Aoto, Shuji Takabayashi, Hiroki Mutoh and Hirotomo Saitsu
Int. J. Mol. Sci. 2022, 23(19), 11915; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231911915 - 07 Oct 2022
Cited by 3 | Viewed by 2531
Abstract
Specific antibodies are necessary for cellular and tissue expression, biochemical, and functional analyses of protein complexes. However, generating a specific antibody is often time-consuming and effort-intensive. The epitope tagging of an endogenous protein at an appropriate position can overcome this problem. Here, we [...] Read more.
Specific antibodies are necessary for cellular and tissue expression, biochemical, and functional analyses of protein complexes. However, generating a specific antibody is often time-consuming and effort-intensive. The epitope tagging of an endogenous protein at an appropriate position can overcome this problem. Here, we investigated epitope tag position using AlphaFold2 protein structure prediction and developed Flag/DYKDDDDK tag knock-in CaMKIIα and CaMKIIβ mice by combining CRISPR-Cas9 genome editing with electroporation (i-GONAD). With i-GONAD, it is possible to insert a small fragment of up to 200 bp into the genome of the target gene, enabling efficient and convenient tagging of a small epitope. Experiments with commercially available anti-Flag antibodies could readily detect endogenous CaMKIIα and β proteins by Western blotting, immunoprecipitation, and immunohistochemistry. Our data demonstrated that the generation of Flag/DYKDDDDK tag knock-in mice by i-GONAD is a useful and convenient choice, especially if specific antibodies are unavailable. Full article
(This article belongs to the Special Issue Gene Editing and Delivery in Animal Genetic Engineering)
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12 pages, 2196 KiB  
Article
Gender-Difference in Hair Length as Revealed by Crispr-Based Production of Long-Haired Mice with Dysfunctional FGF5 Mutations
by Ryo Takahashi, Gou Takahashi, Yuichi Kameyama, Masahiro Sato, Masato Ohtsuka and Kenta Wada
Int. J. Mol. Sci. 2022, 23(19), 11855; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231911855 - 06 Oct 2022
Cited by 2 | Viewed by 1908
Abstract
Fibroblast growth factor 5 (FGF5) is an important molecule required for the transition from anagen to catagen phase of the mammalian hair cycle. We previously reported that Syrian hamsters harboring a 1-bp deletion in the Fgf5 gene exhibit excessive hair growth in males. [...] Read more.
Fibroblast growth factor 5 (FGF5) is an important molecule required for the transition from anagen to catagen phase of the mammalian hair cycle. We previously reported that Syrian hamsters harboring a 1-bp deletion in the Fgf5 gene exhibit excessive hair growth in males. Herein, we generated Fgf5 mutant mice using genome editing via oviductal nucleic acid delivery (GONAD)/improved GONAD (i-GONAD), an in vivo genome editing system used to target early embryos present in the oviductal lumen, to study gender differences in hair length in mutant mice. The two lines (Fgf5go-malc), one with a 2-bp deletion (c.552_553del) and the other with a 1-bp insertion (c.552_553insA) in exon 3 of Fgf5, were successfully established. Each mutation was predicted to disrupt a part of the FGF domain through frameshift mutation (p.Glu184ValfsX128 or p.Glu184ArgfsX128). Fgf5go-malc1 mice had heterogeneously distributed longer hairs than wild-type mice (C57BL/6J). Notably, this change was more evident in males than in females (p < 0.0001). Immunohistochemical analysis revealed the presence of FGF5 protein in the dermal papilla and outer root sheath of the hair follicles from C57BL/6J and Fgf5go-malc1 mice. Histological analysis revealed that the prolonged anagen phase might be the cause of accelerated hair growth in Fgf5go-malc1 mice. Full article
(This article belongs to the Special Issue Gene Editing and Delivery in Animal Genetic Engineering)
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13 pages, 2754 KiB  
Communication
Successful i-GONAD in Mice at Early Zygote Stage through In Vivo Electroporation Three Min after Intraoviductal Instillation of CRISPR-Ribonucleoprotein
by Shuji Takabayashi, Kenta Iijima, Masumi Tsujimura, Takuya Aoshima, Hisayoshi Takagi, Kazushi Aoto and Masahiro Sato
Int. J. Mol. Sci. 2022, 23(18), 10678; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms231810678 - 14 Sep 2022
Cited by 1 | Viewed by 1431
Abstract
Improved genome editing via oviductal nucleic acids delivery (i-GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are [...] Read more.
Improved genome editing via oviductal nucleic acids delivery (i-GONAD) is a new technology enabling in situ genome editing of mammalian zygotes exiting the oviductal lumen, which is now available in mice, rats, and hamsters. In this method, CRISPR/Cas9 genome-editing reagents are delivered directly to the oviducts of pregnant animals (corresponding to late zygote stage). After intraoviductal instillation, electric shock to the entire oviduct was provided with a specialized electroporation (EP) device to drive the genome editing reagents into the zygotes present in the oviductal lumen. i-GONAD toward early zygotes has been recognized as difficult, because they are tightly surrounded by a cumulus cell layer, which often hampers effective transfer of nucleic acids to zygotes. However, in vivo EP three min after intraoviductal instillation of the genome-editing reagents enabled genome editing of early zygotes with an efficiency of 70%, which was in contrast with the rate of 18% when in vivo EP was performed immediately after intraoviductal instillation at Day 0.5 of pregnancy (corresponding to 13:00–13:30 p.m. on the day when vaginal plug was recognized after natural mating). We also found that addition of hyaluronidase, an enzyme capable of removing cumulus cells from a zygote, slightly enhanced the efficiency of genome editing in early zygotes. These findings suggest that cumulus cells surrounding a zygote can be a barrier for efficient generation of genome-edited mouse embryos and indicate that a three-minute interval before in vivo EP is effective for achieving i-GONAD-mediated genome editing at the early zygote stage. These results are particularly beneficial for researchers who want to perform genome editing experiments targeting early zygotes. Full article
(This article belongs to the Special Issue Gene Editing and Delivery in Animal Genetic Engineering)
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19 pages, 2502 KiB  
Article
A Pan-RNase Inhibitor Enabling CRISPR-mRNA Platforms for Engineering of Primary Human Monocytes
by Kanut Laoharawee, Matthew J. Johnson, Walker S. Lahr, Christopher J. Sipe, Evan Kleinboehl, Joseph J. Peterson, Cara-lin Lonetree, Jason B. Bell, Nicholas J. Slipek, Andrew T. Crane, Beau R. Webber and Branden S. Moriarity
Int. J. Mol. Sci. 2022, 23(17), 9749; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23179749 - 28 Aug 2022
Viewed by 2487
Abstract
Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune [...] Read more.
Monocytes and their downstream effectors are critical components of the innate immune system. Monocytes are equipped with chemokine receptors, allowing them to migrate to various tissues, where they can differentiate into macrophage and dendritic cell subsets and participate in tissue homeostasis, infection, autoimmune disease, and cancer. Enabling genome engineering in monocytes and their effector cells will facilitate a myriad of applications for basic and translational research. Here, we demonstrate that CRISPR-Cas9 RNPs can be used for efficient gene knockout in primary human monocytes. In addition, we demonstrate that intracellular RNases are likely responsible for poor and heterogenous mRNA expression as incorporation of pan-RNase inhibitor allows efficient genome engineering following mRNA-based delivery of Cas9 and base editor enzymes. Moreover, we demonstrate that CRISPR-Cas9 combined with an rAAV vector DNA donor template mediates site-specific insertion and expression of a transgene in primary human monocytes. Finally, we demonstrate that SIRPa knock-out monocyte-derived macrophages have enhanced activity against cancer cells, highlighting the potential for application in cellular immunotherapies. Full article
(This article belongs to the Special Issue Gene Editing and Delivery in Animal Genetic Engineering)
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14 pages, 3303 KiB  
Article
Correction of Beta-Thalassemia IVS-II-654 Mutation in a Mouse Model Using Prime Editing
by Haokun Zhang, Ruilin Sun, Jian Fei, Hongyan Chen and Daru Lu
Int. J. Mol. Sci. 2022, 23(11), 5948; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23115948 - 25 May 2022
Cited by 10 | Viewed by 2540
Abstract
Prime editing was used to insert and correct various pathogenic mutations except for beta-thalassemia variants, which disrupt functional beta-globin and prevent hemoglobin assembly in erythrocytes. This study investigated the effect of gene correction using prime editor version 3 (PE3) in a mouse model [...] Read more.
Prime editing was used to insert and correct various pathogenic mutations except for beta-thalassemia variants, which disrupt functional beta-globin and prevent hemoglobin assembly in erythrocytes. This study investigated the effect of gene correction using prime editor version 3 (PE3) in a mouse model with the human beta-thalassemia IVS-II-654 mutation (C > T). The T conversion generates a 5′ donor site at intron 2 of the beta-globin gene resulting in aberrant splicing of pre-mRNA, which affects beta-globin expression. We microinjected PE3 components (pegRNA, nick sgRNA, and PE2 mRNA) into the zygotes from IVS-II-654 mice to generate mutation-edited mice. Genome sequencing of the IVS-II-654 site showed that PE3 installed the correction (T > C), with an editing efficiency of 14.29%. Reverse transcription-PCR analysis showed that the PE3-induced conversion restored normal splicing of beta-globin mRNA. Subsequent comprehensive phenotypic analysis of thalassemia symptoms, including anemic hematological parameters, anisocytosis, splenomegaly, cardiac hypertrophy, extramedullary hematopoiesis, and iron overload, showed that the corrected IVS-II-654 mice had a normal phenotype identical to the wild type mice. Off-target analysis of pegRNA and nick sgRNA additionally showed the genomic safety of PE3. These results suggest that correction of beta-thalassemia mutation by PE3 may be a straightforward therapeutic strategy for this disease. Full article
(This article belongs to the Special Issue Gene Editing and Delivery in Animal Genetic Engineering)
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15 pages, 3420 KiB  
Communication
Direct Injection of Recombinant AAV-Containing Solution into the Oviductal Lumen of Pregnant Mice Caused In Situ Infection of Both Preimplantation Embryos and Oviductal Epithelium
by Masahiro Sato, Nami Sato-Yamamoto, Ai Wakita, Misako Haraguchi, Manabu Shimonishi and Hiroyuki Okuno
Int. J. Mol. Sci. 2022, 23(9), 4897; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23094897 - 28 Apr 2022
Cited by 2 | Viewed by 2635
Abstract
Adeno-associated virus (AAV) vector is an efficient viral-based gene delivery tool used with many types of cells and tissues, including neuronal cells and muscles. AAV serotype 6 (AAV-6), one of numerous AAV serotypes, was recently found to efficiently transduce mouse preimplantation embryos. Furthermore, [...] Read more.
Adeno-associated virus (AAV) vector is an efficient viral-based gene delivery tool used with many types of cells and tissues, including neuronal cells and muscles. AAV serotype 6 (AAV-6), one of numerous AAV serotypes, was recently found to efficiently transduce mouse preimplantation embryos. Furthermore, through coupling with a clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system—a modern genome editing technology—AAV-6 has been shown to effectively create a mutation at a target locus, which relies on isolation of zygotes, in vitro viral infection, and transplantation of the infected embryos to recipient females. Unfortunately, this procedure, termed “ex vivo handling of embryos”, requires considerable investment of capital, time, and effort. Direct transduction of preimplantation embryos through the introduction of AAV-6 into the oviductal lumen of pregnant females would be an ideal approach. In this study, we injected various types of recombinant AAV vectors (namely, rAAV-CAG-EGFP-1, -2, -5, and -6, each carrying an enhanced green fluorescent protein [EGFP] cDNA whose expression is under the influence of a cytomegalovirus enhancer + chicken β-actin promoter) into the ampulla region of oviducts in pregnant female mice at Day 0.7 of pregnancy (corresponding to the late 1-cell stage), and EGFP-derived green fluorescence was assessed in the respective morulae. The highest levels of fluorescence were observed in rAAV-CAG-EGFP-6. The oviductal epithelium was distinctly fluorescent. The fluorescence in embryos peaked at the morula stage. Our results indicate that intra-oviductal injection of AAV-6 vectors is the most effective method for transducing zona pellucida-enclosed preimplantation embryos in situ. AAV-6 vectors could be a useful tool in the genetic manipulation of early embryos, as well as oviductal epithelial cells. Full article
(This article belongs to the Special Issue Gene Editing and Delivery in Animal Genetic Engineering)
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Review

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14 pages, 1212 KiB  
Review
Enhancing Animal Disease Resistance, Production Efficiency, and Welfare through Precise Genome Editing
by Zhiguo Liu, Tianwen Wu, Guangming Xiang, Hui Wang, Bingyuan Wang, Zheng Feng, Yulian Mu and Kui Li
Int. J. Mol. Sci. 2022, 23(13), 7331; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23137331 - 30 Jun 2022
Cited by 3 | Viewed by 2625
Abstract
The major goal of animal breeding is the genetic enhancement of economic traits. The CRISPR/Cas system, which includes nuclease-mediated and base editor mediated genome editing tools, provides an unprecedented approach to modify the mammalian genome. Thus, farm animal genetic engineering and genetic manipulation [...] Read more.
The major goal of animal breeding is the genetic enhancement of economic traits. The CRISPR/Cas system, which includes nuclease-mediated and base editor mediated genome editing tools, provides an unprecedented approach to modify the mammalian genome. Thus, farm animal genetic engineering and genetic manipulation have been fundamentally revolutionized. Agricultural animals with traits of interest can be obtained in just one generation (and without long time selection). Here, we reviewed the advancements of the CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas (CRISPR associated proteins) genome editing tools and their applications in animal breeding, especially in improving disease resistance, production performance, and animal welfare. Additionally, we covered the regulations on genome-edited animals (GEAs) and ways to accelerate their use. Recommendations for how to produce GEAs were also discussed. Despite the current challenges, we believe that genome editing breeding and GEAs will be available in the near future. Full article
(This article belongs to the Special Issue Gene Editing and Delivery in Animal Genetic Engineering)
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58 pages, 1951 KiB  
Review
Recent Advances in the Production of Genome-Edited Rats
by Masahiro Sato, Shingo Nakamura, Emi Inada and Shuji Takabayashi
Int. J. Mol. Sci. 2022, 23(5), 2548; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23052548 - 25 Feb 2022
Cited by 11 | Viewed by 3306
Abstract
The rat is an important animal model for understanding gene function and developing human disease models. Knocking out a gene function in rats was difficult until recently, when a series of genome editing (GE) technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases [...] Read more.
The rat is an important animal model for understanding gene function and developing human disease models. Knocking out a gene function in rats was difficult until recently, when a series of genome editing (GE) technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the type II bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Cas9 (CRISPR/Cas9) systems were successfully applied for gene modification (as exemplified by gene-specific knockout and knock-in) in the endogenous target genes of various organisms including rats. Owing to its simple application for gene modification and its ease of use, the CRISPR/Cas9 system is now commonly used worldwide. The most important aspect of this process is the selection of the method used to deliver GE components to rat embryos. In earlier stages, the microinjection (MI) of GE components into the cytoplasm and/or nuclei of a zygote was frequently employed. However, this method is associated with the use of an expensive manipulator system, the skills required to operate it, and the egg transfer (ET) of MI-treated embryos to recipient females for further development. In vitro electroporation (EP) of zygotes is next recognized as a simple and rapid method to introduce GE components to produce GE animals. Furthermore, in vitro transduction of rat embryos with adeno-associated viruses is potentially effective for obtaining GE rats. However, these two approaches also require ET. The use of gene-engineered embryonic stem cells or spermatogonial stem cells appears to be of interest to obtain GE rats; however, the procedure itself is difficult and laborious. Genome-editing via oviductal nucleic acids delivery (GONAD) (or improved GONAD (i-GONAD)) is a novel method allowing for the in situ production of GE zygotes existing within the oviductal lumen. This can be performed by the simple intraoviductal injection of GE components and subsequent in vivo EP toward the injected oviducts and does not require ET. In this review, we describe the development of various approaches for producing GE rats together with an assessment of their technical advantages and limitations, and present new GE-related technologies and current achievements using those rats in relation to human diseases. Full article
(This article belongs to the Special Issue Gene Editing and Delivery in Animal Genetic Engineering)
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