• CRISPR/Cas9‐mediated knockin
• Fluorescent reporter rats
• knockin rats
• rat ROSA26 locus
• rat genome editing

• RIKEN Intramural funding

• AAV vectors
• Danon disease
• Lysosomal storage disorders
• Maroteaux-Lamy syndrome
• Morquio A syndrome
• Pompe disease
• Rat model

• CRISPR
• Embryo
• Porcine
• SCNT

• Animals
• Gene Editing/methods
• Swine/genetics
• CRISPR-Cas Systems
• Nuclear Transfer Techniques
• Gene Transfer Techniques
• Male
• Embryo, Mammalian/metabolism

• artificial insemination
• engineered sperm
• genome editing
• genome-edited animals
• in vitro fertilization
• sperm-mediated gene transfer

• Bacteria
• CRISPR-Cas9
• Genome editing
• Metabolic engineering
• Terpenoids

• Embryo implantation
• Estradiol
• Mammalian reproduction
• Progesterone
• Uterine receptivity

• Female
• Animals
• Embryo Implantation/physiology
• Uterus/metabolism
• Uterus/physiology
• Humans
• Pregnancy
• Endometrium/metabolism
• Signal Transduction
• Mice
• Mammals

• CRISPR-Cas
• Diagnosis
• Genome
• Therapeutic
• Vaccine

• Gene Editing/methods
• Humans
• CRISPR-Cas Systems
• Animals
• Vaccines/immunology
• Vaccines/genetics
• Vaccine Development

• CRISPR/Cas9 gene editing
• Drug screening
• Gene mutations
• Induced pluripotent stem cells
• Myeloid malignancy
• Reprogramming

• Humans
• Induced Pluripotent Stem Cells/metabolism
• Myeloproliferative Disorders/genetics
• Myeloproliferative Disorders/therapy
• Myeloproliferative Disorders/metabolism
• Myelodysplastic Syndromes/genetics
• Myelodysplastic Syndromes/therapy
• Myelodysplastic Syndromes/metabolism
• Cell Differentiation
• Leukemia, Myeloid, Acute/genetics
• Leukemia, Myeloid, Acute/therapy
• Leukemia, Myeloid, Acute/metabolism

• Alzheimer’s diseases
• Cell-based therapies
• Disease modeling
• Drug development
• Induced pluripotent stem cells
• Mechanism of diseases
• Regenerative medicine
• iPSCs

• Humans
• Alzheimer Disease/drug therapy
• Alzheimer Disease/genetics
• Induced Pluripotent Stem Cells/physiology
• Drug Evaluation, Preclinical
• Neurons
• Drug Discovery

• Mansoura University

• Cornea
• Disease
• Eye
• Gene editing
• Gene therapy

• Humans
• Cornea/metabolism
• Corneal Diseases/etiology
• Genetic Therapy/methods
• Corneal Transplantation/adverse effects
• Tissue Donors

• R01 EY034319 NEI NIH HHS
• R01 EY030774 NEI NIH HHS
• IK6 BX005646 BLRD VA
• U01 EY031650 NEI NIH HHS
• I01 BX000357 BLRD VA

• bacterial pathogens
• biotechnology
• disease control and pest management
• disease resistance
• host−parasite interactions

• Humans
• Epigenesis, Genetic/genetics
• Epigenome
• Hemoglobinopathies/genetics
• Hemoglobinopathies/therapy
• beta-Globins/genetics
• beta-Globins/metabolism
• Chromatin
• DNA/metabolism

• CRISPR/Cas9
• Electroporation
• Genome editing
• In vitro fertilization
• Rat

• Animals
• Female
• Humans
• Male
• Pregnancy
• Rats
• Embryo Transfer
• Fertilization in Vitro/methods
• Gene Editing/methods
• Horses
• Oocytes
• Semen

• CRISPR-Cas9
• Electroporation
• Embryo transfer
• Embryos
• In vitro fertilization
• Microinjection
• Rat

• NDDs
• brain organoids
• differentiation
• disease modeling
• gene-editing
• genomic stability
• hIPSCs
• neurodevelopment
• protocols
• reprogramming
• stem cells

• CRISPR/Cas9
• Foxn1
• Pronuclear injection
• Rat blastocysts
• Ribonucleoprotein

• CRISPR/Cas9
• Electroporation
• Embryo transfer
• Fluorescent
• In vitro fertilization
• Intact embryos
• Mouse
• Rat

• Rats
• Mice
• Animals
• Gene Editing/methods
• CRISPR-Cas Systems
• Mice, Knockout
• Electroporation/methods
• Electroporation Therapies
• Endonucleases/genetics
• Endonucleases/metabolism
• Craniocerebral Trauma

• Animal cloning
• Animal transgenesis
• Genetic engineering
• Genome editing
• Synthetic biology

• BAC
• CRISPR
• Genetics
• Genome editing
• Knockout
• TALEN
• Transgenic
• YAC
• ZFN

• crispr-cas9 genome editing
• embryonic stem cells
• genetic transduction
• genetic engineering

• CRISPR-Cas9
• disease modeling
• gene therapy
• genome editing

Gene editing (GE) has become one of the mainstream bioengineering technologies over the past two decades, mainly fueled by the rapid development of the CRISPR/Cas system since 2012. To date, plenty of articles related to the progress and applications of GE have been published globally, but the objective, quantitative and comprehensive investigations of them are relatively few. Here, 13,980 research articles and reviews published since 1999 were collected by using GE-related queries in the Web of Science. We used bibliometric analysis to investigate the competitiveness and cooperation of leading countries, influential affiliations, and prolific authors. Text clustering methods were used to assess technical trends and research hotspots dynamically. The global application status and regulatory framework were also summarized. This analysis illustrates the bottleneck of the GE innovation and provides insights into the future trajectory of development and application of the technology in various fields, which will be helpful for the popularization of gene editing technology.

• Bibliometrics
• CRISPR-Cas Systems/genetics
• Gene Editing/methods
• Publications

Immune homeostasis requires fully functional Tregs with a stable phenotype to control autoimmunity. Although IL‐34 is a cytokine first described as mainly involved in monocyte cell survival and differentiation, we recently described its expression by CD8⁺ Tregs in a rat model of transplantation tolerance and by activated FOXP3⁺ CD4⁺ and CD8⁺ Tregs in human healthy individuals. However, its role in autoimmunity and potential in human diseases remains to be determined.

We generated Il34^−/− rats and using both Il34^−/− rats and mice, we investigated their phenotype under inflammatory conditions. Using Il34^−/− rats, we further analyzed the impact of the absence of expression of IL‐34 for CD4⁺ Tregs suppressive function. We investigated the potential of IL‐34 in human disease to prevent xenogeneic GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, taking advantage of a biocollection, we investigated the correlation between presence of IL‐34 in the serum and kidney transplant rejection.

Here we report that the absence of expression of IL‐34 in Il34^−/− rats and mice leads to an unstable immune phenotype, with production of multiple auto‐antibodies, exacerbated under inflammatory conditions with increased susceptibility to DSS‐ and TNBS‐colitis in Il34^−/− animals. Moreover, we revealed the striking inability of Il34^−/− CD4⁺ Tregs to protect Il2rg^−/− rats from a wasting disease induced by transfer of pathogenic cells, in contrast to Il34^+/+ CD4⁺ Tregs. We also showed that IL‐34 treatment delayed EAE in mice as well as GVHD and human skin allograft rejection in immune humanized immunodeficient NSG mice. Finally, we show that presence of IL‐34 in the serum is associated with a longer rejection‐free period in kidney transplanted patients.

Altogether, our data emphasize on the crucial necessity of IL‐34 for immune homeostasis and for CD4⁺ Tregs suppressive function. Our data also shows the therapeutic potential of IL‐34 in human transplantation and auto‐immunity.

–

Absence of expression of IL‐34 in Il34^−/− rats and mice leads to an unstable immune phenotype, with a production of multiple auto‐antibodies and exacerbated immune pathology under inflammatory conditions.

• CRISPR/Cas9
• Foxp3
• IL-34
• Treg
• autoimmunity
• immunotherapy
• knockout
• rat
• tolerance

• Animals
• Glutamate Decarboxylase/metabolism
• Interneurons/metabolism
• Peripheral Nerve Injuries/metabolism
• Rats
• Rats, Transgenic
• Somatosensory Cortex/metabolism

• R01 NS072171 NINDS NIH HHS
• R01 NS098231 NINDS NIH HHS
• R01NS098231 NIH HHS
• R01NS072171 NIH HHS

• CRISPR/Cas9
• Genome editing
• Livestock
• Meganucleases
• TALENs
• ZFNs

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.

• CRISPR/Cas9
• GONAD/i-GONAD
• TALENs
• ZFNs
• adeno-associated virus
• electroporation
• embryonic stem cell
• genome editing
• microinjection
• rats

• Animals
• CRISPR-Cas Systems/genetics
• Female
• Gene Editing/methods
• Genome/genetics
• Humans
• Nucleic Acids
• Rats
• Transcription Activator-Like Effector Nucleases/genetics
• Zinc Finger Nucleases/genetics

• CRISPR/Cas9
• Genome editing
• laboratory animals

• Animals
• Animals, Laboratory
• CRISPR-Associated Protein 9/genetics
• CRISPR-Associated Protein 9/metabolism
• CRISPR-Cas Systems
• Gene Editing/methods
• RNA, Guide, CRISPR-Cas Systems/genetics
• RNA, Guide, CRISPR-Cas Systems/metabolism

• MC_U117562207 Medical Research Council

• CRISPR
• Cloning
• Genomic editing
• Knockout animals
• Nuclear transfer
• Transgenesis

• Animals
• CRISPR-Cas Systems/genetics
• DNA/genetics
• Endonucleases/genetics
• Gene Editing/methods
• Gene Knockout Techniques
• Mammals/genetics
• Mice

Technique for Animal Knockout system by Electroporation (TAKE) is a simple and efficient method to generate genetically modified (GM) mice using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems. To reinforce the versatility of electroporation used for gene editing in mice, the electric condition was optimized for vitrified-warmed mouse embryos, and applied to the fresh embryos from widely used inbred strains (C57BL/6NCr, BALB/cCrSlc, FVB/NJcl, and C3H/HeJJcl). The electric pulse settings (poring pulse: voltage, 150 V; pulse width, 1.0 ms; pulse interval, 50 ms; number of pulses, +4; transfer pulse: voltage, 20 V; pulse width, 50 ms; pulse interval, 50 ms; number of pulses, ±5) were optimal for vitrified-warmed mouse embryos, which could efficiently deliver the gRNA/Cas9 complex into the zygotes without zona pellucida thinning process and edit the target locus. These electric condition efficiently generated GM mice in widely used inbred mouse strains. In addition, electroporation using the electrode with a 5 mm gap could introduce more than 100 embryos within 5 min without specific pretreatment and sophisticated technical skills, such as microinjection, and exhibited a high developmental rate of embryos and genome-editing efficiency in the generated offspring, leading to the rapid and efficient generation of genome editing mice. The electric condition used in this study is highly versatile and can contribute to understanding human diseases and gene functions by generating GM mice more easily and efficiently.

• CRISPR/Cas9
• electroporation
• frozen-thawed embryos
• genome editing

• R01 CA194864 NCI NIH HHS
• R01 CA212608 NCI NIH HHS
• R21 AI158997 NIAID NIH HHS
• National Cancer Institute
• National Institute of Allergy and Infectious Diseases
• American Cancer Society

Leishmania infection causes diverse clinical manifestations in humans. The disease outcome is complicated by the combination of many host and parasite factors. Inbred mouse strains vary in resistance to Leishmania major but are highly susceptible to Leishmania amazonensis infection. However, rats are highly resistant to L. amazonensis infection due to unknown mechanisms. We use the inducible nitric oxide synthase (Nos2) gene knockout rat model (Nos2^−/− rat) to investigate the role of NOS2 against leishmania infection in rats. Our results demonstrated that diversion toward the NOS2 pathway is the key factor explaining the resistance of rats against L. amazonensis infection. Rats deficient in NOS2 are susceptible to L. amazonensis infection even though their immune response to infection is still strong. Moreover, adoptive transfer of NOS2 competent macrophages into Nos2^−/− rats significantly reduced disease development and parasite load. Thus, we conclude that the distinct L-arginine metabolism, observed in rat macrophages, is the basis of the strong innate resistance to Leishmania. These data highlight that macrophages from different hosts possess distinctive properties and produce different outcomes in innate immunity to Leishmania infections.

• Leishmania (L.) amazonensis
• adoptive transfer
• iNOS knock out
• nitric oxide
• rat peritoneal macrophages

• CRISPR
• brain aging
• genome editing technology
• human induced pluripotent stem cells (hiPSCs)
• induced neurons (iNs)
• neuronal senescence

• MOST 108-2321-B-010-013-MY2 Ministry of Science and Technology
• MOST 108-2320-B-010-042- Ministry of Science and Technology
• MOST 110-2321-B-010-007 Ministry of Science and Technology

• CRISPR
• CRISPRa
• CRISPRi
• disease modeling
• gene editing
• induced pluripotent stem cells
• neurological disorders
• precision treatment

Architectural proteins alter the shape of DNA. Some distort the double helix by introducing sharp kinks. This can serve to relieve strain in tightly-bent DNA structures. Here, we design and test artificial architectural proteins based on a sequence-specific Transcription Activator-like Effector (TALE) protein, either alone or fused to a eukaryotic high mobility group B (HMGB) DNA-bending domain. We hypothesized that TALE protein binding would stiffen DNA to bending and twisting, acting as an architectural protein that antagonizes the formation of small DNA loops. In contrast, fusion to an HMGB domain was hypothesized to generate a targeted DNA-bending architectural protein that facilitates DNA looping. We provide evidence from Escherichia coli Lac repressor gene regulatory loops supporting these hypotheses in living bacteria. Both data fitting to a thermodynamic DNA looping model and sophisticated molecular modeling support the interpretation of these results. We find that TALE protein binding inhibits looping by stiffening DNA to bending and twisting, while the Nhp6A domain enhances looping by bending DNA without introducing twisting flexibility. Our work illustrates artificial approaches to sculpt DNA geometry with functional consequences. Similar approaches may be applicable to tune the stability of small DNA loops in eukaryotes.

• CRISPR/Cas9
• SOX2
• embryo
• pig

• Animals
• Blastocyst/cytology
• Blastocyst/metabolism
• CRISPR-Cas Systems/genetics
• Cell Lineage
• Cell Proliferation
• Embryo, Mammalian/cytology
• Embryo, Mammalian/metabolism
• Embryonic Development
• Female
• RNA, Guide, CRISPR-Cas Systems/metabolism
• SOXB1 Transcription Factors/genetics
• SOXB1 Transcription Factors/metabolism
• Swine
• Transcription Factors/genetics
• Transcription Factors/metabolism
• Transcriptome

• 20012411 Ministry of Trade, Industry and Energy
• 2021R1A6A3A13038516 National Research Foundation of Korea
• Ministry of Trade, Industry and Energy
• National Research Foundation of Korea

• Disease model
• Fish
• Genome editing
• Traits

The emergence of an array of genome-editing tools in recent years has facilitated the introduction of genetic modifications directly into the embryo, increasing the ease, efficiency and catalogue of alleles accessible to researchers across a range of species. Bypassing the requirement for a selection cassette and resulting in a broad range of outcomes besides the desired allele, genome editing has altered the allele validation process both temporally and technically. Whereas traditional gene targeting relies upon selection and allows allele validation at the embryonic stem cell modification stage, screening for the presence of the intended allele now occurs in the (frequently mosaic) founder animals. Final confirmation of the edited allele can only take place at the subsequent G1 generation and the validation strategy must differentiate the desired allele from a range of unintended outcomes. Here we present some of the challenges posed by gene editing, strategies for validation and considerations for animal colony management.

• Animal model
• GM
• PCR
• Quality assurance / control

• Animals
• CRISPR-Cas Systems/genetics
• Electroporation
• Endonucleases/genetics
• Endonucleases/metabolism
• Gene Editing
• Gene Knockout Techniques
• Mice
• Mice, Knockout
• Zygote/metabolism
• RNA, Guide, CRISPR-Cas Systems

• 3D cell culture systems
• CRISPR-Cas9
• cardiovascular disease modeling
• cardiovascular tissue engineering
• human induced pluripotent stem cells (hiPSCs)
• regenerative medicine

• CRISPR/Cas9
• Diabetes
• Disease modeling
• Insulin gene therapy
• Programmable nucleases
• Stem cells

Mucosal damage, barrier breach, inflammation, and iron-deficiency anemia (IDA) typify ulcerative colitis (UC) in humans. The anemia in UC appears to mainly relate to systemic inflammation. The pathogenesis of this ‘anemia of inflammation’ (AI) involves cytokine-mediated transactivation of hepatic Hamp (encoding the iron-regulatory hormone, hepcidin). In AI, high hepcidin represses iron absorption (and iron release from stores), thus lowering serum iron, and restricting iron for erythropoiesis (causing anemia). In less-severe disease states, inflammation may be limited to the intestine, but whether this perturbs iron homeostasis is uncertain. We hypothesized that localized gut inflammation will increase overall iron demand (to support the immune response and tissue repair), and that hepatic Hamp expression will decrease in response, thus derepressing (i.e., enhancing) iron absorption. Accordingly, we developed a rat model of mild, acute colitis, and studied iron absorption and homeostasis. Rats exposed (orally) to DSS (4%) for 7 days had intestinal (but not systemic) inflammation, and biomarker analyses demonstrated that iron utilization was elevated. Iron absorption was enhanced (by 2-3-fold) in DSS-treated, WT rats of both sexes, but unexpectedly, hepatic Hamp expression was not suppressed. Therefore, to gain a better understanding of regulation of iron absorption during acute colitis, Hamp KO rats were used for further experimentation. The severity of DSS-colitis was similar in Hamp KOs as in WT controls. In the KOs, increased iron requirements associated with the physiological response to colitis were satisfied by mobilizing hepatic storage iron, rather than by increasing absorption of enteral iron (as occurred in WT rats). In conclusion then, in both sexes and genotypes of rats, iron absorption was appropriately modulated to match physiological demand for dietary iron during acute intestinal inflammation, but regulatory mechanisms may not involve hepcidin.

• Animals
• Colitis/chemically induced
• Colitis/genetics
• Colitis/metabolism
• Colitis/physiopathology
• Dextran Sulfate/adverse effects
• Disease Models, Animal
• Female
• Gene Knockout Techniques
• Hepcidins/genetics
• Hepcidins/metabolism
• Intestinal Absorption
• Iron/metabolism
• Liver/metabolism
• Male
• Rats
• Rats, Sprague-Dawley
• Severity of Illness Index

• R01 DK074867 NIDDK NIH HHS
• R01 DK109717 NIDDK NIH HHS
• National Institute of Diabetes and Digestive and Kidney Diseases
• Office of Dietary Supplements

• chromatin accessibility
• rat cerebral cortex
• regulatory element
• snATAC-seq
• transcription factor

• synthetic biology

• Animals
• Animals, Genetically Modified
• Caenorhabditis elegans/genetics
• Caenorhabditis elegans/microbiology
• Escherichia coli/genetics
• Escherichia coli/physiology
• Gene Expression Regulation
• Gene Regulatory Networks/genetics
• Genetic Engineering/methods
• Green Fluorescent Proteins/genetics
• Green Fluorescent Proteins/metabolism
• Microscopy, Fluorescence
• RNA Interference
• Reverse Transcriptase Polymerase Chain Reaction
• Synthetic Biology/methods

• CRISPR-Cas9
• genetic diseases
• immune genes
• knockin
• knockout
• rat
• transgenesis

• CRISPR-Cas systems
• Diseases
• Drug
• Knock in
• Knock out
• Mutations
• Therapeutic

• CRISPR-Cas
• Ethics
• Gene editing
• History
• TALENs
• Zinc finger

Rabbits are one of the most used experimental animals for investigating the mechanisms of human cardiovascular disease and lipid metabolism because they are phylogenetically closer to human than rodents (mice and rats). Cholesterol-fed wild-type rabbits were first used to study human atherosclerosis more than 100 years ago and are still playing an important role in cardiovascular research. Furthermore, transgenic rabbits generated by pronuclear microinjection provided another means to investigate many gene functions associated with human disease. Because of the lack of both rabbit embryonic stem cells and the genome information, for a long time, it has been a dream for scientists to obtain knockout rabbits generated by homologous recombination-based genomic manipulation as in mice. This obstacle has greatly hampered using genetically modified rabbits to disclose the molecular mechanisms of many human diseases. The advent of genome editing technologies has dramatically extended the applications of experimental animals including rabbits. In this review, we will update genetically modified rabbits, including transgenic, knock-out, and knock-in rabbits during the past decades regarding their use in cardiovascular research and point out the perspectives in future.

• CRISPR-Cas9
• atherosclerosis
• hypercholesterolemia
• knock-out rabbits
• transgenic rabbits

• CRISPR-Cas9
• De novo gene targeting
• Developmental biology
• In utero electroporation
• Visualization of protein subcellular localization

• Animals
• Brain/embryology
• Brain/metabolism
• CRISPR-Associated Protein 9/genetics
• CRISPR-Associated Protein 9/metabolism
• CRISPR-Cas Systems
• Clustered Regularly Interspaced Short Palindromic Repeats
• Electroporation
• Female
• Gene Expression Regulation, Developmental
• Gene Knock-In Techniques
• Gene Targeting
• Gene Transfer Techniques
• Genes, Reporter
• Gestational Age
• Green Fluorescent Proteins/genetics
• Green Fluorescent Proteins/metabolism
• Mice
• Mice, Inbred ICR
• Pregnancy
• RNA, Guide, CRISPR-Cas Systems/genetics
• RNA, Guide, CRISPR-Cas Systems/metabolism