Gene-edited Animals Research Articles

Opportunities for CRISPR-Cas9 application in farm animal genetic improvement.

CRISPR-Cas9 has emerged as a powerful tool in livestock breeding, enabling precise genetic modifications to address genetic diseases, enhance productivity, and develop disease-resistant animal breeds. A thorough analysis of previous research highlights the potential of CRISPR-Cas9 in overcoming genetic disorders by targeting specific mutations in genes. Furthermore, its integration with reproductive biotechnologies and genomic selection facilitates the production of gene-edited animals with high genomic value, contributing to genetic enhancement and improved productivity. Additionally, CRISPR-Cas9 opens new avenues for developing disease-resistant livestock and creating innovative breeding models for high-quality production. A key trend in the field is the development of multi-sgRNA vectors to correct mutations in various genes linked to productivity traits or certain diseases within individual genomes, thereby increasing resistance in animals. However, despite the potential advantages of CRISPR-Cas9, public acceptance of genetically modified agricultural products remains uncertain. Would consumers be willing to purchase such products? It is essential to advocate for bold and innovative research into genetically edited animals, with a focus on safety, careful promotion, and strict regulatory oversight to align with long-term goals and public acceptance. Continued advancements in this technology and its underlying mechanisms promise to improve poultry products and genetically modified livestock. Overall, CRISPR-Cas9 technology offers a promising pathway for advancing livestock breeding practices, with opportunities for genetic improvement, enhanced disease resistance, and greater productivity.

Generation of Anti-Mastitis Gene-Edited Dairy Goats with Enhancing Lysozyme Expression by Inflammatory Regulatory Sequence using ISDra2-TnpB System.

Gene-editing technology has become a transformative tool for the precise manipulation of biological genomes and holds great significance in the field of animal disease-resistant breeding. Mastitis, a prevalent disease in animal husbandry, imposes a substantial economic burden on the global dairy industry. In this study, a regulatory sequence gene editing breeding strategy for the successful creation of a gene-edited dairy (GED) goats with enhanced mastitis resistance using the ISDra2-TnpB system and dairy goats as the model animal is proposed. This included the targeted integration of an innate inflammatory regulatory sequence (IRS) into the promoter region of the lysozyme (LYZ) gene. Upon Escherichia Coli (E. coli) mammary gland infection, GED goats exhibited increased LYZ expression, showing robust anti-mastitis capabilities, mitigating PANoptosis activation, and alleviating blood-milk-barrier (BMB) damage. Notably, LYZ is highly expressed only in E. coli infection. This study marks the advent of anti-mastitis gene-edited animals with exogenous-free gene expression and demonstrates the feasibility of the gene-editing strategy proposed in this study. In addition, it provides a novel gene-editing blueprint for developing disease-resistant strains, focusing on disease specificity and biosafety while providing a research basis for the widespread application of the ISDra2-TnpB system.

Consequences of gene editing of PRLR on thermotolerance, growth, and male reproduction in cattle.

Global warming is a major challenge to the sustainable and humane production of food because of the increased risk of livestock to heat stress. Here, the example of the prolactin receptor (PRLR) gene is used to demonstrate how gene editing can increase the resistance of cattle to heat stress by the introduction of mutations conferring thermotolerance. Several cattle populations in South and Central America possess natural mutations in PRLR that result in affected animals having short hair and being thermotolerant. CRISPR/Cas9 technology was used to introduce variants of PRLR in two thermosensitive breeds of cattle - Angus and Jersey. Gene-edited animals exhibited superior ability to regulate vaginal temperature (heifers) and rectal temperature (bulls) compared to animals that were not gene-edited. Moreover, gene-edited animals exhibited superior growth characteristics and had larger scrotal circumference. There was no evidence for deleterious effects of the mutation on carcass characteristics or male reproductive function. These results indicate the potential for reducing heat stress in relevant environments to enhance cattle productivity.

118 Gene editing: A European perspective

Abstract Gene editing by CRISPR/Cas has revolutionized numerous facets of biotechnology within a remarkably short period of time. Its combined use with either somatic cell nuclear transfer or microinjection into fertilized oocytes is highly useful for mutating genes in a very precise and specific manner. This technology has profoundly transformed the world of animal research, particularly for biomedical and productive applications. Gene editing can now be applied to animal modeling and therapeutic research, bringing us closer to the possibility of using animals as organ donors for xenotransplantation. Furthermore, it has become an effective tool in animal production for purposes such as increasing disease tolerance or resistance and enhancing animal welfare. In this presentation, we will explore how this genome editing tool has influenced the research conducted in Europe, examining the current landscape and addressing the challenges, with a special focus on pigs. We will also discuss the regulation of gene-edited animals in the EU.

119 Bentley Lecture: Gene editing: A Japanese perspective

Abstract Development of useful gene editing technologies, such as ZFN, TALEN and CRISPR/Cas9 systems, has made it possible to efficiently produce knock-out and knock-in mammals, including domestic animals. In particular, the number of scientific papers on gene editing by using the CRISPR/Cas9 system has increased rapidly since 2013, although it has begun to decline from 2022. Among those researchers, the contribution of Japanese scientists accounts for 5 to 10% of the total number of published papers in many species, including laboratory and domestic animals, while those in rats account for nearly 20 % of the total. Whereas research results in rodents, such as mice and rats, have been published from many Japanese institutions, those in domestic animals, such as pigs, appears to be from only limited numbers. With regard to the application of gene editing technology to livestock species, only pigs have different objectives compared with other species, and are used for xenotransplantation and disease models rather than for industrial applications. Whereas this trend is also true for research in Japan, publications on the attempts of new methodologies seem to stand out. A distinctive feature of Japanese research on mammalian gene editing may be the development of methods to introduce gene editors into oocytes with electroporation, for example, GEEP and iGonad, and the applications. These methods seem to be used to generate knock-out animals efficiently, and production of pigs with simultaneous knockout of two or three genes has been reported. To produce knock-in pigs, however, it seems that cloned animals still need to be produced by nuclear transplantation of somatic cells in which the interested gene has been previously modified. Although the attempts to introduce editing tools directly into zygotes by microinjection, which is much simpler than somatic cell nuclear transplantation, have been reported, it has not been controlled yet to reduce the occurrence of mosaics, and it would be necessary to wait for further improvement. In addition, some methods for gene editing chemically or by lipofection have also been attempted, but the efficiencies may still need to be improved. Arrangements for the proper handling of gene-edited animals and food products by Japanese government, especially whether or not such animals and food products are genetically modified organisms, etc. as defined by law, will have a significant impact on the direction and promotion of research on gene editing in Japan.

Interplay between lipid dysregulation and ferroptosis in chondrocytes and the targeted therapy effect of metformin on osteoarthritis

IntroductionOsteoarthritis (OA) is a devastating whole-joint disease affecting a large population worldwide; the role of lipid dysregulation in OA and mechanisms underlying targeted therapy effect of lipid-lowering metformin on OA remains poorly defined. ObjectivesTo investigate the effects of lipid dysregulation on OA progression and to explore lipid dysregulation-targeting OA treatment of metformin. MethodsRNA-Seq data, biochemical, and histochemical assays in human and murine OA cartilage as well as primary chondrocytes were utilized to determine lipid dysregulation. Effects of metformin, a potent lipid-lowering medication, on ACSL4 expression and chondrocyte metabolism were determined. Further molecular experiments, including RT-qPCR, western blotting, flow cytometry, and immunofluorescence staining, were performed to investigate underlying mechanisms. Mice with intra-articular injection of metformin were utilized to determine the effects on ACLT-induced OA progression. ResultsACSL4 and 4-HNE expressions were elevated in human and ACLT-induced mouse OA cartilage and IL-1β-treated chondrocytes (P < 0.05). Ferrostatin-1 largely rescued IL-1β-induced MDA, lipid peroxidation, and ferroptotic mitochondrial morphology (P < 0.05). Metformin decreased the levels of OA-related genes (P < 0.05) and increased the levels of p-AMPK and p-ACC in IL-1β-treated chondrocytes. Intra-articular injection of metformin alleviated ACLT-induced OA lesions in mice, and reverted the percentage of chondrocytes positive for MMP13, Col2a1, ACSL4 and 4-HNE in ACLT mice (P < 0.05). Ferroptotic chondrocytes promoted the recruitment and chemotaxis of RAW264.7 cells via CCL2, which was blocked by metformin in vitro (P < 0.05). ConclusionWe establish a critical role of polyunsaturated fatty acids metabolic process in OA cartilage degradation and define metformin as a potential OA treatment. Metformin reshapes lipid availability and ameliorates chondrocyte ferroptosis sensitivity via the AMPK/ACC pathway. In the future, gene-edited animals and extensive omics technologies will be utilized to reveal detailed lipids’ involvement in cartilage lesions.

An oocyte-specific Cas9-expressing mouse for germline CRISPR/Cas9-mediated genome editing.

Cas9 transgenes can be employed for genome editing in mouse zygotes. However, using transgenic instead of exogenous Cas9 to produce gene-edited animals creates unique issues including ill-defined transgene integration sites, the potential for prolonged Cas9 expression in transgenic embryos, and increased genotyping burden. To overcome these issues, we generated mice harboring an oocyte-specific, Gdf9 promoter driven, Cas9 transgene (Gdf9-Cas9) targeted as a single copy into the Hprt1 locus. The X-linked Hprt1 locus was selected because it is a defined integration site that does not influence transgene expression, and breeding of transgenic males generates obligate transgenic females to serve as embryo donors. Using microinjections and electroporation to introduce sgRNAs into zygotes derived from transgenic dams, we demonstrate that Gdf9-Cas9 mediates genome editing as efficiently as exogenous Cas9 at several loci. We show that genome editing efficiency is independent of transgene inheritance, verifying that maternally derived Cas9 facilitates genome editing. We also show that paternal inheritance of Gdf9-Cas9 does not mediate genome editing, confirming that Gdf9-Cas9 is not expressed in embryos. Finally, we demonstrate that off-target mutagenesis is equally rare when using transgenic or exogenous Cas9. Together, these results show that the Gdf9-Cas9 transgene is a viable alternative to exogenous Cas9.

Molecular breeding of farm animals through gene editing

The rapid development of biotechnology has promoted people's understanding of the biological functions of candidate genes for important economic traits in farm animals. Molecular breeding by gene editing has greatly revolutionized the breeding of farm animals. Through gene editing and embryo manipulation, breeds with designed economic or disease-resistant traits can be readily generated. Along with this fast progress, the safety assessment of gene-edited farm animals has attracted public and regulatory attention. This review summarizes the research progress of gene editing in farm animals, focusing on performance improvement, disease resistance, bioreactors, animal welfare, and environmental friendliness. The limitations and future development of gene editing technology in farm animal breeding are also discussed.

Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for “correcting” variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.

Utilization of Genome Editing for Livestock Resilience in Changing Environment

Climate change poses a significant threat to livestock production systems, including changes in temperature and rainfall patterns, increased frequency of extreme weather events, and the spread of diseases. The use of genome editing technologies presents a potential solution to mitigate the impacts of climate change on livestock. This paper reviewed the prospects of utilizing genome editing in mitigating the impact of climate change in livestock. Applications of genome editing in development of heat-tolerant, and disease-resistant as well as animals with improved feed and water use efficiency and reduced methane emissions are explored. Additionally, a potential breeding program for gene edited animals is proposed. There are several different genome editing techniques that can be used in livestock breeding, including CRISPR/Cas9, TALENs, and zinc-finger nucleases. These techniques involve introducing specific changes to the animal's genome, such as deleting or replacing genes, or introducing new ones. The technology has enormous potential for improving livestock breeding, as it allows for the creation of animals with desirable traits in a much shorter time frame than traditional breeding methods. Generally, it may take years or even decades to breed an animal with a specific trait using traditional breeding methods, whereas genome editing can achieve the same result in just a few generations. Genome editing can be used to mitigate the impact of climate change on livestock production by reducing the methane emissions by improving the efficiency of feed conversion and modifying the genes responsible for methane production. Technology can be utilized to improve livestock feeds by modifying genes involved in plant growth, development, and nutrient use. This lead to the creation of forages that are high yielding, more nutritious and better adapted to diverse production environments. Genome editing allows development of animals that are more resistant to diseases, which can help reduce the need for antibiotics and other treatments. This is particularly important given the growing problem of antibiotic resistance, which is a major concern in both human and animal health. Genome editing has the potential of developing animals that are thermo-tolerant, as well as animals with improved feed and water use efficiency. The proposed breeding program for gene-edited animals will ensure that the animals produced are healthy, genetically diverse, and meet the desired traits. In terms of ethical concerns, policies for genome editing ought to consider the potential for unintended consequences or the creation of animals with characteristics that are viewed as undesirable or unethical. Overall, genome editing technology has the potential to revolutionize livestock production and contribute to the global effort to mitigate the impact of climate change.

What Is a Drug? Enabling the Bioeconomy Through Updated Food and Drug Administration Policy

There are several emerging biotechnology and biomanufacturing innovations designed to improve the sustainability of the livestock and related commercial sectors. These include gene-edited animals, feed additives to reduce methane production, and cultivated meats. In the United States, disparate regulatory policies govern the commercialization of these products. Intentional genomic alterations (IGAs) in animals, and feed additives intended to reduce methane emissions, trigger federal regulatory oversight using the expensive and time-consuming new animal drug-approval pathway. Conversely, animal cells, with or without IGAs, cultured for human food production are evaluated using a “voluntary pre-market consultation” process. The current drug-centric regulatory policies for bioengineered animals and feed additives disincentivize investment, and the United States is starting to lag behind the rest of the world when it comes to the approval, and subsequent adoption of these innovations by farmers and ranchers at a time of increasing concern regarding the environmental footprint of animal agriculture.

GTKO rabbit: A novel animal model for preclinical assessment of decellularized xenogeneic grafts via in situ implantation.

Wild type (WT) animals cannot be used to objectively assess the immunogenicity of animal tissue-derived biomaterials when used as recipients due to difference with human in α-Gal expression. The purpose of this study is to compare the differences of immunological responses between the GGTA1 gene-knockout (GTKO) rabbits and WT rabbits after implantation with animal tissue-derived biomaterials. The porcine-derived decellularized bone matrix (natural bone material, NBM) and fresh porcine cancellous bone (PCB) were implanted in GTKO rabbits and WT rabbits, respectively, and sham operation was used as control (Con). At 2- and 6-week post-implantation, the related immunological items including antibody levels, serum-mediated cell lysis, cytokines, lymphocyte subtypes, and histopathological changes were assessed. GTKO rabbits exhibited more sensitive immune responses than WT rabbits after PCB implantation, resulted from a significant increase of antibodies (except total antibodies) and cytokines levels, cell lysis ratios, CD4/CD8 proportions, and inflammatory cells infiltration. Immunological factors and inflammatory cells infiltrate in GTKO rabbits after NBM implantation were significantly lower than those in the PCB group. Among the three groups, the NBM group showed the highest contents of new bone formation elements. In conclusion, the GTKO rabbit is a more sensitive alternative model than WT rabbit for preclinical study of xenografts via in situ implantation. Studies on multiple gene-edited animals are also necessary for more comprehensively evaluating xenoimmunologen risks of animal tissue-derived biomaterials in the future. Additionally, the immunogenicity of NBM was remarkably decreased compared to PCB.

- Invited Review - Gene-editing techniques and their applications in livestock and beyond.

Genetic modification enables modification of target genes or genome structure in livestock and experimental animals. These technologies have not only advanced bioscience but also improved agricultural productivity. To introduce a foreign transgene, the piggyBac transposon element/transposase system could be used for production of transgenic animals and specific target protein-expressing animal cells. In addition, the clustered regularly interspaced short palindromic repeat-CRISPR associated protein 9 (CRISPR-Cas9) system have been utilized to generate chickens with knockout of G0/G1 switch gene 2 (G0S2) and myostatin, which are related to lipid deposition and muscle growth, respectively. These experimental chickens could be the invaluable genetic resources to investigate the regulatory pathways and mechanisms of improvement of economic traits such as fat quantity and growth. The gene-edited animals could also be applicable to the livestock industry.

Genome-edited zebrafish model of ABCC8 loss-of-function disease

ABSTRACT ATP-sensitive potassium channel (KATP)gain- (GOF) and loss-of-function (LOF) mutations underlie human neonatal diabetes mellitus (NDM) and hyperinsulinism (HI), respectively. While transgenic mice expressing incomplete KATP LOF do reiterate mild hyperinsulinism, KATP knockout animals do not exhibit persistent hyperinsulinism. We have shown that islet excitability and glucose homeostasis are regulated by identical KATP channels in zebrafish. SUR1 truncation mutation (K499X) was introduced into the abcc8 gene to explore the possibility of using zebrafish for modeling human HI. Patch-clamp analysis confirmed the complete absence of channel activity in β-cells from K499X (SUR1−/−) fish. No difference in random blood glucose was detected in heterozygous SUR1+/- fish nor in homozygous SUR1−/− fish, mimicking findings in SUR1 knockout mice. Mutant fish did, however, demonstrate impaired glucose tolerance, similar to partial LOF mouse models. In paralleling features of mammalian diabetes and hyperinsulinism resulting from equivalent LOF mutations, these gene-edited animals provide valid zebrafish models of KATP -dependent pancreatic diseases.

Kidney ECM Pregel Nanoarchitectonics for Microarrays to Accelerate Harvesting Gene-Edited Porcine Primary Monoclonal Spheres

One of the key steps of using CRISPR/Cas9 to obtain gene-edited cells used in generating gene-edited animals combined with somatic cell nuclear transplantation (SCNT) is to harvest monoclonal cells with genetic modifications. However, primary cells used as nuclear donors always grow slowly and fragile after a series of gene-editing operations. The extracellular matrix (ECM) formulated directly from different organs comprises complex proteins and growth factors that can improve and regulate the cellular functions of primary cells. Herein, sodium lauryl ether sulfate (SLES) detergent was first used to perfuse porcine kidney ECM, and the biological properties of the kidney ECM were optimized. Then, we used a porcine kidney ECM pregel to pattern the microarray and developed a novel strategy to shorten the time of obtaining gene-edited monoclonal cell spheroids with low damage in batches. Our results showed that the SLES-perfused porcine kidney ECM pregel displayed superior biological activities in releasing growth factors and promoting cell proliferation. Finally, combined with microarray technology, we quickly obtained monoclonal cells in good condition, and the cells used as nuclear donors to construct recombinant embryos showed a significantly higher success rate than those of the traditional method. We further successfully produced genetically edited pigs.

Moo-re Gene-edited Animals Get Regulatory Clearance: U.S. FDA Approves Genetically Engineered Cattle for Human Consumption

Moo-re Gene-edited Animals Get Regulatory Clearance: U.S. FDA Approves Genetically Engineered Cattle for Human Consumption

Optimized Cas9:sgRNA delivery efficiently generates biallelic MSTN knockout sheep without affecting meat quality.

BackgroundCRISPR/Cas9-based genome-editing systems have been used to efficiently engineer livestock species with precise genetic alterations intended for biomedical and agricultural applications. Previously, we have successfully generated gene-edited sheep and goats via one-cell-stage embryonic microinjection of a Cas9 mRNA and single-guide RNAs (sgRNAs) mixture. However, most gene-edited animals produced using this approach were heterozygotes. Additionally, non-homozygous gene-editing outcomes may not fully generate the desired phenotype in an efficient manner.ResultsWe report the optimization of a Cas9 mRNA-sgRNA delivery system to efficiently generate homozygous myostatin (MSTN) knockout sheep for improved growth and meat production. Firstly, an sgRNA selection software (sgRNAcas9) was used to preliminarily screen for highly efficient sgRNAs. Ten sgRNAs targeting the MSTN gene were selected and validated in vitro using sheep fibroblast cells. Four out of ten sgRNAs (two in exon 1 and two in exon 2) showed a targeting efficiency > 50%. To determine the optimal CRISPR/Cas9 microinjection concentration, four levels of Cas9 mRNA and three levels of sgRNAs in mixtures were injected into sheep embryos. Microinjection of 100 ng/μL Cas9 mRNA and 200 ng/μL sgRNAs resulted in the most improved targeting efficiency. Additionally, using both the highly efficient sgRNAs and the optimal microinjection concentration, MSTN-knockout sheep were generated with approximately 50% targeting efficiency, reaching a homozygous knockout efficiency of 25%. Growth rate and meat quality of MSTN-edited lambs were also investigated. MSTN-knockout lambs exhibited increased body weight and average daily gain. Moreover, pH, drip loss, intramuscular fat, crude protein, and shear force of gluteal muscles of MSTN-knockout lambs did not show changes compared to the wild-type lambs.ConclusionsThis study highlights the importance of in vitro evaluation for the optimization of sgRNAs and microinjection dosage of gene editing reagents. This approach enabled efficient engineering of homozygous knockout sheep. Additionally, this study confirms that MSTN-knockout lambs does not negatively impact meat quality, thus supporting the adoption of gene editing as tool to improve productivity of farm animals.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-022-08594-6.

Analysis of the gut microbiota composition of myostatin mutant cattle prepared using CRISPR/Cas9

Myostatin (MSTN) negatively regulates muscle development and positively regulates metabolism through various pathways. Although MSTN function in cattle has been widely studied, the changes in the gut microbiota due to MSTN mutation, which contribute to host health by regulating its metabolism, remain unclear. Here, high-throughput sequencing of the 16S rRNA gene was conducted to analyze the gut microbiota of wild-type (WT) and MSTN mutant (MT) cattle. A total of 925 operational taxonomic units (OTUs) were obtained, which were classified into 11 phyla and 168 genera. Alpha diversity results showed no significant differences between MT and WT cattle. Beta diversity analyses suggested that the microbial composition of WT and MT cattle was different. Three dominant phyla and 21 dominant genera were identified. The most abundant bacterial genus had a significant relationship with the host metabolism. Moreover, various bacteria beneficial for health were found in the intestines of MT cattle. Analysis of the correlation between dominant gut bacteria and serum metabolic factors affected by MSTN mutation indicated that MSTN mutation affected the metabolism mainly by three metabolism-related bacteria, Ruminococcaceae_UCG-013, Clostridium_sensu_stricto_1, and Ruminococcaceae_UCG-010. This study provides further insight into MSTN mutation regulating the host metabolism by gut microbes and provides evidence for the safety of gene-edited animals.

A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection.

Gene-edited pigs for agricultural and biomedical applications are typically generated using somatic cell nuclear transfer (SCNT). However, SCNT requires the use of monoclonal cells as donors, and the time-consuming and laborious monoclonal selection process limits the production of large populations of gene-edited animals. Here, we developed a rapid and efficient method named RE-DSRNP (reporter RNA enriched dual-sgRNA/CRISPR-Cas9 ribonucleoproteins) for generating gene-edited donor cells. RE-DSRNP takes advantage of the precise and efficient editing features of dual-sgRNA and the high editing efficiency, low off-target effects, transgene-free nature, and low cytotoxic characteristics of reporter RNA enriched RNPs (CRISPR-Cas9 ribonucleoproteins), thus eliminating the need for the selection of monoclonal cells and thereby greatly reducing the generation time of donor cells from 3–4 weeks to 1 week, while also reducing the extent of apoptosis and chromosomal aneuploidy of donor cells. We applied RE-DSRNP to produce cloned pigs bearing a deletion edit of the wild-type p53-induced phosphatase 1 (WIP1) gene: among 32 weaned cloned pigs, 31 (97%) carried WIP1 edits, and 15 (47%) were homozygous for the designed fragment deletion, and no off-target event was detected. The WIP1 knockout (KO) pigs exhibited male reproductive disorders, illustrating the utility of RE-DSRNP for rapidly generating precisely edited animals for functional genomics and disease research. RE-DSRNP’s strong editing performance in a large animal and its marked reduction in the required time for producing SCNT donor cells support its application prospects for rapidly generating populations of transgene-free cloned animals.Supporting InformationThe supporting information is available online at 10.1007/s11427-021-2058-2. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

No apparent p53 activation in CRISPR-engineered gene-edited rabbits.

Clustered regularly interspaced short palindromic repeats‐CRISPR‐associated 9 (CRISPR‐Cas9) and base editors (BEs) are revolutionary gene‐editing technology that has been widely utilized in biology, biotechnology and medicine. However, recent reports show that CRISPR‐Cas9‐mediated genome editing can induce a p53‐mediated stress response and cell cycle arrest in human cells, while not illustrated in gene‐editing animals. In the study, to verify whether there is a phenomenon of p53 activation, by analysing nine gene‐edited rabbits using CRISPR‐Cas9 and BEs, we provide the first evidence that no apparent p53 expression changes in those rabbits generated by Cas9 or BE‐edited, suggesting that p53 may not need to consider for application in gene‐edited animals.