RESUMO
Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here, we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro, both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing ß-thalassemic phenotype. In an immunodeficient female mouse model, transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease.
Assuntos
Anemia Falciforme , Edição de Genes , Terapia Genética , Células-Tronco Hematopoéticas , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Anemia Falciforme/terapia , Anemia Falciforme/genética , Edição de Genes/métodos , Animais , Células-Tronco Hematopoéticas/metabolismo , Humanos , Feminino , Camundongos , Terapia Genética/métodos , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Transplante de Células-Tronco Hematopoéticas , Globinas beta/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Reparo do DNA , Mutação , Talassemia beta/terapia , Talassemia beta/genética , Modelos Animais de Doenças , Técnicas de Transferência de GenesRESUMO
ß-Thalassemia is the world's number 1 single-gene genetic disorder and is characterized by suppressed or impaired production of ß-pearl protein chains. This results in intramedullary destruction and premature lysis of red blood cells in peripheral blood. Among them, patients with transfusion-dependent ß-thalassemia face the problem of long-term transfusion and iron chelation therapy, which leads to clinical complications and great economic stress. As gene editing technology improves, we are seeing the dawn of a cure for the disease, with its reduction of ineffective erythropoiesis and effective prolongation of survival in critically ill patients. Here, we provide an overview of ß-thalassemia distribution and pathophysiology. In addition, we focus on gene therapy and gene editing advances. Nucleic acid endonuclease tools currently available for gene editing fall into 3 categories: zinc finger nucleases, transcription activator-like effector nucleases, and regularly interspaced short palindromic repeats (CRISPR-Cas9) nucleases. This paper reviews the exploratory applications and exploration of emerging therapeutic tools based on 3 classes of nucleic acid endonucleases in the treatment of ß-thalassemia diseases.
Assuntos
Edição de Genes , Terapia Genética , Talassemia beta , Talassemia beta/terapia , Talassemia beta/genética , Humanos , Edição de Genes/métodos , Terapia Genética/métodos , Sistemas CRISPR-Cas , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases de Dedos de Zinco/genéticaRESUMO
Programmable nucleases-based genome editing systems offer several advantages, such as high editing efficiency, high product purity, and fewer editing by-products. They have been widely used in biopharmaceutical research and crop engineering. Given the diverse needs for research and application, developing functional base editors has become a major focus in the field of genome editing. Currently, genome editing systems derived from clustered regularly interspaced short palindromic repeats and CRISPR-associated (CRISPR-Cas) and transcription activator-like effector (TALE) systems include single base editors, dual base editors, mitochondrial base editors, and CRISPR-related transposase systems. This review provides a comprehensive overview of the development of base editing systems, summarizes the characteristics, off-target effects, optimization, and improvement strategies of various base editors, and provides insights for further improvement and application of genome editing systems.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Engenharia Genética , HumanosRESUMO
Gene therapy in hematopoietic stem and progenitor cells (HSPCs) shows great potential for the treatment of inborn metabolic diseases. Typical HSPC gene therapy approaches rely on constitutive promoters to express a therapeutic transgene, which is associated with multiple disadvantages. Here, we propose a novel promoterless intronic gene editing approach that triggers transgene expression only after cellular differentiation into the myeloid lineage. We integrated a splicing-competent eGFP cassette into the first intron of CD11b and observed expression of eGFP in the myeloid lineage but minimal to no expression in HSPCs or differentiated non-myeloid lineages. In vivo, edited HSPCs successfully engrafted in immunodeficient mice and displayed transgene expression in the myeloid compartment of multiple tissues. Using the same approach, we expressed alpha-L-iduronidase (IDUA), the defective enzyme in Mucopolysaccharidosis type I, and observed a 10-fold supraendogenous IDUA expression exclusively after myeloid differentiation. Edited cells efficiently populated bone marrow, blood, and spleen of immunodeficient mice, and retained the capacity to secrete IDUA ex vivo. Importantly, cells edited with the eGFP and IDUA transgenes were also found in the brain. This approach may unlock new therapeutic strategies for inborn metabolic and neurological diseases that require the delivery of therapeutics in brain.
Assuntos
Edição de Genes , Células-Tronco Hematopoéticas , Íntrons , Células Mieloides , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Transgenes , Animais , Edição de Genes/métodos , Camundongos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células Mieloides/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Diferenciação Celular/genética , Terapia Genética/métodos , Iduronidase/genética , Iduronidase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Expressão Gênica , Linhagem da Célula/genética , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Transplante de Células-Tronco Hematopoéticas/métodos , Mucopolissacaridose I/terapia , Mucopolissacaridose I/genéticaRESUMO
Precision genome editing is a rapidly evolving field in gene therapy, allowing for the precise modification of genetic material. The CRISPR and Cas systems, particularly the CRISPRCas9 system, have revolutionized genetic research and therapeutic development by enabling precise changes like single-nucleotide substitutions, insertions, and deletions. This technology has the potential to correct disease-causing mutations at their source, allowing for the treatment of various genetic diseases. Programmable nucleases like CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs) can be used to restore normal gene function, paving the way for novel therapeutic interventions. However, challenges, such as off-target effects, unintended modifications, and ethical concerns surrounding germline editing, require careful consideration and mitigation strategies. Researchers are exploring innovative solutions, such as enhanced nucleases, refined delivery methods, and improved bioinformatics tools for predicting and minimizing off-target effects. The prospects of precision genome editing in gene therapy are promising, with continued research and innovation expected to refine existing techniques and uncover new therapeutic applications.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Terapia Genética , Humanos , Terapia Genética/métodos , Terapia Genética/tendências , Edição de Genes/métodos , Edição de Genes/tendências , Nucleases de Dedos de Zinco/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Medicina de Precisão/métodosRESUMO
Cytoplasmic male sterility (CMS), encoded by the mitochondrial open reading frames (ORFs), has long been used to economically produce crop hybrids. However, the utilization of CMS also hinders the exploitation of sterility and fertility variation in the absence of a restorer line, which in turn narrows the genetic background and reduces biodiversity. Here, we used a mitochondrial targeted transcription activator-like effector nuclease (mitoTALENs) to knock out ORF138 from the Ogura CMS broccoli hybrid. The knockout was confirmed by the amplification and re-sequencing read mapping to the mitochondrial genome. As a result, knockout of ORF138 restored the fertility of the CMS hybrid, and simultaneously manifested a cold-sensitive male sterility. ORF138 depletion is stably inherited to the next generation, allowing for direct use in the breeding process. In addition, we proposed a highly reliable and cost-effective toolkit to accelerate the life cycle of fertile lines from CMS-derived broccoli hybrids. By applying the k-mean clustering and interaction network analysis, we identified the central gene networks involved in the fertility restoration and cold-sensitive male sterility. Our study enables mitochondrial genome editing via mitoTALENs in Brassicaceae vegetable crops and provides evidence that the sex production machinery and its temperature-responsive ability are regulated by the mitochondria.
Assuntos
Brassica , Infertilidade Masculina , Masculino , Humanos , Brassica/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Melhoramento Vegetal , Mitocôndrias/genética , Fertilidade/genética , Infertilidade das Plantas/genéticaRESUMO
Plastids (including chloroplasts) and mitochondria are remnants of endosymbiotic bacteria, yet they maintain their own genomes, which encode vital components for photosynthesis and respiration, respectively. Organellar genomes have distinctive features, such as being present as multicopies, being mostly inherited maternally, having characteristic genomic structures and undergoing frequent homologous recombination. To date, it has proven to be challenging to modify these genomes. For example, while CRISPR/Cas9 is a widely used system for editing nuclear genes, it has not yet been successfully applied to organellar genomes. Recently, however, precise gene-editing technologies have been successfully applied to organellar genomes. Protein-based enzymes, especially transcription activator-like effector nucleases (TALENs) and artificial enzymes utilizing DNA-binding domains of TALENs (TALEs), have been successfully used to modify these genomes by harnessing organellar-targeting signals. This short review introduces and discusses the use of targeted nucleases and base editors in organellar genomes, their effects and their potential applications in plant science and breeding.
Assuntos
Edição de Genes , Genoma de Cloroplastos , Genoma Mitocondrial , Genoma de Planta , Edição de Genes/métodos , Genoma de Cloroplastos/genética , Genoma de Planta/genética , Genoma Mitocondrial/genética , Sistemas CRISPR-Cas , Plantas/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Cloroplastos/genéticaRESUMO
Gene knockout is a widely used method in biology for investigating gene function. Several technologies are available for gene knockout, including zinc-finger nuclease technology (ZFN), suicide plasmid vector systems, transcription activator-like effector protein nuclease technology (TALEN), Red homologous recombination technology, CRISPR/Cas, and others. Of these, Red homologous recombination technology, CRISPR/Cas9 technology, and suicide plasmid vector systems have been the most extensively used for knocking out bacterial drug resistance genes. These three technologies have been shown to yield significant results in researching bacterial gene functions in numerous studies. This study provides an overview of current gene knockout methods that are effective for genetic drug resistance testing in bacteria. The study aims to serve as a reference for selecting appropriate techniques.
Assuntos
Traumatismos Craniocerebrais , Farmacorresistência Bacteriana/genética , Técnicas de Inativação de Genes , Genes Bacterianos , Tecnologia , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Nucleases de Dedos de Zinco , AnimaisRESUMO
BACKGROUND: Echinoderms have long been utilized as experimental materials to study the genetic control of developmental processes and their evolution. Among echinoderms, the molecular study of starfish embryos has received considerable attention across research topics such as gene regulatory network evolution and larval regeneration. Recently, experimental techniques to manipulate gene functions have been gradually established in starfish as the feasibility of genome editing methods was reported. However, it is still unclear when these techniques cause genome cleavage during the development of starfish, which is critical to understand the timeframe and applicability of the experiment during early development of starfish. RESULTS: We herein reported that gene functions can be analyzed by the genome editing method TALEN in early embryos, such as the blastula of the starfish Patiria pectinifera. We injected the mRNA of TALEN targeting rar, which was previously constructed, into eggs of P. pectinifera and examined the efficiency of genome cleavage through developmental stages from 6 to 48 hours post fertilization. CONCLUSION: The results will be key knowledge not only when designing TALEN-based experiments but also when assessing the results.
Assuntos
Estrelas-do-Mar , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Animais , Estrelas-do-Mar/genética , Desenvolvimento Embrionário/genética , BlastocistoRESUMO
Adoptive cell therapy based on chimeric antigen receptor (CAR)-engineered T-cells has proven to be lifesaving for many cancer patients. However, its therapeutic efficacy has so far been restricted to only a few malignancies, with solid tumors proving to be especially recalcitrant to efficient therapy. Poor intra-tumor infiltration by T cells and T cell dysfunction due to a desmoplastic, immunosuppressive microenvironment are key barriers for CAR T-cell success against solid tumors. Cancer-associated fibroblasts (CAFs) are critical components of the tumor stroma, evolving specifically within the tumor microenvironment (TME) in response to tumor cell cues. The CAF secretome is a significant contributor to the extracellular matrix and a plethora of cytokines and growth factors that induce immune suppression. Together they form a physical and chemical barrier which induces a T cell-excluding 'cold' TME. CAF depletion in stroma rich solid tumors can thus provide an opportunity to convert immune evasive tumors susceptible to tumor-antigen CAR T-cell cytotoxicity. Using our TALEN-based gene editing platform we engineered non-alloreactive, immune evasive CAR T-cells (termed UCAR T-cells) targeting the unique CAF marker Fibroblast Activation Protein, alpha (FAP). In an orthotopic mouse model of triple-negative breast cancer (TNBC) composed of patient derived-CAFs and tumor cells, we demonstrate the efficacy of our engineered FAP UCAR T-cells in CAF depletion, reduction of desmoplasia and successful tumor infiltration. Furthermore, while previously resistant, pre-treatment with FAP UCAR T-cells now sensitized these tumors to Mesothelin (Meso) UCAR T-cell infiltration and anti-tumor cytotoxicity. Combination therapy of FAP UCAR, Meso UCAR T cells and the checkpoint inhibitor anti-PD-1 significantly reduced tumor burden and prolonged mice survival. Our study thus proposes a novel treatment paradigm for successful CAR T-cell immunotherapy against stroma-rich solid tumors.
Assuntos
Receptores de Antígenos Quiméricos , Animais , Camundongos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Imunoterapia , Linfócitos T , Antígenos de NeoplasiasRESUMO
Gene integration at site-specific loci is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor for human and mouse studies. In this study, we found an AAVS1-like sequence (pAAVS1) in the porcine genome using the Genome Browser and designed TALEN and CRISPR/Cas9 to target the pAAVS1. The efficiency of CRISPR/Cas9 in porcine cells was superior to that of TALEN. We added a loxP-lox2272 sequences to the pAAVS1 targeting donor vector containing GFP for further exchange of various transgenes via recombinase-mediated cassette exchange (RMCE). The donor vector and CRISPR/Cas9 components were transfected into porcine fibroblasts. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in was confirmed by PCR. To induce RMCE, another donor vector containing the loxP-lox2272 and inducible Cre recombinase was cloned. The Cre-donor vector was transfected into the pAAVS1 targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR. In conclusion, gene targeting at the pAAVS1 and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.
Assuntos
Sistemas CRISPR-Cas , Recombinases , Animais , Suínos/genética , Humanos , Camundongos , Recombinases/genética , Recombinases/metabolismo , Sistemas CRISPR-Cas/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Animais Geneticamente Modificados/metabolismo , Marcação de GenesRESUMO
Nowadays, agricultural production is strongly affected by both climate change and pathogen attacks which seriously threaten global food security. For a long time, researchers have been waiting for a tool allowing DNA/RNA manipulation to tailor genes and their expression. Some earlier genetic manipulation methods such as meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) allowed site directed modification but their successful rate was limited due to lack of flexibility when targeting a 'site-specific nucleic acid'. The discovery of clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has revolutionized genome editing domain in different living organisms during the past 9 years. Based on RNA-guided DNA/RNA recognition, CRISPR/Cas9 optimizations have offered an unrecorded scientific opportunity to engineer plants resistant to diverse pathogens. In this report, we describe the main characteristics of the primary reported-genome editing tools ((MNs, ZFNs, TALENs) and evaluate the different CRISPR/Cas9 methods and achievements in developing crop plants resistant to viruses, fungi and bacteria.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Edição de Genes/métodos , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Resistência à Doença/genética , Plantas/genética , Genoma de PlantaRESUMO
Among the major egg allergens, ovomucoid (OVM) is very stable against heat and digestive enzymes, making it difficult to remove physiochemically and inactivate allergens. However, recent genome editing technology has made it possible to generate OVM-knockout chicken eggs. To use this OVM-knockout chicken egg as food, it is important to evaluate its safety as food. Therefore, in this study, we examined the presence or absence of mutant protein expression, vector sequence insertion, and off-target effects in chickens knocked out with OVM by platinum TALENs. The eggs laid by homozygous OVM-knockout hens showed no evident abnormalities, and immunoblotting showed that the albumen contained neither the mature OVM nor the OVM truncated variant. Whole genome sequencing (WGS) revealed that the potential TALEN-induced off-target effects in OVM-knockout chickens were localized in the intergenic and intron regions. The WGS information confirmed that plasmid vectors used for genome editing were only transiently present and did not integrate into the genome of edited chickens. These results indicate the importance of safety evaluation and reveal that the eggs laid by this OVM knockout chicken solve the allergy problem in food and vaccines.
Assuntos
Hipersensibilidade a Ovo , Ovomucina , Animais , Feminino , Galinhas , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Alérgenos/genética , Hipersensibilidade a Ovo/genéticaRESUMO
Achromatopsia is an autosomal recessive disorder, in which cone photoreceptors undergo progressive degeneration, causing color blindness and poor visual acuity, among other significant eye affectations. It belongs to a group of inherited retinal dystrophies that currently have no treatment. Although functional improvements have been reported in several ongoing gene therapy studies, more efforts and research should be carried out to enhance their clinical application. In recent years, genome editing has arisen as one of the most promising tools for personalized medicine. In this study, we aimed to correct a homozygous PDE6C pathogenic variant in hiPSCs derived from a patient affected by achromatopsia through CRISPR/Cas9 and TALENs technologies. Here, we demonstrate high efficiency in gene editing by CRISPR/Cas9 but not with TALENs approximation. Despite a few of the edited clones displaying heterozygous on-target defects, the proportion of corrected clones with a potentially restored wild-type PDE6C protein was more than half of the total clones analyzed. In addition, none of them presented off-target aberrations. These results significantly contribute to advances in single-nucleotide gene editing and the development of future strategies for the treatment of achromatopsia.
Assuntos
Sistemas CRISPR-Cas , Defeitos da Visão Cromática , Edição de Genes , Humanos , Defeitos da Visão Cromática/genética , Defeitos da Visão Cromática/terapia , Edição de Genes/métodos , Mutação , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Células-Tronco Pluripotentes InduzidasRESUMO
Therapeutic genome editing is currently reshaping and transforming the development of advanced therapies as more ex vivo and in vivo gene editing-based technologies are used to treat a broad range of debilitating and complex disorders. With first-generation gene editing modalities (notably those based on ZFNs, TALENs and CRISPR/Cas9), comes a new second-generation of gene editing-based therapeutics including base editing, prime editing and other nuclease-free genome editing modalities. Such ground-breaking innovative products warrant careful considerations from a product development and regulatory perspective, that take into account not only the common development considerations that apply to standard gene and cell therapy products, but also other specific considerations linked with the technology being used. This article sheds light into specific considerations for developing safe and effective in vivo and ex vivo genome editing medicines that will continue to push barriers even further for the cell and gene therapy field.
Assuntos
Edição de Genes , Terapia Genética , Endonucleases/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases de Dedos de Zinco/genética , Sistemas CRISPR-CasRESUMO
Zinc finger nucleases (ZFNs) are programmable nucleases that have contributed significantly to past genome-editing research. They are now utilized much less owing to the advent of transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein system (CRISPR-Cas). These new methods allow for easier generation of reagents that target genomic sequences of interest and are less labor-intensive than ZFNs at targeting desired sequences. However, fundamental ZFN patents have expired, enabling a wide range of their distribution for clinical and industrial applications. This article introduces a ZFN construction protocol that uses bacterial one-hybrid (B1H) selection and single-strand annealing (SSA) assay.
Assuntos
Sistemas CRISPR-Cas , Nucleases de Dedos de Zinco , Nucleases de Dedos de Zinco/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Endonucleases/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genéticaRESUMO
Transcription activator-like effector (TALE) nuclease (TALEN) is the second-generation genome editing tool consisting of TALE protein containing customizable DNA-binding repeats and nuclease domain of FokI enzyme. Each DNA-binding repeat recognizes one base of double-strand DNA, and functional TALEN can be created by a simple modular assembly of these repeats. To easily and efficiently assemble the highly repetitive DNA-binding repeat arrays, various construction systems such as Golden Gate assembly, serial ligation, and ligation-independent cloning have been reported. In this chapter, we summarize the updated situation of these systems and publicly available reagents and protocols, enabling optimal selection of best suited systems for every researcher who wants to utilize TALENs in various research fields.
Assuntos
Edição de Genes , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismoRESUMO
Many genetically engineered rat strains have been produced by the development of genome editing technology, although it used to be technical difficulty and low production efficiency. Knockout and knock-in strains can be simple and quick produced using zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), or clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Presently, genome edited strains have been produced by microinjection and a new electroporation method named technique for animal knockout system by electroporation (TAKE). This chapter presents the latest protocols for producing genome edited rats.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Ratos , Animais , Edição de Genes/métodos , Engenharia Genética/métodos , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismoRESUMO
Pigs have anatomical and physiological characteristics similar to humans; therefore, genetically modified pigs have the potential to become a valuable bioresource in biomedical research. In fact, considering the increasing need for translational research, pigs are useful for studying intractable diseases, organ transplantation, and regenerative medicine as large-scale experimental animals with excellent potential for extrapolation to humans. With the advent of zinc finger nucleases (ZFNs), breakthroughs in genome editing tools such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) have facilitated the efficient generation of genetically modified pigs. Genome editing has been used in pigs for more than 10 years; now, along with knockout pigs, knock-in pigs are also gaining increasing importance. In this chapter, we describe the establishment of gene-modified cells (nuclear donor cells), which are necessary for gene knockout and production of knock-in pigs via somatic cell nuclear transplantation, as well as the production of gene knockout pigs using a simple cytoplasmic injection method.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Humanos , Animais , Suínos/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes , Técnicas de Transferência Nuclear , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genéticaRESUMO
Silkworm is a lepidopteran insect that has been used as a model for a wide variety of biological studies. The microinjection technique is available, and it is possible to cause transgenesis as well as target gene disruption via the genome editing technique. TALEN-mediated knockout is especially effective in this species. We also succeeded in the precise and efficient integration of a donor vector using the precise integration into target chromosome (PITCh) method. Here we describe protocols for ZFN (zinc finger nuclease)-, TALEN (transcription activator-like effector nuclease)-, and CRISPR/Cas9-mediated genome editing as well as the PITCh technique in the silkworm. We consider that all of these techniques can contribute to the further promotion of various biological studies in the silkworm and other insect species.