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Gene Editing in Mouse Zygotes Using the CRISPR/Cas9 System.
Engineering of the mouse germline is a key technology in biomedical research for studying the function of genes in health and disease. Since the first knockout mouse was described in 1989, gene targeting was based on recombination of vector encoded sequences in mouse embryonic stem cell lines and their introduction into preimplantation embryos to obtain germline chimeric mice. This approach has been replaced in 2013 by the application of the RNA-guided CRISPR/Cas9 nuclease system, which is introduced into zygotes and directly creates targeted modifications in the mouse genome. Upon the introduction of Cas9 nuclease and guide RNAs into one-cell embryos, sequence-specific double-strand breaks are created that are highly recombinogenic and processed by DNA repair enzymes. Gene editing commonly refers to the diversity of DSB repair products that include imprecise deletions or precise sequence modifications copied from repair template molecules. Since gene editing can now be easily applied directly in mouse zygotes, it has rapidly become the standard procedure for generating genetically engineered mice. This article covers the design of guide RNAs, knockout and knockin alleles, options for donor delivery, preparation of reagents, microinjection or electroporation of zygotes, and the genotyping of pups derived from gene editing projects.
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Gene Therapy and Gene Editing: Current Trends and Future Prospects of Molecular Medicine.
Gene therapy and genome editing have emerged as transformative approaches in the management of a diverse range of genetic and acquired diseases. This evaluation offers a thorough examination of the present state and prospects of these innovative technologies. Gene therapy is a prospective approach to the treatment and prevention of a variety of conditions, including complex cancers and inherited genetic disorders, which entail the introduction, removal, or modification of genetic material within a patient's cells. Genome editing, particularly through techniques such as CRISPR-Cas9, enables targeted corrections of genetic defects and opens new possibilities for personalized medicine by allowing for precise modifications at the DNA level. The review addresses the ethical implications, clinical applications, and significant advancements of these technologies. This article endeavors to underscore the substantial influence of gene therapy and genome editing on contemporary medicine by assessing the most recent research and clinical trials, thereby emphasizing their potential to revolutionize disease treatment and management.
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Precision Genome Editing with CRISPR-Cas9.
The CRISPR/Cas9 system is a revolutionary technology for genome editing that allows for precise and efficient modifications of DNA sequences. The system is composed of two main components, the Cas9 enzyme and a guide RNA (gRNA). The gRNA is designed to specifically target a desired DNA sequence, while the Cas9 enzyme acts as molecular scissors to cut the DNA at that specific location. The cell then repairs the digested DNA, either through nonhomologous end joining (NHEJ) or homology-directed repair (HDR), resulting in either indels or precise modifications of DNA sequences with broad implications in biotechnology, agriculture, and medicine. This chapter provides a practical approach for utilizing CRISPR/Cas9 in precise genome editing, including identifying the target gene sequence, designing gRNA and protein (Cas9), and delivering the CRISPR components to target cells.
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CRISPR/Cas9 therapeutics for liver diseases.
The development of innovative genome editing techniques in recent years has revolutionized the field of biomedicine. Among the novel approaches, the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (CRISPR/Cas9) technology has become the most popular, in part due to its matchless ability to carry out gene editing at the target site with great precision. With considerable successes in animal and preclinical studies, CRISPR/Cas9-mediated gene editing has paved the way for its use in human trials, including patients with a variety of liver diseases. Gene editing is a logical therapeutic approach for liver diseases because many metabolic and acquired disorders are caused by mutations within a single gene. In this review, we provide an overview on current and emerging therapeutic strategies for the treatment of liver diseases using the CRISPR/Cas9 technology.
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Combining multiplex gene editing and doubled haploid technology in maize.
A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.
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Human embryo gene editing: God's scalpel or Pandora's box?
Gene editing refers to the site-specific modification of the genome, which mainly focuses on basic research, model organism construction and treatment and prevention of disease. Since the first application of CRISPR/Cas9 on the human embryo genome in 2015, the controversy over embryo gene editing (abbreviated as EGE in the following text) has never stopped. At present, the main contradictions focus on (1) ideal application prospects and immature technologies; (2) scientific progress and ethical supervision; and (3) definition of reasonable application scope. In fact, whether the EGE is 'God's scalpel' or 'Pandora's box' depends on the maturity of the technology and ethical supervision. This non-systematic review included English articles in NCBI, technical documents from the Human Fertilization and Embryology Authority as well as reports in the media, which performed from 1980 to 2018 with the following search terms: 'gene editing, human embryo, sequence-specific nuclease (SSN) (CRISPR/Cas, TALENT, ZFN), ethical consideration, gene therapy.' Based on the research status of EGE, this paper summarizes the technical defects and ethical controversies, enumerates the optimization measures and looks forward to the application prospect, aimed at providing some suggestions for the development trend. We should regard the research and development of EGE optimistically, improve and innovate the technology boldly and apply its clinical practice carefully.
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[Methods of Evaluating the Efficiency of CRISPR/Cas Genome Editing].
The CRISPR/Cas system is currently widely used for genome editing. The procedure of genome editing includes two necessary steps: (i) searching for the most effective guide RNA, and (ii) analyzing clones for presence of the desired mutation. This review presents the methods used to assess the efficiency of the CRISPR/Cas system and to confirm mutation in the target locus and discusses their advantages and disadvantages. It aims to provide information that could help researchers to choose a technique most appropriate for their specific tasks and available resources.
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CRISPR-Cas9-Mediated Knockout of MLO3 Confers Enhanced Resistance to Reniform Nematode in Upland Cotton.
Upland cotton (Gossypium hirsutum L.) is a major global commodity crop whose production is threatened by the reniform nematode (Rotylenchulus reniformis Linford and Oliveira), a plant-parasitic pest that causes substantial yield losses. Host-plant resistance offers a sustainable management strategy, but currently available resistant cotton cultivars provide only partial protection and often require supplemental control methods. In this study, Clustered Regularly Interspaced Palindromic Repeats (CRISPR)-CRISPR-associated 9 (Cas9) gene editing was used to generate targeted knockouts of Mildew Resistance Locus O (GhiMLO3) in cotton and assess its role in resistance to R. reniformis. Four independent knockout lines (A1, D3, E1, and P3) were developed, confirmed by sequencing, and evaluated for nematode resistance under controlled greenhouse conditions. Nematode reproduction was significantly reduced on lines D3 and E1, with lower egg counts and fewer vermiform life stages compared with the control genotypes, Coker 312 (WT), Delta Pearl, and Jin668. The edited lines also showed characteristic mesophyll cell-death phenotypes, suggesting potential pleiotropic effects associated with MLO-mediated resistance. Sequence analysis confirmed multiple homozygous and heterozygous mutations in MLO3 alleles from both the A and D subgenomes, with D3 and E1 lines displaying the strongest resistance profiles. These findings demonstrate that MLO3 gene editing is a promising approach for improving R. reniformis resistance in cotton.
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SpCas9-mediated gene editing in bovine embryo via single adeno-associated virus infection using a novel micro-sized promoter.
Genome editing in livestock offers practical solutions to address challenges related to land use, climate change, and food production. However, conventional delivery methods such as electroporation and microinjection impose physical stress on embryos, limiting scalability. This study aimed to develop a simplified, non-invasive, and scalable genome editing system for bovine embryos by designing an all-in-one adeno-associated virus (AAV) vector. A novel micro-sized promoter (50 bp), derived from the core regulatory region upstream of the bovine MSTN gene, was constructed to enable expression of Streptococcus pyogenes Cas9 (spCas9) within the AAV packaging limit (∼4.7 kb). This promoter was incorporated into an AAV cassette containing spCas9, a polyadenylation signal, a U6 promoter, and a single-guide RNA (sgRNA) targeting the bovine ALB gene. After confirming editing activity in bovine fibroblasts, the AAV6 vector was added directly to in vitro fertilization (IVF) cultures without physical manipulation. Genome editing was successfully induced, with insertion/deletion (indel) mutations detected in 33.8 ± 23.2 % of the blastocysts. Although blastocyst development was moderately reduced, gene editing was achieved without invasive techniques. These results demonstrate that a micro-promoter-based AAV system can support spCas9-mediated genome editing in bovine embryos through a single-vector infection strategy. The system presents a promising platform for producing gene-edited livestock and may contribute to more efficient and less labor-intensive applications in animal biotechnology.
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First-in-human Phase 1 CRISPR Gene Editing Cancer Trials: Are We Ready?
A prospective first-in-human Phase 1 CRISPR gene editing trial in the United States for patients with melanoma, synovial sarcoma, and multiple myeloma offers hope that gene editing tools may usefully treat human disease. An overarching ethical challenge with first-in-human Phase 1 clinical trials, however, is knowing when it is ethically acceptable to initiate such trials on the basis of safety and efficacy data obtained from pre-clinical studies. If the pre-clinical studies that inform trial design are themselves poorly designed - as a result of which the quality of pre-clinical evidence is deficient - then the ethical requirement of scientific validity for clinical research may not be satisfied. In turn, this could mean that the Phase 1 clinical trial will be unsafe and that trial participants will be exposed to risk for no potential benefit. To assist sponsors, researchers, clinical investigators and reviewers in deciding when it is ethically acceptable to initiate first-in-human Phase 1 CRISPR gene editing clinical trials, structured processes have been developed to assess and minimize translational distance between pre-clinical and clinical research. These processes draw attention to various features of internal validity, construct validity, and external validity. As well, the credibility of supporting evidence is to be critically assessed with particular attention to optimism bias, financial conflicts of interest and publication bias. We critically examine the pre-clinical evidence used to justify the first-inhuman Phase 1 CRISPR gene editing cancer trial in the United States using these tools. We conclude that the proposed trial cannot satisfy the ethical requirement of scientific validity because the supporting pre-clinical evidence used to inform trial design is deficient.
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