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Generation of genome-edited chicken and duck lines by adenovirus-mediated in vivo genome editing.
Conventional avian genome editing is mediated by isolation, culture, and genome editing of primordial germ cells (PGCs); screening and propagating the genome-edited PGCs; and transplantation of the PGCs into recipient embryos. The PGC-mediated procedures, however, are technically difficult, and therefore, the conventional method has previously been utilized only in chickens. Here, we generated germline mosaic founder chicken and duck lines without the PGC-mediated procedures by injecting an adenovirus containing the CRISPR-Cas9 system into avian blastoderms. Genome-edited chicken and duck offspring produced from the founders carried different insertion or deletion mutations without mutations in the potential off-target sites. Our data demonstrate successful applications of the adenovirus-mediated method for production of genome-edited chicken and duck lines.
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[Establishment and Application of Efficient Gene Editing Method for Classical HLA-I Molecules].
OBJECTIVE: To establish an efficient gene editing method of HLA-I gene to prepare HLA-I universal hematopoietic stem cells. METHODS: The easyedit small guide RNA(sgRNA) was designed according to the sequences of β2 microglobulin gene and synthesized by GenScript company. RNP complexes were formed by NLS-Cas9-NLS nuclease and Easyedit sgRNA according to different molar ratios (1∶1~1∶4). Control group and four transfection groups were performed respectively. HEK-293 cells and CD34+ hematopoietic stem cells were nucleotransfected with RNP complex by Lonza 4D Nucleofector system. The expression of HLA-I on the surface of HEK-293 cells was detected by flow cytometry after transfection for 72 hours, the cleavage effect was determined by T7E1 enzyme digestion reaction and the presence of nested peak in the DNA sequence was identified by direct sequencing. RESULTS: The transfection groups had different levels of HLA-I negative expression cell populations by flow cytometry after transient transfection of HEK-293 cells and CD34+ hematopoietic stem cells with different molar concentrations of RNP complex for 72 hours. There were nested peaks proximal to the sgRNA PAM sequence in the transfection groups by direct DNA sequencing, indicating that sgRNA had obvious editing effect. In the transfection of HEK-293 cells, the highest proportion of HLA-I negative expression cells was (87.69±0.83)% when the molar ratio of NLS-Cas9-NLS nuclease to Easyedit sgRNA was 1∶4. The cutting efficiency of T7E1 was the highest up to (38±2.0)% when the molar ratio was 1∶3. In the transfection of CD34+ hematopoietic stem cells, the proportion of HLA-I negative expression cells was (91.56±3.39)% when the molar ratio was 1∶2, and the cutting efficiency of T7E1 was (64±8.45)% when the molar ratio was 1∶1. CONCLUSION: This study provides an efficient gene editing method for classical HLA-I molecules, which can effectively silence the expression of class HLA-I molecules on the cell surface, and is suitable for stem cell system with difficult transfection.
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Avenues of genome editing for color trait improvement in ornamental fishes: current status and future perspectives.
Ornamental fish industries are growing sectors contributing significantly to livelihood, trade and export, driven by the worldwide demand for colourful and unique species. Pigmentation is the focal point of the visual appeal of ornamental fish, market value, and species-specific interaction. The pigment cells are called chromatophores, derived from neural crest cells and controlled by sophisticated genetic mechanisms, conferring these fish with distinctive colours and patterns. Historically, selective breeding and dietary pigment supplementation have been applied to enhance colouration. Such traditional practices, however, are prone to disadvantages such as slow development, genetic ambiguity, and unforeseen consequences. With the invention of genome editing, such as CRISPR-Cas9, researchers now have a sensitive and powerful tool to control pigmentation traits at the genetic level. Central pigmentation gene manipulation, such as Tyr, Mc1r, and Slc45a2, can allow researchers to introduce stable and uniform color changes. Such enhancements confer unparalleled control of fish colour, a promising avenue in the ornamental fish industry. This review discusses the genetic nature of fish pigmentation. It reports recent advances in CRISPR-based modifications and describes their possible applications and implications for future ornamental fish breeding.
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Genetically modified humans: the X-Men of scientific research.
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Challenges in Gene Therapy for Somatic Reverted Mosaicism in X-Linked Combined Immunodeficiency by CRISPR/Cas9 and Prime Editing.
X-linked severe combined immunodeficiency (X-SCID) is a primary immunodeficiency that is caused by mutations in the interleukin-2 receptor gamma (IL2RG) gene. Some patients present atypical X-SCID with mild clinical symptoms due to somatic revertant mosaicism. CRISPR/Cas9 and prime editing are two advanced genome editing tools that paved the way for treating immune deficiency diseases. Prime editing overcomes the limitations of the CRISPR/Cas9 system, as it does not need to induce double-strand breaks (DSBs) or exogenous donor DNA templates to modify the genome. Here, we applied CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs) and prime editing methods to generate an in vitro model of the disease in K-562 cells and healthy donors' T cells for the c. 458T>C point mutation in the IL2RG gene, which also resulted in a useful way to optimize the gene correction approach for subsequent experiments in patients' cells. Both methods proved to be successful and were able to induce the mutation of up to 31% of treated K-562 cells and 26% of treated T cells. We also applied similar strategies to correct the IL2RG c. 458T>C mutation in patient T cells that carry the mutation with revertant somatic mosaicism. However, both methods failed to increase the frequency of the wild-type sequence in the mosaic T cells of patients due to limited in vitro proliferation of mutant cells and the presence of somatic reversion. To the best of our knowledge, this is the first attempt to treat mosaic cells from atypical X-SCID patients employing CRISPR/Cas9 and prime editing. We showed that prime editing can be applied to the formation of specific-point IL2RG mutations without inducing nonspecific on-target modifications. We hypothesize that the feasibility of the nucleotide substitution of the IL2RG gene using gene therapy, especially prime editing, could provide an alternative strategy to treat X-SCID patients without revertant mutations, and further technological improvements need to be developed to correct somatic mosaicism mutations.
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Is Duchenne gene therapy a suitable treatment despite its immunogenic class effect?
INTRODUCTION: Duchenne muscular dystrophy (DMD) is a severe X-linked disorder characterized by progressive muscle weakness and eventual death due to cardiomyopathy or respiratory complications. Currently, there is no cure for DMD, with standard treatments primarily focusing on symptom management. Using immunosuppressive measures and optimized vector designs allows for gene therapies to better address the genetic cause of the disease. AREAS COVERED: This review evaluates the efficacy and safety of emerging DMD gene therapies as of 2024. It also discusses the potential of utrophin upregulation, gene editing, and truncated dystrophin as therapeutic strategies. It highlights safety concerns associated with these therapies, including adverse events and patient deaths. A comprehensive overview of developments covers topics such as CRISPR-Cas9 therapies, micro-dystrophin, and the potential delivery of full-length dystrophin. EXPERT OPINION: The FDA's recent approval of delandistrogene moxeparvovec (Elevidys) underscores the promise of gene replacement therapies for DMD patients. Understanding the mechanisms behind the adverse effects and excluding patients with specific pathogenic variants may enhance the safety profiles of these therapies. CRISPR/Cas9 therapies, while promising, face significant regulatory and safety challenges that hinder their clinical application. Optimal DMD therapies should target both skeletal and cardiac muscles to be effective.
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In vivo CRISPR-Cas9 expression in Candida glabrata, Candida bracarensis, and Candida nivariensis: A versatile tool to study chromosomal break repair.
The CRISPR-Cas9 system is extremely useful for genome editing in many species, including the model yeast Saccharomyces cerevisiae, and other yeast species. We have previously reported the use of an inducible CRISPR-Cas9 system in Candida glabrata, which allows genome editing but also the study of double-strand break (DSB) repair. We report, in this study, a comparable system for C. glabrata, relying on a new plasmid, which is more stable than the previous one. We also report the use of this plasmid to induce DSBs in two additional human pathogens, Candida bracarensis and Candida nivariensis. We examine lethality induced by an in vivo DSB in the three species and describe the different types of nonhomologous end-joining (NHEJ) events detected in these three pathogens.
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A Decade of CRISPR-Cas Gnome Editing in C. elegans.
CRISPR-Cas allows us to introduce desired genome editing, including mutations, epitopes, and deletions, with unprecedented efficiency. The development of CRISPR-Cas has progressed to such an extent that it is now applicable in various fields, with the help of model organisms. C. elegans is one of the pioneering animals in which numerous CRISPR-Cas strategies have been rapidly established over the past decade. Ironically, the emergence of numerous methods makes the choice of the correct method difficult. Choosing an appropriate selection or screening approach is the first step in planning a genome modification. This report summarizes the key features and applications of CRISPR-Cas methods using C. elegans, illustrating key strategies. Our overview of significant advances in CRISPR-Cas will help readers understand the current advances in genome editing and navigate various methods of CRISPR-Cas genome editing.
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Improved split prime editors enable efficient in vivo genome editing.
Efficient prime editor (PE) delivery in vivo is critical for realizing its full potential in disease modeling and therapeutic correction. Although PE has been divided into two halves and delivered using dual adeno-associated viruses (AAVs), the editing efficiency at different gene loci varies among split sites. Furthermore, efficient split sites within Cas9 nickase (Cas9n) are limited. Here, we verified that 1115 (Asn) is an efficient split site when delivering PEs by dual AAVs. Additionally, we utilized a feature in which reverse transcriptase could be detached from the Cas9n and designed split sites in the first half of Cas9n. We found that split-PE-367 enabled high editing efficiency with Rma intein. To test the editing efficiency in vivo, split-ePE3-367 was packaged in AAV9 and achieved 17.5% precise editing in mice. Our findings establish an alternative split-PE architecture that enables robust editing efficiency, facilitating potential utility in disease modeling and correction.
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Supramolecular Genome Editing: Targeted Delivery and Endogenous Activation of CRISPR/Cas9 by Dynamic Host-Guest Recognition.
We synthesize supramolecular poly(disulfide) (CPS) containing covalently attached cucurbit[7]uril (CB[7]), which is exploited not only as a carrier to deliver plasmid DNA encoding destabilized Cas9 (dsCas9), but also as a host to include trimethoprim (TMP) by CB[7] moieties through the supramolecular complexation to form TMP@CPS/dsCas9. Once the plasmid is transfected into tumor cells by CPS, the presence of polyamines can competitively trigger the decomplexation of TMP@CPS, thereby displacing and releasing TMP from CB[7] to stabilize dsCas9 that can target and edit the genomic locus of PLK1 to inhibit the growth of tumor cells. Following the systemic administration of TMP@CPS/dsCas9 decorated with hyaluronic acid (HA), tumor-specific editing of PLK1 is detected due to the elevated polyamines in tumor microenvironment, greatly minimizing off-target editing in healthy tissues and non-targeted organs. As the metabolism of polyamines is dysregulated in a wide range of disorders, this study offers a supramolecular approach to precisely control CRISPR/Cas9 functions under particular pathological contexts.
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