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Страница 787 из 1000-
Wake-up call from Hong Kong.
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Racing for academic glory and patents: Lessons from CRISPR.
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CASGEVY Makes History as FDA Approves First CRISPR/Cas9 Genome Edited Therapy.
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Preclinical Assessment of a Gene-Editing Approach in a Mouse Model of Mitochondrial Neurogastrointestinal Encephalomyopathy.
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare disease caused by recessive mutations in the TYMP gene, which encodes the enzyme thymidine phosphorylase (TP). In this study, the efficient integration of a TYMP transgene into introns of the Tymp and Alb loci of hepatocytes in a murine model of MNGIE was achieved by the coordinated delivery and activity of CRISPR/Cas9 and a TYMP cDNA. CRISPR/Cas9 was delivered either as mRNA using lipid nanoparticle (LNP) or polymeric nanoparticle, respectively, or in an AAV2/8 viral vector; the latter was also used to package the TYMP cDNA. Insertion of the cDNA template downstream of the Tymp and Alb promoters ensured transgene expression. The best in vivo results were obtained using LNP carrying the CRISPR/Cas9 mRNAs. Treated mice showed a consistent long-term (1 year) reduction in plasma nucleoside (thymidine and deoxyuridine) levels that correlated with the presence of TYMP mRNA and functional enzyme in liver cells. In mice with an edited Alb locus, the transgene produced a hybrid Alb-hTP protein that was secreted, with supraphysiological levels of TP activity detected in the plasma. Equivalent results were obtained in mice edited at the Tymp locus. Finally, some degree of gene editing was found in animals treated only with AAV vectors containing the DNA templates, in the absence of nucleases, although there was no impact on plasma nucleoside levels. Overall, these results demonstrate the feasibility of liver-directed genome editing in the long-term correction of MNGIE, with several advantages over other methods.
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Genome Editing of Pigs for Agriculture and Biomedicine.
Pigs serve as an important agricultural resource and animal model in biomedical studies. Efficient and precise modification of pig genome by using recently developed gene editing tools has significantly broadened the application of pig models in various research areas. The three types of site-specific nucleases, namely, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein, are the main gene editing tools that can efficiently introduce predetermined modifications, including knockouts and knockins, into the pig genome. These modifications can confer desired phenotypes to pigs to improve production traits, such as optimal meat production, enhanced feed digestibility, and disease resistance. Besides, given their genetic, anatomic, and physiologic similarities to humans, pigs can also be modified to model human diseases or to serve as an organ source for xenotransplantation to save human lives. To date, many genetically modified pig models with agricultural or biomedical values have been established by using gene editing tools. These pig models are expected to accelerate research progress in related fields and benefit humans.
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Sabotage of CRISPR-Cas.
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Packaging and delivery of genome-editing tools.
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Generation of epitope tag knock-in mice with CRISPR-Cas9 to study the function of endogenous proteins.
Studying endogenous proteins in mice has provided numerous insights into the physiological and pathological roles of these proteins. However, the availability and specificity of protein-specific antibodies often limit such studies. Here we present a protocol for generating epitope tag knock-in mice with CRISPR-Cas9-mediated gene editing. We discuss key considerations for tag selection and knock-in location and provide insights into CRISPR design. Subsequently, we outline the sequential steps involved in knock-in mouse generation, genotyping, and validation and explore potential applications. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).1.
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Microfluidic-assisted biomineralization of CRISPR/Cas9 in near-infrared responsive metal-organic frameworks for programmable gene-editing.
Ribonucleoprotein (RNP) based CRISPR/Cas9 gene-editing system shows great potential in biomedical applications. However, due to the large size, charged surface and high biological sensitivity of RNP, its efficient delivery with precise control remains highly challenging. Herein, a microfluidic-assisted metal-organic framework (MOF) based biomineralization strategy is designed and utilized for the efficient delivery and remote regulation of CRISPR/Cas9 RNP gene editing. The strategy is realized by biomimetic growing of thermo-responsive EuMOFs onto photothermal template Prussian blue (PB). The RNP is loaded during MOFs crystallization in microfluidic channels. By adjusting different microfluidic parameters, well-defined and comparable RNP encapsulated nanocarrier (PB@RNP-EuMOFs) are obtained with high loading efficiency (60%), remarkable RNP protection and NIR-stimulated release capacity. Upon laser exposure, the nanocarrier induces effective endosomal escape (4 h) and precise gene knockout of green fluorescent protein by 40% over 2 days. Moreover, the gene-editing activity can be programmed by tuning exposure times (42% for three times and 47% for four times), proving more controllable and inducible editing modality compared to control group without laser irradiation. This novel microfluidic-assisted MOFs biomineralization strategy thus offers an attractive route to optimize delivery systems and reduce off-target side effects by NIR-triggered remote control of CRISPR/Cas9 RNP, improving the potential for its highly efficient and precise therapeutic application.
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Rapid generation of novel models of RAG1 deficiency by CRISPR/Cas9-induced mutagenesis in murine zygotes.
Mutations in the Recombination Activating Gene 1 (RAG1) can cause a wide variety of clinical and immunological phenotypes in humans, ranging from absence of T and B lymphocytes to occurrence of autoimmune manifestations associated with expansion of oligoclonal T cells and production of autoantibodies. Although the mechanisms underlying this phenotypic heterogeneity remain poorly understood, some genotype-phenotype correlations can be made. Currently, mouse models of Rag deficiency are restricted to RAG1-/- mice and to knock-in models carrying severe missense mutations. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system is a novel and powerful gene-editing strategy that permits targeted introduction of DNA double strand breaks with high efficiency through simultaneous delivery of the Cas9 endonuclease and a guide RNA (gRNA). Here, we report on CRISPR-based, single-step generation and characterization of mutant mouse models in which gene editing was attempted around residue 838 of RAG1, a region whose functional role had not been studied previously.
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