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Ключевые слова, автор, журнал, годы — результаты из PubMed, аннотации с переводом на русский, закладки в браузере.
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CRISPR-based technologies for large DNA insertions.
While the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based technologies has democratized the genesis of precise mutations, there is a need for more sophisticated tools to enable large-scale DNA manipulations, advancing genome editing across medicine, biotechnology, and agriculture. The success of Cas9 and Cas12 has hinged on the generation of precise DNA nicks and double-stranded breaks (DSBs), enabling local sequence mutagenesis, albeit of a limited size range. Emerging effectors combining Cas with other enzymatic functions, such as CRISPR-associated transposons and site-specific recombinases, enable larger integrations. Sophisticated combinations such as programmable addition via site-specific targeting element (PASTE), prime-editing-assisted site-specific integrase gene editing (PASSIGE), and prime-editing-mediated recombination of opportune target (PrimeRoot) expand payload options and DSB-free editing modalities, with translational potential for next-generation crop breeding in sustainable agriculture and the development of gene and cell therapies in personalized medicine.
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Wake-up call from Hong Kong.
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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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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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Racing for academic glory and patents: Lessons from CRISPR.
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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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Restore natural fertility of Kitw/Kitwv mouse with nonobstructive azoospermia through gene editing on SSCs mediated by CRISPR-Cas9.
BACKGROUND: Male infertility is a serious social problem in modern society. Nonobstructive azoospermia (NOA) caused by germ cell gene defects is an important reason for male infertility, but effective clinical treatment for this disease has not been established. METHODS: We choose Kitw/Kitwv mouse as a research model and try to develop a new treatment strategy and "cure" its infertility. Mutant spermatogonial stem cells (SSCs) were isolated from one single unilateral testis of a 14-day-old Kitw/Kitwv mouse and propagated in vitro. The C to T point mutation on Kitwv site of these SSCs was corrected through CRISPR-Cas9-mediated homology-directed repair (HDR) in vitro. Then, the repaired SSCs were screened out, proliferated, and transplanted into the remaining testis, and complete spermatogenesis was established in the recipient testis. RESULTS: Healthy offsprings with wild type Kit gene or Kitw mutation were obtained through natural mating 4 months after SSC transplantation. CONCLUSION: In this study, we established an effective new treatment strategy for NOA caused by germ cell gene defects through a combination of SSC isolation, CRISPR-Cas9-mediated gene editing, and SSC transplantation, which brought hope for these NOA patients to restore their natural fertility.
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CASGEVY Makes History as FDA Approves First CRISPR/Cas9 Genome Edited Therapy.
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Sabotage of CRISPR-Cas.
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Packaging and delivery of genome-editing tools.
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