Your browser does not allow saving data (e.g. private mode): bookmarks will be lost when the tab closes.
Search PubMed
Keywords, author, journal, years — results from PubMed, abstracts with Russian translation, bookmarks in your browser.
Found: 26 178
Page 834 of 1000-
A first step toward in vivo gene editing in patients.
-
Gene therapy for hereditary hearing loss.
Gene therapy is a technique by which exogenous genetic material is introduced into target cells to treat or prevent diseases caused by genetic mutations. Hearing loss is the most common sensory disorder. Genetic factors contribute to approximately 50 % of all cases of profound hearing loss, and more than 150 independent genes have been reported as associated with hearing loss. Recent advances in CRISPR/Cas based gene-editing tools have facilitated the development of gene therapies for hereditary hearing loss (HHL). Viral delivery vectors, and especially adeno-associated virus (AAV) vectors, have been demonstrated as safe and efficient carriers for the delivery of transgenes into inner ear cells in animal models. More importantly, AAV-mediated gene therapy can restore hearing in some children with hereditary deafness. However, there are many different types of HHL that need to be identified and evaluated to determine appropriate gene therapy options. In the present review, we summarize recent animal model-based advances in gene therapy for HHL, as well as gene therapy strategies, gene-editing tools, delivery vectors, and administration routes. We also discuss the strengths and limitations of different gene therapy methods and describe future challenges for the eventual clinical application of gene therapy for HHL.
-
Oligonucleotide-based CRISPR-Cas9 toolbox for efficient engineering of Komagataella phaffii.
Komagataella phaffii (Pichia pastoris) is a methylotrophic yeast that is favored by industry and academia mainly for expression of heterologous proteins. However, its full potential as a host for bioproduction of valuable compounds cannot be fully exploited as genetic tools are lagging behind those that are available for baker's yeast. The emergence of CRISPR-Cas9 technology has significantly improved the efficiency of gene manipulations of K. phaffii, but improvements in gene-editing methods are desirable to further accelerate engineering of this yeast. In this study, we have developed a versatile vector-based CRISPR-Cas9 method and showed that it works efficiently at different genetic loci using linear DNA fragments with very short targeting sequences including single-stranded oligonucleotides. Notably, we performed site-specific point mutations and full gene deletions using short (90 nt) single-stranded oligonucleotides at very high efficiencies. Lastly, we present a strategy for transient inactivation of nonhomologous end-joining (NHEJ) pathway, where KU70 gene is disrupted by a visual marker (uidA gene). This system enables precise CRISPR-Cas9-based editing (including multiplexing) and facilitates simple reversion to NHEJ-proficient genotype. In conclusion, the tools presented in this study can be applied for easy and efficient engineering of K. phaffii strains and are compatible with high-throughput automated workflows.
-
An episomal CRISPR/Cas9 system to derive vector-free gene modified mammalian cells.
-
Insights into the compact CRISPR-Cas9d system.
Cas9d, the smallest known member of the Cas9 family, employs a compact domain architecture for effective target cleavage. However, the underlying mechanism remains unclear. Here, we present the cryo-EM structures of the Cas9d-sgRNA complex in both target-free and target-bound states. Biochemical assays elucidated the PAM recognition and DNA cleavage mechanisms of Cas9d. Structural comparisons revealed that at least 17 base pairs in the guide-target heteroduplex is required for nuclease activity. Beyond its typical role as an adaptor between Cas9 enzymes and targets, the sgRNA also provides structural support and functional regulation for Cas9d. A segment of the sgRNA scaffold interacts with the REC domain to form a functional target recognition module. Upon target binding, this module undergoes a coordinated conformational rearrangement, enabling heteroduplex propagation and facilitating nuclease activity. This hybrid functional module precisely monitors heteroduplex complementarity, resulting in a lower mismatch tolerance compared to SpyCas9. Moreover, structure-guided engineering in both the sgRNA and Cas9d protein led to a more compact Cas9 system with well-maintained nuclease activity. Altogether, our findings provide insights into the target recognition and cleavage mechanisms of Cas9d and shed light on the development of high-fidelity mini-CRISPR tools.
-
Prime Editing: An Overview.
CRISPR tools are revolutionizing the landscape of genetic therapies, with the potential to cure a range of previously untreatable diseases. Among all the available genome editing technologies, prime editing is an especially versatile tool that enables precise genetic modifications, including point mutations, insertions, and deletions, without inducing double-strand breaks or requiring a donor DNA template. Through structural modifications and the development of novel systems with additional functionalities, prime editing has expanded its applicability with improved precision, efficacy, and safety. It is already being tested in clinical trials for chronic granulomatous disease, and many preclinical studies are underway. However, significant challenges remain for its broad applicability as a potential curative therapy for human genetic diseases, mainly related to ensuring efficient and safe delivery to target tissues.
-
Production of genetically engineered mice with higher efficiency, lower mosaicism, and multiplexing capability using maternally expressed Cas9.
The CRISPR/Cas9 system is widely used to generate gene-edited animals. Here, we developed an efficient system for generating genetically modified mice using maternal Cas9 from Cas9 transgenic mice. Using this system, we achieved lower mosaicism and higher rates of knock-in success, gene-editing, and birth compared to the similar parameters obtained using exogenously administered Cas9 (mRNA/protein) system. Furthermore, we successfully induced simultaneous mutations at multiple loci (a maximum of nine). Our novel gene-editing system based on maternal Cas9 could potentially facilitate the generation of mice with single and multiple gene modifications.
-
Spatiotemporal Control of CRISPR/Cas9 Function in Cells and Zebrafish using Light-Activated Guide RNA.
We developed a new method for the conditional regulation of CRISPR/Cas9 activity in mammalian cells and zebrafish embryos using photochemically activated, caged guide RNAs (gRNAs). Caged gRNAs are generated by substituting four nucleobases evenly distributed throughout the 5'-protospacer region with caged nucleobases during synthesis. Caging confers complete suppression of gRNA:dsDNA-target hybridization and rapid restoration of CRISPR/Cas9 function upon optical activation. This tool offers simplicity and complete programmability in design, high spatiotemporal specificity in cells and zebrafish embryos, excellent off-to-on switching, and stability by preserving the ability to form Cas9:gRNA ribonucleoprotein complexes. Caged gRNAs are novel tools for the conditional control of gene editing, thereby enabling the investigation of spatiotemporally complex physiological events by obtaining a better understanding of dynamic gene regulation.
-
In Vitro Packaging Mediated One-Step Targeted Cloning of Natural Product Pathway.
Direct cloning of natural product pathways for efficient refactoring and heterologous expression has become an important strategy for microbial natural product research and discovery, especially for those kept silent or poorly expressed in the original strains. Accordingly, the development of convenient and efficient cloning approaches is becoming increasingly necessary. Here we presented an in vitro packaging mediated cloning approach that combines CRISPR/Cas9 system with in vitro λ packaging system, for targeted cloning of natural product pathways. In such a scheme, pathways of Tü3010 (27.4 kb) and sisomicin (40.7 kb) were respectively cloned, and stuR was further depicted to positively regulate Tü3010 production. In vitro packaging mediated approach not only enables to activate cryptic pathways, but also facilitates refactoring or interrogating the pathways in conjunction with various gene editing systems. This approach features an expedited, convenient, and generic manner, and it is conceivable that it may be widely adopted for targeted cloning of the natural product pathways.
-
Establishment of Agrobacterium-mediated genetic transformation and CRISPR/Cas9-guided gene editing in Elymus nutans.
Elymus nutans, an allohexaploid (2n = 6x = 42) species with a StStHHYY genome, is a native perennial in the alpine grasslands of the Qinghai-Xizang Plateau, and has been widely used for artificial pasture and ecological restoration as a forage grass with highest yield on the plateau. Nevertheless, the lack of a stable transformation system has impeded further efforts to trait improvement of E. nutans. In the present study, we established a reliable Agrobacterium-mediated genetic transformation system for E. nutans, and successfully generated EnTCP4-edited plants using the CRISPR/Cas9 system. The editing efficiency achieved 19.23 % in E. nutans. Knocking out EnTCP4 significantly delayed flowering and enhanced water-deficit stress resistance. This research represents a significant breakthrough in the genetic transformation and gene editing of E. nutans, laying a technological foundation to gain insight into gene functions and molecular breeding in E. nutans.
PubMed returns only the first 10,000 results — refine the query to see the rest.