Браузер не даёт сохранять данные (например, приватный режим): закладки пропадут после закрытия вкладки.
Поиск статей PubMed
Ключевые слова, автор, журнал, годы — результаты из PubMed, аннотации с переводом на русский, закладки в браузере.
Найдено: 26 219
Страница 736 из 1000-
ARL13B regulates Sonic hedgehog signaling from outside primary cilia.
ARL13B is a regulatory GTPase highly enriched in cilia. Complete loss of Arl13b disrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To determine whether ARL13B is required within cilia, we knocked in a cilia-excluded variant of ARL13B (V358A) and showed it retains all known biochemical function. We found that ARL13BV358A protein was expressed but could not be detected in cilia, even when retrograde ciliary transport was blocked. We showed Arl13bV358A/V358A mice are viable and fertile with normal Shh signal transduction. However, in contrast to wild type cilia, Arl13bV358A/V358A cells displayed short cilia and lacked ciliary ARL3 and INPP5E. These data indicate that ARL13B's role within cilia can be uncoupled from its function outside of cilia. Furthermore, these data imply that the cilia defects upon complete absence of ARL13B do not underlie the alterations in Shh transduction, which is unexpected given the requirement of cilia for Shh transduction.
-
Development of a gRNA-tRNA array of CRISPR/Cas9 in combination with grafting technique to improve gene-editing efficiency of sweet orange.
Here, we developed a reliable protocol for the fast and efficient gene-edited Anliu sweet orange plants production. The application of in vitro shoot grafting technology significantly reduced the growth cycle of transgenic seedlings, and the survival rate of cleft grafting was more than 90%. In addition, the mutation efficiency of the grafted geneedited sweet orange was significantly improved by short-term heat stress treatments. Thus, the combination strategy of grafting and heat stress treatments provided a reference for the fast and efficient multiplex gene editing of sweet orange.
-
DNAzyme activated protein-scaffolded CRISPR-Cas9 nanoassembly for genome editing.
A novel self-assembled protein-scaffolded CRISPR-Cas9 nanosystem for facile and efficient gene editing in a DNAzyme-controlled manner has been developed.
-
Optimized prime editing of the Alzheimer's disease-associated APOE4 mutation.
Gene editing strategies to safely and robustly modify the Alzheimer's disease-associated APOE4 isoform are still lacking. Prime editing (PE) enables the precise introduction of genetic variants with minimal unintended editing and without donor templates. However, it requires optimization for each target site and has not yet been applied to APOE4 gene editing. Here, we screened PE guide RNA (pegRNA) parameters and PE systems for introducing the APOE4 variant and applied the optimized PE strategy to generate disease-relevant human induced pluripotent stem cell models. We show that introducing a single-nucleotide difference required for APOE4 correction inhibits PE activity. To advance efficient and robust genome engineering of precise genetic variants, we further present a reliable PE enrichment strategy based on diphtheria toxin co-selection. Our work provides an optimized and reproducible genome engineering pipeline to generate APOE4 disease models and outlines novel strategies to accelerate genome editing in cellular disease model generation.
-
Generation of albino medaka (Oryzias latipes) by CRISPR/Cas9.
CRISPR/Cas9 system is a powerful tool to produce the genetic modification in plants and animals such as mouse and zebrafish. However, this technique was less reported in fish model medaka (Oryzias latipes). Here, we describe an efficient and rapid procedure for genome editing in medaka tyr and generate a stable albino strain. The Cas9 mRNA and gRNA for tyr gene were injected into the embryos of orange-red medaka, and the tyr gene was disrupted in more than 90% of embryos in F0 and F1, which were validated by observation and sequencing of targeted locus. The pigment cells were largely decreased in the mutant medaka because open reading frames of tyr were shifted near the targeted locus, generating albino medaka. Taken together, this method provides a detailed procedure to generate the genetic modification medaka by using an optimized CRISPR/Cas9 system, and the new albino medaka provides an important research platform to study the pigmentation.
-
Enhancing the CRISPR/Cas9 system based on multiple GmU6 promoters in soybean.
Small guide RNA (sgRNA) is an important component of the CRISPR/Cas9 system. The gene editing efficiency of the CRISPR/Cas9 system could be enhanced by using highly active U6 promoters to drive the expression of sgRNA. Therefore, we constructed various expression vectors based on the 11 GmU6 promoters predicted and cloned in the whole soybean genome. The expression of truncated GUS driven by 11 GmU6 promoters was tested in hairy roots and by Arabidopsis thaliana transformation. The results indicated that higher transcriptional levels were driven by 5 GmU6 promoters (GmU6-4, GmU6-7, GmU6-8, GmU6-10 and GmU6-11) in both soybean hairy roots and Arabidopsis thaliana. In addition, three genes, Glyma03g36470, Glyma14g04180 and Glyma06g136900, were selected as targets to detect the transcriptional levels of multiple GmU6 promoters. Mutations in these three genes were detected in soybean hairy roots after Agrobacterium rhizogenes infection, indicating efficient target gene editing, including nucleotide insertion, deletion, and substitution. Mutation efficiencies differed among the 11 GmU6 promoters, ranging from 2.8% to 20.6%, and markedly higher efficiencies were obtained with all three genes using the GmU6-8 (20.3%) and GmU6-10 (20.6%) promoters. These two GmU6 promoters also showed higher ability to drive truncated GUS transcription in both soybean hairy roots and transformed Arabidopsis thaliana. These results will help to construct an efficient CRISPR-Cas9 gene editing system and promote the application of the CRISPR-Cas9 genome editing system in soybean molecular breeding.
-
Toward the correction of muscular dystrophy by gene editing.
Recent advances in gene editing technologies are enabling the potential correction of devastating monogenic disorders through elimination of underlying genetic mutations. Duchenne muscular dystrophy (DMD) is an especially severe genetic disorder caused by mutations in the gene encoding dystrophin, a membrane-associated protein required for maintenance of muscle structure and function. Patients with DMD succumb to loss of mobility early in life, culminating in premature death from cardiac and respiratory failure. The disease has thus far defied all curative strategies. CRISPR gene editing has provided new opportunities to ameliorate the disease by eliminating DMD mutations and thereby restore dystrophin expression throughout skeletal and cardiac muscle. Proof-of-concept studies in rodents, large mammals, and human cells have validated the potential of this approach, but numerous challenges remain to be addressed, including optimization of gene editing, delivery of gene editing components throughout the musculature, and mitigation of possible immune responses. This paper provides an overview of recent work from our laboratory and others toward the genetic correction of DMD and considers the opportunities and challenges in the path to clinical translation. Lessons learned from these studies will undoubtedly enable further applications of gene editing to numerous other diseases of muscle and other tissues.
-
CRISPR/Cas9-mediated Genomic Editing of Cluap1/IFT38 Reveals a New Role in Actin Arrangement.
CRISPR/Cas9-mediated gene editing allows manipulation of a gene of interest in its own chromosomal context. When applied to the analysis of protein interactions and in contrast to exogenous expression of a protein, this can be studied maintaining physiological stoichiometry, topology, and context. We have used CRISPR/Cas9-mediated genomic editing to investigate Cluap1/IFT38, a component of the intraflagellar transport complex B (IFT-B). Cluap1 has been implicated in human development as well as in cancer progression. Cluap1 loss of function results in early developmental defects with neural tube closure, sonic hedgehog signaling and left-right defects. Herein, we generated an endogenously tagged Cluap1 for protein complex analysis, which was then correlated to the corresponding interactome determined by ectopic expression. Besides IFT-B complex components, new interacting proteins like Ephrin-B1 and TRIP6, which are known to be involved in cytoskeletal arrangement and protein transport, were identified. With the identification of platelet-derived growth factor A (PDGFA) and coiled-coil domain-containing protein 6 (CCDC6) two new interactions were discovered, which link Cluap1 to ciliogenesis and cancer development. The CRISPR/Cas9-mediated knockout of Cluap1 revealed a new phenotype affecting the actin cytoskeleton. Together, these data provide first evidence for a role of Cluap1 not only for cilia assembly and maintenance but also for cytoskeletal rearrangement and intracellular transport processes.
-
Efficient generation of Knock-in/Knock-out marmoset embryo via CRISPR/Cas9 gene editing.
Genetically modified nonhuman primates (NHP) are useful models for biomedical research. Gene editing technologies have enabled production of target-gene knock-out (KO) NHP models. Target-gene-KO/knock-in (KI) efficiency of CRISPR/Cas9 has not been extensively investigated in marmosets. In this study, optimum conditions for target gene modification efficacies of CRISPR/mRNA and CRISPR/nuclease in marmoset embryos were examined. CRISPR/nuclease was more effective than CRISPR/mRNA in avoiding mosaic genetic alteration. Furthermore, optimal conditions to generate KI marmoset embryos were investigated using CRISPR/Cas9 and 2 different lengths (36 nt and 100 nt) each of a sense or anti-sense single-strand oligonucleotide (ssODN). KIs were observed when CRISPR/nuclease and 36 nt sense or anti-sense ssODNs were injected into embryos. All embryos exhibited mosaic mutations with KI and KO, or imprecise KI, of c-kit. Although further improvement of KI strategies is required, these results indicated that CRISPR/Cas9 may be utilized to produce KO/KI marmosets via gene editing.
-
Detecting Methylation Changes Induced by Prime Editing.
While prime editing offers improved precision compared to traditional CRISPR-Cas9 systems, concerns remain regarding potential off-target effects, including epigenetic changes such as DNA methylation. In this study, we investigated whether prime editing induces aberrant CpG methylation patterns. Whole-genome bisulfite sequencing revealed overall methylation similarity between Cas9-edited, and PE2-edited cells. However, localized epigenetic changes were observed, particularly in CpG islands and exon regions. The PE2-edited group showed a higher proportion of differentially methylated regions (DMRs) in some coding sequences compared to controls and Cas9-edited samples. Notably, CpG island methylation reached 0.18% in the PE2 vs. Cas9 comparison, indicating a higher susceptibility of these regulatory elements to epigenetic alterations by prime editing. Molecular function analyses including Gene Ontology and KEGG pathway analyses further revealed enrichment in molecular functions related to transcriptional regulation and redox activity in PE2-edited cells. These findings suggest that prime editing, while precise, may introduce subtle but functionally relevant methylation changes that could influence gene expression and cellular pathways. In summary, prime editing can induce localized DNA methylation changes in human cells, particularly within regulatory and coding regions. Understanding these epigenetic consequences is critical for the development of safer and more effective therapeutic applications of genome editing technologies.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.