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: 26 131
Страница 281 из 1000-
High-throughput genome editing and phenotyping facilitated by high resolution melting curve analysis.
With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.
-
Unintended CRISPR-Cas9 editing outcomes: a review of the detection and prevalence of structural variants generated by gene-editing in human cells.
Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas) gene-editing system (CRISPR-Cas) is a valuable tool for fundamental and applied research applications. Significant improvements in editing efficacy have advanced genome editing strategies into phase 3 human clinical trials. However, recent studies suggest that our understanding of editing outcomes has lagged behind the developments made in generating the edits themselves. While many researchers have analyzed on- and off-target events through the lens of small insertions or deletions at predicted sites, screens for larger structural variants (SVs) and chromosomal abnormalities are not routinely performed. Full and comprehensive validation of on- and off-target effects is required to ensure reproducibility and to accurately assess the safety of future editing applications. Here we review SVs associated with CRISPR-editing in cells of human origin and highlight the methods used to detect and avoid them.
-
Mechanistic insights into the versatile class II CRISPR toolbox.
The constantly expanding group of class II CRISPR-Cas (clustered regularly interspaced short palindromic repeats-associated) effectors and their engineered variants exhibit distinct editing modes and efficiency, fidelity, target range, and molecular size. Their enormous diversity of capabilities provides a formidable toolkit for a large array of technologies. We review the structural and biochemical mechanisms of versatile effector proteins from class II CRISPR-Cas systems to provide mechanistic insights into their target specificity, protospacer adjacent motif (PAM) restriction, and activity regulation, and discuss possible strategies to enhance genome-engineering tools in terms of accuracy, efficiency, applicability, and controllability.
-
CRISPR/Cas-based genome engineering in natural product discovery.
Covering: up to February, 2018 This review briefly introduces and summarizes current knowledge about the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) - CRISPR/Cas system and how it was engineered to become one of the most important and versatile genome editing techniques that are currently revolutionizing the whole field of molecular biology. It aims to highlight and discuss the applications and remaining challenges of CRISPR/Cas (mainly focusing on CRISPR/SpCas9)-based genome editing in natural product discovery. The organisms covered include bacteria such as Streptomyces, Corynebacteria, and Myxobacteria; filamentous fungi such as Aspergillus, Beauveria, and Ganoderma; microalgae; and some plants. As closing remarks, the prospects of using CRISPR/Cas in natural product discovery will be discussed.
-
CRISPR-Cas9 gene editing: Delivery aspects and therapeutic potential.
The CRISPR-Cas9 gene editing system has taken the biomedical science field by storm, initiating rumors about future Nobel Prizes and heating up a fierce patent war, but also making significant scientific impact. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with CRISPR-associated proteins (Cas) are a part of the prokaryotic adaptive immune system and have successfully been repurposed for genome editing in mammalian cells. The CRISPR-Cas9 system has been used to correct genetic mutations and for replacing entire genes, opening up a world of possibilities for the treatment of genetic diseases. In addition, recently some new CRISPR-Cas systems have been discovered with interesting mechanistic variations. Despite these promising developments, many challenges have to be overcome before the system can be applied therapeutically in human patients and enabling delivery technology is one of the key challenges. Furthermore, the relatively high off-target effect of the system in its current form prevents it from being safely applied directly in the human body. In this review, the transformation of the CRISPR-Cas gene editing systems into a therapeutic modality will be discussed and the currently most realistic in vivo applications will be highlighted.
-
CRISPR/Cas9-based Gene Therapies for Fighting Drug Resistance Mediated by Cancer Stem Cells.
Cancer stem cells (CSCs) are cancer-initiating cells found in most tumors and hematological cancers. CSCs are involved in cells progression, recurrence of tumors, and drug resistance. Current therapies have been focused on treating the mass of tumor cells and cannot eradicate the CSCs. CSCs drug-specific targeting is considered as an approach to precisely target these cells. Clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) gene-editing systems are making progress and showing promise in the cancer research field. One of the attractive applications of CRISPR/Cas9 as one approach of gene therapy is targeting the critical genes involved in drug resistance and maintenance of CSCs. The synergistic effects of gene editing as a novel gene therapy approach and traditional therapeutic methods, including chemotherapy, can resolve drug resistance challenges and regression of the cancers. This review article considers different aspects of CRISPR/Cas9 ability in the study and targeting of CSCs with the intention to investigate their application in drug resistance.
-
CRISPR/Cas9 mediated genome editing for crop improvement against Abiotic stresses: current trends and prospects.
Abiotic stresses associated with climate change, such as heat, cold, salinity, and drought, represent a serious threat to crop health. To mitigate the risks posed by these environmental challenges, both transgenic technology and conventional breeding methods have been extensively utilized. However, these methods have faced numerous limitations. The development of synthetic nucleases as precise genetic tools allows for the targeted alteration of stress-responsive genes in crop improvement. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas) genome-editing technique has transformed gene editing with its broad applicability, accessibility, adaptability, flexibility, and simplicity. Its application shows promise for the development of crop types that are more able to survive abiotic stress conditions. The present study presents recent scenario and application of CRISPR/Cas genome-editing technology in enhancing crop tolerance to a variety of abiotic stresses.
-
CRISPR/Cas9-Based Gene Dropout Screens.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-based technology enables efficient and precise perturbations of target genomic sites. Combining the endonuclease Cas9 and a pooled guide RNA library allows for systematic screenings of genes associated with a growth disadvantage or lethal phenotype under various conditions in organisms and tissues. Here, we describe a complete protocol for scalable CRISPR/Cas9-based dropout screening for essential genes from focused genomic regions to whole genomes.
-
In vivo gene editing in dystrophic mouse muscle and muscle stem cells.
Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.
-
Prime Editing: An Emerging Tool in Cancer Treatment.
Prime Editing is a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based genome editing technique having promising potential in terms of reducing off target activity. It introduces fragments of DNA sequences into the target site using a guide RNA (gRNA) molecule, composed of both the sequence that is to be inserted into the target site along with an inactive Cas9 nickase and a reverse transcriptase. Prime Editing can cause insertions, deletions, and various point mutations for reverting the phenetic characteristics of a disease specially tested in human adult stem cells and cancer cell lines. The main aim of our review is to explore how Prime Editing and its various forms are being utilized as an emerging tool to cure deleterious diseases like cancer, also as a delivery strategy of the tool into cells. There are almost five generations of Prime Editors (PE) with increasing levels of efficiency from one level to another that have huge clinical potential in correcting mutations; however, the necessity for a pegRNA design is extremely significant. But besides having such advantages, the limitations of this technology particularly include generation of double nicks while optimizing the efficiency of PE3. So, it is important to consider all such consequences and customize PE as per requirements.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.