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: 26 245
Страница 864 из 1000-
An Introduced RNA-Only Approach for Plasmid Curing via the CRISPR-Cpf1 System in Saccharomyces cerevisiae.
The CRISPR-Cas system has been widely used for genome editing due to its convenience, simplicity and flexibility. Using a plasmid-carrying Cas protein and crRNA or sgRNA expression cassettes is an efficient strategy in the CRISPR-Cas genome editing system. However, the plasmid remains in the cells after genome editing. Development of general plasmid-curing strategies is necessary. Based on our previous CRISPR-Cpf1 genome-editing system in Saccharomyces cerevisiae, the crRNA, designed for the replication origin of the CRISPR-Cpf1 plasmid, and the ssDNA, as a template for homologous recombination, were introduced for plasmid curing. The efficiency of the plasmid curing was 96 ± 4%. In addition, we further simplified the plasmid curing system by transforming only one crRNA into S. cerevisiae, and the curing efficiency was about 70%. In summary, we have developed a CRISPR-mediated plasmid-curing system. The RNA-only plasmid curing system is fast and easy. This plasmid curing strategy can be applied in broad hosts by designing crRNA specific for the replication origin of the plasmid. The plasmid curing system via CRISPR-Cas editing technology can be applied to produce traceless products without foreign genes and to perform iterative processes in multiple rounds of genome editing.
-
Fast and efficient CRISPR-mediated genome editing in Aureobasidium using Cas9 ribonucleoproteins.
Species of the genus Aureobasidium are ubiquitous, polyextremotolerant, "yeast-like" ascomycetes used for the industrial production of pullulan and other products and as biocontrol agents in agriculture. Their application potential and wide-spread occurrence make Aureobasidium spp. interesting study objects. The availability of a fast and efficient genome editing method is an obvious advantage for future basic and applied research on Aureobasidium. In this study, we describe the development of a CRISPR/Cas9-based genome editing method using ribonucleoproteins (RNPs) in A. pullulans and A. melanogenum. We demonstrate that this method can be used for single and multiplex genome editing using only RNPs by targeting URA3 (encoding for orotidine-5'-phosphate decarboxylase), ADE2 (encoding for phosphoribosylaminoimidazole carboxylase) and ARG4 (encoding for argininosuccinate lyase). We demonstrate the applicability of Trichoderma reesei pyr4 and Aspergillus fumigatus pyrG to complement the URA3 deficiency. Further, we show that using RNPs improves the homologous recombination rate and 20 bp long homologous flanks are sufficient. Therefore, the repair cassettes can be constructed by a single PCR, abolishing the need for laborious and time-consuming cloning, which is necessary for previously described methods for CRISPR-mediated genome editing in these fungi. The here presented method allows fast and efficient genome editing for gene deletions, modifications, and insertions in Auresobasidium with a minimized risk of off-target effects.
-
MAGIK: A rapid and efficient method to create lineage-specific reporters in human pluripotent stem cells.
Precise insertion of fluorescent proteins into lineage-specific genes in human pluripotent stem cells (hPSCs) presents challenges due to low knockin efficiency and difficulties in isolating targeted cells. To overcome these hurdles, we present the modified mRNA (ModRNA)-based Activation for Gene Insertion and Knockin (MAGIK) method. MAGIK operates in two steps: first, it uses a Cas9-2A-p53DD modRNA with a mini-donor plasmid (without a drug selection cassette) to significantly enhance efficiency. Second, a deactivated Cas9 activator modRNA and a 'dead' guide RNA are used to temporarily activate the targeted gene, allowing for live cell sorting of targeted cells. Consequently, MAGIK eliminates the need for drug selection cassettes or labor-intensive single-cell colony screening, expediting precise gene editing. We showed MAGIK can be utilized to insert fluorescent proteins into various genes, including SOX17, NKX6.1, NKX2.5, and PDX1, across multiple hPSC lines. This underscores its robust performance and offers a promising solution for achieving knockin in hPSCs within a significantly shortened time frame.
-
Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in Yarrowia lipolytica.
Yarrowia lipolytica has fast become a biotechnologically significant yeast for its ability to accumulate lipids to high levels. While there exists a suite of synthetic biology tools for genetic engineering in this yeast, there is a need for multipurposed tools for rapid strain generation. Here, we describe a dual purpose CRISPR-Cpf1 system that is capable of simultaneous gene disruption and gene regulation. Truncating guide RNA spacer length to 16 nt inhibited nuclease activity but not binding to the target loci, enabling gene activation and repression with Cpf1-fused transcriptional regulators. Gene repression was demonstrated using a Cpf1-Mxi1 fusion achieving a 7-fold reduction in mRNA, while CRISPR-activation with Cpf1-VPR increased hrGFP expression by 10-fold. High efficiency disruptions were achieved with gRNAs 23-25 bp in length, and efficiency and repression levels were maintained with multiplexed expression of truncated and full-length gRNAs. The developed CRISPR-Cpf1 system should prove useful in metabolic engineering, genome wide screening, and functional genomics studies.
-
CRISPR/Cas9-Based Methods for Inactivating Actinobacterial Biosynthetic Genes and Elucidating Function.
The CRISPR/Cas9 technology allows fast and marker-less genome engineering that can be employed to study secondary metabolism in actinobacteria. Here, we report a standard experimental protocol for the deletion of a biosynthetic gene in a Streptomyces species, using the vector pCRISPomyces-2 developed by Huimin Zhao and collaborators. We also describe how carrying out metabolite analysis can reveal the putative biosynthetic function of the inactivated gene.
-
Regulating genome editing technologies: a comparison of expert recommendations in the U.K. and in the U.S.A.
The present paper aims to inform the bioethical debate on the regulation of human genome editing technologies with a specific focus on the role of scientific experts and their interactions with the general public in the formulation of policy. It reviews and compares two of the major contributions to this debate in the U.K. and in the U.S.A., comparing expert approaches towards regulation on genome editing technologies. The results of this analysis offer important lessons that should be appreciated in building an international regulatory framework. On the basis of these results, I conclude that the experts should embrace a socially responsible approach and encourage active public engagement.
-
Simultaneous precise editing of multiple genes in human cells.
When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.
-
Gene editing of the wheat homologs of TONNEAU1-recruiting motif encoding gene affects grain shape and weight in wheat.
Grain size and weight are important components of a suite of yield-related traits in crops. Here, we showed that the CRISPR-Cas9 gene editing of TaGW7, a homolog of rice OsGW7 encoding a TONNEAU1-recruiting motif (TRM) protein, affects grain shape and weight in allohexaploid wheat. By editing the TaGW7 homoeologs in the B and D genomes, we showed that mutations in either of the two or both genomes increased the grain width and weight but reduced the grain length. The effect sizes of mutations in the TaGW7 gene homoeologs coincided with the relative levels of their expression in the B and D genomes. The effects of gene editing on grain morphology and weight traits were dosage dependent with the double-copy mutant showing larger effect than the respective single copy mutants. The TaGW7-centered gene co-expression network indicated that this gene is involved in the pathways regulating cell division and organ growth, also confirmed by the cellular co-localization of TaGW7 with α- and β-tubulin proteins, the building blocks of microtubule arrays. The analyses of exome capture data in tetraploid domesticated and wild emmer, and hexaploid wheat revealed the loss of diversity around TaGW7-associated with domestication selection, suggesting that TaGW7 is likely to play an important role in the evolution of yield component traits in wheat. Our study showed how integrating CRISPR-Cas9 system with cross-species comparison can help to uncover the function of a gene fixed in wheat for allelic variants targeted by domestication selection and select targets for engineering new gene variants for crop improvement.
-
A multiplex CRISPR/Cas9 platform for fast and efficient editing of multiple genes in Arabidopsis.
The recently developed CRISPR/Cas9 system is a promising technology for targeted genome editing in a variety of species including plants. However, the first generation systems were designed to target one or two gene loci at a time. We designed a new multiplex CRISPR/Cas9 system that allows the co-expression of six sgRNA modules in one binary vector using a simple (three steps) cloning strategy in Arabidopsis. The transcription of the sgRNA modules is under the control of three different RNA Polymerase III-dependent promoters. We tested the efficiency of the new multiplex system by targeting six of the fourteen PYL families of ABA receptor genes in a single transformation experiment. One line with mutations in all six targeted PYLs was identified from 15 T1 plants. The mutagenesis frequency for the six individual PYL targets in the T1 lines ranged from 13 to 93 %. In the presence of ABA, the transgenic line identified as containing mutations in all six PYL genes produced the highest germination rate in the T2 progeny (37 %). Among these germinated seedlings, half of the analyzed plants (15/30) were homozygous mutants for at least four targeted genes and two plants (6.7 %) contained homozygous mutations in five of the targeted PYLs and the other targeted PYL had biallelic mutations. Homozygous sextuple mutants were identified in the T3 progeny and characterized together with previously described triple and sextuple PYL mutants. We anticipate that the application of this multiplex CRISPR/Cas9 system will strongly facilitate functional analysis of genes pathways and families.
-
Precision in Action: The Role of Clustered Regularly Interspaced Short Palindromic Repeats/Cas in Gene Therapies.
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated enzyme-CAS holds great promise for treating many uncured human diseases and illnesses by precisely correcting harmful point mutations and disrupting disease-causing genes. The recent Food and Drug Association (FDA) approval of the first CRISPR-based gene therapy for sickle cell anemia marks the beginning of a new era in gene editing. However, delivering CRISPR specifically into diseased cells in vivo is a significant challenge and an area of intense research. The identification of new CRISPR/Cas variants, particularly ultra-compact CAS systems with robust gene editing activities, paves the way for the low-capacity delivery vectors to be used in gene therapies. CRISPR/Cas technology has evolved beyond editing DNA to cover a wide spectrum of functionalities, including RNA targeting, disease diagnosis, transcriptional/epigenetic regulation, chromatin imaging, high-throughput screening, and new disease modeling. CRISPR/Cas can be used to engineer B-cells to produce potent antibodies for more effective vaccines and enhance CAR T-cells for the more precise and efficient targeting of tumor cells. However, CRISPR/Cas technology has challenges, including off-target effects, toxicity, immune responses, and inadequate tissue-specific delivery. Overcoming these challenges necessitates the development of a more effective and specific CRISPR/Cas delivery system. This entails strategically utilizing specific gRNAs in conjunction with robust CRISPR/Cas variants to mitigate off-target effects. This review seeks to delve into the intricacies of the CRISPR/Cas mechanism, explore progress in gene therapies, evaluate gene delivery systems, highlight limitations, outline necessary precautions, and scrutinize the ethical considerations associated with its application.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.