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: 26 081
Страница 113 из 1000-
Allele-Specific Knockout by CRISPR/Cas to Treat Autosomal Dominant Retinitis Pigmentosa Caused by the G56R Mutation in NR2E3.
Retinitis pigmentosa (RP) is an inherited retinal dystrophy that causes progressive vision loss. The G56R mutation in NR2E3 is the second most common mutation causing autosomal dominant (ad) RP, a transcription factor that is essential for photoreceptor development and maintenance. The G56R variant is exclusively responsible for all cases of NR2E3-associated adRP. Currently, there is no treatment for NR2E3-related or, other, adRP, but genome editing holds promise. A pertinent approach would be to specifically knockout the dominant mutant allele, so that the wild type allele can perform unhindered. In this study, we developed a CRISPR/Cas strategy to specifically knockout the mutant G56R allele of NR2E3 and performed a proof-of-concept study in induced pluripotent stem cells (iPSCs) of an adRP patient. We demonstrate allele-specific knockout of the mutant G56R allele in the absence of off-target events. Furthermore, we validated this knockout strategy in an exogenous overexpression system. Accordingly, the mutant G56R-CRISPR protein was truncated and mis-localized to the cytosol in contrast to the (peri)nuclear localizations of wild type or G56R NR2E3 proteins. Finally, we show, for the first time, that G56R iPSCs, as well as G56R-CRISPR iPSCs, can differentiate into NR2E3-expressing retinal organoids. Overall, we demonstrate that G56R allele-specific knockout by CRISPR/Cas could be a clinically relevant approach to treat NR2E3-associated adRP.
-
[Genome editing in plants directed by CRISPR/Cas ribonucleoprotein complexes].
The CRISPR/Cas system is the most popular genome editing technology in recent years and has been widely used in crop improvement. Compared with introducing the CRISPR/Cas system into plant cells with DNA constructs, introducing CRISPR/Cas ribonucleoprotein (RNP) to perform genome editing excels in rapid action, low off-target rates and is free of DNA insertions in editing plants. However, efficient delivery of CRISPR/Cas RNP into plant cells and achieving high editing frequency are still very challenging, which limits the extensive implementation of CRISPR/Cas RNP-mediated genome editing in plants. In this review, we summarize the progress of protein and RNP delivery methods in plant cells, and provide new perspectives of further development and future applications of the CRISPR/Cas RNP technology in plant genome editing.
-
A highly efficient in vivo plasmid editing tool based on CRISPR-Cas12a and phage λ Red recombineering.
-
gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing.
The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is one of the most powerful tools for genome engineering. However, some of the steps are laborious, reducing its usability. In this study, we have developed a simplified method, called the guide RNA-transient expression system (gRNA-TES), to deliver gRNA in yeast. In gRNA-TES, a DNA fragment containing the promoter and gRNA is prepared by two simple PCR steps and co-transformed with a DNA module into the host strain; all steps including PCR steps and yeast transformation are completed within 5-6 h in a single day, in contrast to conventional plasmid-based gRNA delivery systems, which require at least 3-4 days to construct and verify the gRNA-expressing plasmids. The performance of gRNA-TES was evaluated by the replacement of 150-kb, 200-kb, 300-kb, 400-kb, and 500-kb regions of yeast chromosome 4 with a DNA module. Increased numbers of transformants with a high frequency of expected replacement of even the 500-kb region were obtained with gRNA-TES as compared with transformation without gRNA-TES. In addition, the integrity of the replaced region was verified in 67%-100% of transformants tested by colony PCR. We believe that gRNA-TES will vastly increase the accessibility of CRISPR/Cas9 technology to biologists and biotechnologists by offering a simple, fast, and cost-effective tool to deliver gRNA in genome engineering. Furthermore, it might be applied to plant and animal systems if appropriate gene promoters are incorporated in the technology.
-
Multiplex genome editing in Ashbya gossypii using CRISPR-Cpf1.
CRISPR/Cas technologies constitute essential tools for rapid genome engineering of many organisms, including fungi. The CRISPR/Cas9 system adapted for the industrial fungus Ashbya gossypii enables efficient genome editing for the introduction of deletions, insertions and nucleotide substitutions. However, the Cas9 system is constrained by the existence of a specific 5'-NGG-3' PAM sequence in the target site. Here we present a new CRISPR/Cas system for A. gossypii that expands the molecular toolbox available for microbial engineering of this fungus. The use of Cpf1 nuclease from Lachnospiraceae bacterium allows a T-rich PAM sequence (5'-TTTN-3') to be employed and facilitates implementation of a multiplexing CRISPR/Cpf1 system adapted for A. gossypii. The system has been validated for the introduction of large deletions with five different auxotrophic markers (HIS3, ADE2, TRP1, LEU2 and URA3). The use of both crRNA and dDNA arrays in a multi-CRISPR/Cpf1 system is demonstrated to be an efficient strategy for multiplex gene deletion of up to four genes using a single multi-CRISPR/Cpf1 plasmid. Our results also suggest that the selection of the target sequence may affect significantly the editing efficiency of the system.
-
A Hitchhiker's guide to CRISPR editing tools in bacteria : CRISPR can help unlock the bacterial world, but technical and regulatory barriers persist.
Join us on a journey through the complex and ever-expanding universe of CRISPR approaches for genome editing in bacteria. Discover what is available, current technical challenges, successful implementation of these tools and the regulatory framework around their use. [Image: see text]
-
Manipulation of a New Non-model Insect Genome Using Targeted CRISPR-Era Approaches.
Site-specific genome editing is the most direct way to test gene function. When CRISPR-Cas9 was introduced for the editing of eukaryotic genomes, entomologists were ready with questions but had many methodologies to forge for the approach to be useful. Now, roughly 45 non-model insect genomes have been edited to study processes such as insecticide resistance, olfaction, immunity, and development. A useful first step for gene editing in an insect species of interest is identification and targeted editing of a gene with a visible phenotype. Visible markers increase the efficiency of detection of a genetic change; a wide availability of markers is one reason why model insects are so easy to manipulate and so have been key in understanding many biological processes. Here we will describe with detailed protocols how to approach a new insect species with CRISPR-era approaches by targeting a visual marker with Cas9-editing.
-
Targeted DNA methylation in pericentromeres with genome editing-based artificial DNA methyltransferase.
To study the impact of epigenetic changes on biological functions, the ability to manipulate the epigenetic status of certain genomic regions artificially could be an indispensable technology. "Epigenome editing" techniques have gradually emerged that apply TALE or CRISPR/Cas9 technologies with various effector domains isolated from epigenetic code writers or erasers such as DNA methyltransferase, 5-methylcytosine oxidase, and histone modification enzymes. Here we demonstrate that a TALE recognizing a major satellite, consisting of a repeated sequence in pericentromeres, could be fused with the bacterial CpG methyltransferase, SssI. ChIP-qPCR assays demonstrated that the fusion protein TALMaj-SssI preferentially bound to major chromosomal satellites in cultured cell lines. Then, TALMaj-SssI was expressed in fertilized mouse oocytes with hypomethylated major satellites (10-20% CpG islands). Bisulfite sequencing revealed that the DNA methylation status was increased specifically in major satellites (50-60%), but not in minor satellites or other repeat elements, such as Intracisternal A-particle (IAP) or long interspersed nuclear elements-1 (Line1) when the expression level of TALMaj-SssI is optimized in the cell. At a microscopic level, distal ends of chromosomes at the first mitotic stage were dramatically highlighted by the mCherry-tagged methyl CpG binding domain of human MBD1 (mCherry-MBD-NLS). Moreover, targeted DNA methylation to major satellites did not interfere with kinetochore function during early embryonic cleavages. Co-injection of dCas9 fused with SssI and guide RNA (gRNA) recognizing major satellite sequences enabled increment of the DNA methylation in the satellites, but a few off-target effects were also observed in minor satellites and retrotransposons. Although CRISPR can be applied instead of the TALE system, technical improvements to reduce off-target effects are required. We have demonstrated a new method of introducing DNA methylation without the need of other binding partners using the CpG methyltransferase, SssI.
-
Engineering of Multiple Modules to Improve Amorphadiene Production in Bacillus subtilis Using CRISPR-Cas9.
Engineering strategies to improve terpenoids' production in Bacillus subtilis mainly focus on 2C-methyl-d-erythritol-4-phosphate (MEP) pathway overexpression. To systematically engineer the chassis strain for higher amorphadiene (precursor of artemisinin) production, a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system was established in B. subtilis to facilitate precise and efficient genome editing. Then, this system was employed to engineer three more modules to improve amorphadiene production, including the terpene synthase module, the branch pathway module, and the central metabolic pathway module. Finally, our combination of all of the useful strategies within one strain significantly increased extracellular amorphadiene production from 81 to 116 mg/L after 48 h flask fermentation without medium optimization. For the first time, we attenuated the FPP-derived competing pathway to improve amorphadiene biosynthesis and investigated how the TCA cycle affects amorphadiene production in B. subtilis. Overall, this study provides a universal strategy for further increasing terpenoids' production in B. subtilis by comprehensive and systematic metabolic engineering.
-
CRAGE-CRISPR facilitates rapid activation of secondary metabolite biosynthetic gene clusters in bacteria.
With the advent of genome sequencing and mining technologies, secondary metabolite biosynthetic gene clusters (BGCs) within bacterial genomes are becoming easier to predict. For subsequent BGC characterization, clustered regularly interspaced short palindromic repeats (CRISPR) has contributed to knocking out target genes and/or modulating their expression; however, CRISPR is limited to strains for which robust genetic tools are available. Here we present a strategy that combines CRISPR with chassis-independent recombinase-assisted genome engineering (CRAGE), which enables CRISPR systems in diverse bacteria. To demonstrate CRAGE-CRISPR, we select 10 polyketide/non-ribosomal peptide BGCs in Photorhabdus luminescens as models and create their deletion and activation mutants. Subsequent loss- and gain-of-function studies confirm 22 secondary metabolites associated with the BGCs, including a metabolite from a previously uncharacterized BGC. These results demonstrate that the CRAGE-CRISPR system is a simple yet powerful approach to rapidly perturb expression of defined BGCs and to profile genotype-phenotype relationships in bacteria.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.