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: 26 084
Page 114 of 1000-
Hypercompact CRISPR-Cas12j2 (CasΦ) enables genome editing, gene activation, and epigenome editing in plants.
-
CRISPR/Cas9-based genome editing for simultaneous interference with gene expression and protein stability.
Interference with genes is the foundation of reverse genetics and is key to manipulation of living cells for biomedical and biotechnological applications. However, classical genetic knockout and transcriptional knockdown technologies have different drawbacks and offer no control over existing protein levels. Here, we describe an efficient genome editing approach that affects specific protein abundances by changing the rates of both RNA synthesis and protein degradation, based on the two cross-kingdom control mechanisms CRISPRi and the N-end rule for protein stability. In addition, our approach demonstrates that CRISPRi efficiency is dependent on endogenous gene expression levels. The method has broad applications in e.g. study of essential genes and antibiotics discovery.
-
Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.
Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage specificity, we generated fusions of catalytically inactive Cas9 and FokI nuclease (fCas9). DNA cleavage by fCas9 requires association of two fCas9 monomers that simultaneously bind target sites ∼15 or 25 base pairs apart. In human cells, fCas9 modified target DNA sites with >140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 'nickases', recently engineered variants that cleave only one DNA strand per monomer. The specificity of fCas9 was at least fourfold higher than that of paired nickases at loci with highly similar off-target sites. Target sites that conform to the substrate requirements of fCas9 occur on average every 34 bp in the human genome, suggesting the versatility of this approach for highly specific genome-wide editing.
-
Preclinical modeling highlights the therapeutic potential of hematopoietic stem cell gene editing for correction of SCID-X1.
Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all IL2RG (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the IL2RG-edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of IL2RG editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.
-
CRISPR-Cas9 Application in Canadian Public and Private Plant Breeding.
Plant-breeding technologies have expanded, accelerating breeding research beyond the confines of current regulations. The application of genome editing, such as CRISPR-Cas9, do not neatly fit into existing regulatory frameworks, creating uncertainty as to whether they can be regarded as conventionally developed varieties without further regulation. This research presents the current views of Canadian plant breeders based on a national survey of plant breeders. There is evidence that a review of existing regulations is required, as >60% anticipate the use of genome-editing technologies in the next few years. This paper reviews plant-breeding practices under the context of present plants with novel trait (PNT) regulations and where plant breeders place the use of CRISPR-Cas9 within the suite of available genome-editing options. This paper establishes when and why, or why not, breeders choose to introduce CRISPR-Cas9 into their research over other plant-breeding applications.
-
Evolving Regulatory Landscape for Genome-Edited Plants.
New plant breeding innovations, including CRISPR-mediated genome editing, are enhancing the development of new plant varieties, many of which are on the cusp of entering market places internationally. The lack of clear and consistent regulatory policies globally for genome-edited plants, and their products, may limit the adoption and commercialization of these products; In contrast to the positive trends of regulatory agencies in Argentina, Brazil, Colombia, Chile, and the United States, which are at the forefront of a wave of countries that are implementing policies facilitating the development of genome-edited plants, the European Union has adopted a more restrictive approach that considers any new mutagenesis technique developed after 2001 as subject to regulation as genetically modified plants.
-
Generating a Genome Editing Nuclease for Targeted Mutagenesis in Human Cells.
Gene targeting and editing is an essential tool for both basic research and clinical application such as gene therapy. Several endonucleases have been invented to fulfill these purposes, including zinc finger nucleases, TALEN, and CRISPR/Cas9. Although all of these systems can target DNA sequence with high efficiency, they also exert off-target effects and genotoxicity. The off-target effects might not hinder their usage in animal models because the correctly targeted cells can be selected for further studies. However, the off-target effects could cause mutations which may be damaging or cancerous to the patients. In this chapter, we describe a genome-editing nuclease method which relies on modifying specific amino acids on a monomeric endonuclease, I-SceI, to recognize a targeted sequence in the human genome. This nuclease is small in size and shows a much lower genotoxicity compared to other nucleases including CRISPR/Cas9.
-
One Cut to Change Them All: CRISPR/Cas, a Groundbreaking Tool for Genome Editing in Botrytis cinerea and Other Fungal Plant Pathogens.
CRISPR/Cas is a genome editing technology that has opened new dimensions in functional biology. In a recent publication, we presented a highly efficient CRISPR/Cas technique for Botrytis cinerea, which dramatically increases our options to mutagenize and modify single or multiple genes. In this Perspectives article, we describe the essential features of the method and demonstrate with several examples how it opens new avenues for unraveling the virulence mechanisms of Botrytis and other plant pathogenic fungi and can accelerate research for the identification of new antifungal compounds.
-
CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus.
CRISPR/Cas9 is a valuable tool for both basic and applied research that has been widely applied to different plant species. Nonetheless, a systematical assessment of the efficiency of this method is not available for the allotetraploid Brassica napus-an important oilseed crop. In this study, we examined the mutation efficiency of the CRISPR/Cas9 method for 12 genes and also determined the pattern, specificity and heritability of these gene modifications in B. napus. The average mutation frequency for a single-gene targeted sgRNA in the T0 generation is 65.3%. For paralogous genes located in conserved regions that were targeted by sgRNAs, we observed mutation frequencies that ranged from 27.6% to 96.6%. Homozygotes were readily found in T0 plants. A total of 48.2% of the gene mutations, including homozygotes, bi-alleles, and heterozygotes were stably inherited as classic Mendelian alleles in the next generation (T1) without any new mutations or reversions. Moreover, no mutation was found in the putative off-target sites among the examined T0 plants. Collectively, our results demonstrate that CRISPR/Cas9 is an efficient tool for creating targeted genome modifications at multiple loci that are stable and inheritable in B. napus. These findings open many doors for biotechnological applications in oilseed crops.
-
CRISPR Double Cutting through the Labyrinthine Architecture of 3D Genomes.
The genomes are organized into ordered and hierarchical topological structures in interphase nuclei. Within discrete territories of each chromosome, topologically associated domains (TADs) play important roles in various nuclear processes such as gene regulation. Inside TADs separated by relatively constitutive boundaries, distal elements regulate their gene targets through specific chromatin-looping contacts such as long-distance enhancer-promoter interactions. High-throughput sequencing studies have revealed millions of potential regulatory DNA elements, which are much more abundant than the mere ∼20,000 genes they control. The recently emerged CRISPR-Cas9 genome editing technologies have enabled efficient and precise genetic and epigenetic manipulations of genomes. The multiplexed and high-throughput CRISPR capabilities facilitate the discovery and dissection of gene regulatory elements. Here, we describe the applications of CRISPR for genome, epigenome, and 3D genome editing, focusing on CRISPR DNA-fragment editing with Cas9 and a pair of sgRNAs to investigate topological folding of chromatin TADs and developmental gene regulation.
PubMed returns only the first 10,000 results — refine the query to see the rest.