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: 26 149
Page 566 of 1000-
CRISPR/Cas9 editing of three CRUCIFERIN C homoeologues alters the seed protein profile in Camelina sativa.
BACKGROUND: The oilseed Camelina sativa is grown for a range of applications, including for biofuel, biolubricants, and as a source of omega-3 fatty acids for the aquaculture feed industry. The seed meal co-product is used as a source of protein for animal feed; however, the low value of the meal hinders profitability and more widespread application of camelina. The nutritional quality of the seed meal is largely determined by the abundance of specific seed storage proteins and their amino acid composition. Manipulation of seed storage proteins has been shown to be an effective means for either adjustment of nutritional content of seeds or for enhancing accumulation of high-value recombinant proteins in seeds. RESULTS: CRISPR/Cas9 gene editing technology was used to generate deletions in the first exon of the three homoeologous genes encoding the seed storage protein CRUCIFERIN C (CsCRUC), creating an identical premature stop-codon in each and resulting in a CsCRUC knockout line. The mutant alleles were detected by applying a droplet digital PCR drop-off assay. The quantitative nature of this technique is particularly valuable when applied to polyploid species because it can accurately determine the number of mutated alleles in a gene family. Loss of CRUC protein did not alter total seed protein content; however, the abundance of other cruciferin isoforms and other seed storage proteins was altered. Consequently, seed amino acid content was significantly changed with an increase in the proportion of alanine, cysteine and proline, and decrease of isoleucine, tyrosine and valine. CsCRUC knockout seeds did not have changed total oil content, but the fatty acid profile was significantly altered with increased relative abundance of all saturated fatty acids. CONCLUSIONS: This study demonstrates the plasticity of the camelina seed proteome and establishes a CRUC-devoid line, providing a framework for modifying camelina seed protein composition. The results also illustrate a possible link between the composition of the seed proteome and fatty acid profile.
-
Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression.
BACKGROUND: Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice over a TFBS called a CArG box in the Tspan2 promoter. RESULTS: Quantitative RT-PCR showed loss of Tspan2 mRNA in aorta and bladder, but not heart or brain, of mice homozygous for an HDR-mediated three base pair substitution in the Tspan2 CArG box. Using the same protospacer, mice homozygous for a PE2-mediated single-base substitution in the Tspan2 CArG box displayed similar cell-specific loss of Tspan2 mRNA; expression of an overlapping long noncoding RNA was also nearly abolished in aorta and bladder. Immuno-RNA fluorescence in situ hybridization validated loss of Tspan2 in vascular smooth muscle cells of HDR and PE2 CArG box mutant mice. Targeted sequencing demonstrated variable frequencies of on-target editing in all PE2 and HDR founders. However, whereas no on-target indels were detected in any of the PE2 founders, all HDR founders showed varying levels of on-target indels. Off-target analysis by targeted sequencing revealed mutations in many HDR founders, but none in PE2 founders. CONCLUSIONS: PE2 directs high-fidelity editing of a single base in a TFBS leading to cell-specific loss in expression of an mRNA/long noncoding RNA gene pair. The PE2 platform expands the genome editing toolbox for modeling and correcting relevant noncoding SNVs in the mouse.
-
CRISPR Applications in Alzheimer's Disease: From High-Throughput Genetic Screening to Precision Editing and CNS Delivery.
Alzheimer's disease is a devastating progressive neurodegenerative disorder characterized by extracellular amyloid-beta plaques and intracellular tau tangles. Despite recent advancements in amyloid-beta-targeting immunotherapies, achieving safe and definitive disease control remains a profound clinical challenge. The CRISPR/Cas9 system has emerged as a powerful technology for precision neurogenetics, offering significant potential to address the fundamental questions behind Alzheimer's disease. This comprehensive review delineates the trajectory of CRISPR applications in Alzheimer's disease research and therapeutics. First, we explore the integration of CRISPR in engineering high-fidelity in vitro models, such as isogenic induced pluripotent stem cells and three-dimensional cerebral organoids, alongside advanced in vivo mammalian models. Second, we examine how these platforms facilitate unbiased high-throughput genetic screening to uncover molecular underpinnings regulating tau, lipid metabolism, and neuroinflammation. Third, we critically evaluate precision editing strategies targeting core risk genes (APP, MAPT, APOE, and TREM2), explicitly highlighting the severe physiopathological trade-offs between therapeutic efficacy and loss-of-function toxicity. Finally, we address the ultimate translational bottlenecks impeding clinical application. By dissecting the packaging limits of adeno-associated viral vectors and the physical barricade of the blood-brain barrier, we underscore the necessity of transitioning toward next-generation base editors and non-viral lipid nanoparticles to realize safe and efficacious in vivo clinical gene therapies against Alzheimer's disease.
-
RNAi and genome editing of sugarcane: Progress and prospects.
Sugarcane, which provides 80% of global table sugar and 40% of biofuel, presents unique breeding challenges due to its highly polyploid, heterozygous, and frequently aneuploid genome. Significant progress has been made in developing genetic resources, including the recently completed reference genome of the sugarcane cultivar R570 and pan-genomic resources from sorghum, a closely related diploid species. Biotechnological approaches including RNA interference (RNAi), overexpression of transgenes, and gene editing technologies offer promising avenues for accelerating sugarcane improvement. These methods have successfully targeted genes involved in important traits such as sucrose accumulation, lignin biosynthesis, biomass oil accumulation, and stress response. One of the main transformation methods-biolistic gene transfer or Agrobacterium-mediated transformation-coupled with efficient tissue culture protocols, is typically used for implementing these biotechnology approaches. Emerging technologies show promise for overcoming current limitations. The use of morphogenic genes can help address genotype constraints and improve transformation efficiency. Tissue culture-free technologies, such as spray-induced gene silencing, virus-induced gene silencing, or virus-induced gene editing, offer potential for accelerating functional genomics studies. Additionally, novel approaches including base and prime editing, orthogonal synthetic transcription factors, and synthetic directed evolution present opportunities for enhancing sugarcane traits. These advances collectively aim to improve sugarcane's efficiency as a crop for both sugar and biofuel production. This review aims to discuss the progress made in sugarcane methodologies, with a focus on RNAi and gene editing approaches, how RNAi can be used to inform functional gene targets, and future improvements and applications.
-
Precision genome editing using combinatorial viral vector delivery of CRISPR-Cas9 nucleases and donor DNA constructs.
Genome editing based on programmable nucleases and donor DNA constructs permits introducing specific base-pair changes and complete transgenes or live-cell reporter tags at predefined chromosomal positions. A crucial requirement for such versatile genome editing approaches is, however, the need to co-deliver in an effective, coordinated and non-cytotoxic manner all the required components into target cells. Here, adenoviral (AdV) and adeno-associated viral (AAV) vectors are investigated as delivery agents for, respectively, engineered CRISPR-Cas9 nucleases and donor DNA constructs prone to homologous recombination (HR) or homology-mediated end joining (HMEJ) processes. Specifically, canonical single-stranded and self-complementary double-stranded AAVs served as sources of ectopic HR and HMEJ substrates, whilst second- and third-generation AdVs provided for matched CRISPR-Cas9 nucleases. We report that combining single-stranded AAV delivery of HR donors with third-generation AdV transfer of CRISPR-Cas9 nucleases results in selection-free and precise whole transgene insertion in large fractions of target-cell populations (i.e. up to 93%) and disclose that programmable nuclease-induced chromosomal breaks promote AAV transduction. Finally, besides investigating relationships between distinct AAV structures and genome-editing performance endpoints, we further report that high-fidelity CRISPR-Cas9 nucleases are critical for mitigating off-target chromosomal insertion of defective AAV genomes known to be packaged in vector particles.
-
Harnessing CRISPR to combat human viral infections.
CRISPR/Cas9 is a technology that allows for targeted and precise genome editing in eukaryotic cells. The technique has changed the landscape of molecular biology and may be applied to repair genetic disorders in future therapies. Besides targeting the human genome, it can be used to cleave and edit viral DNA present in infected cells, and as such provides a promising new strategy for anti-viral therapy. Here, we discuss recent studies on the use of anti-viral CRISPRs to target pathogenic human viruses, with a focus on in vivo studies, challenges, and potential for future clinical applications.
-
Characterization and engineering of a type IV-A3 CRISPR-Cas system for genome editing in Escherichia coli.
CRISPR-Cas systems have revolutionized genome engineering technologies, but type IV CRISPR-Cas systems and their genome engineering potential have been critically underexplored. In this study, we identified a type IV-A3 CRISPR-Cas system from a clinical Klebsiella pneumoniae isolate and characterized its plasmid targeting activity and capacity to suppress chromosomal and plasmid gene expression in Escherichia coli. We revealed the pivotal role of Csf3 (Cas5) and the dispensable roles of Csf1 (Cas8-like) and Csf4 (DinG helicase) subunits in IV-A3 CRISPR-Cas complex formation. The system prevents plasmid propagation via interplay between DinG helicase activity and strategic protospacer positioning relative to plasmid replication and maintenance components. We enabled the IV-A3 CRISPR-Cas system to introduce lethal, sequence-specific double-stranded (ds)DNA breaks in the E. coli chromosome by fusing the nuclease domain of the I-TevI nuclease to the Cas8 N-terminus. Further, we developed a series of base editors, with various editing efficiencies and windows, by fusing the PmCDA1 cytidine deaminase to the Cas8, Cas5, and DinG subunits. Finally, conjugative transfer of the Cas5-PmCDA1 base editor into E. coli deactivated the tryptophan repressor gene, boosting IAA production. Our study provides new insights into type IV-A3 CRISPR-Cas systems and highlights their potential in genome engineering applications.
-
CRISPR-M: Predicting sgRNA off-target effect using a multi-view deep learning network.
Using the CRISPR-Cas9 system to perform base substitutions at the target site is a typical technique for genome editing with the potential for applications in gene therapy and agricultural productivity. When the CRISPR-Cas9 system uses guide RNA to direct the Cas9 endonuclease to the target site, it may misdirect it to a potential off-target site, resulting in an unintended genome editing. Although several computational methods have been proposed to predict off-target effects, there is still room for improvement in the off-target effect prediction capability. In this paper, we present an effective approach called CRISPR-M with a new encoding scheme and a novel multi-view deep learning model to predict the sgRNA off-target effects for target sites containing indels and mismatches. CRISPR-M takes advantage of convolutional neural networks and bidirectional long short-term memory recurrent neural networks to construct a three-branch network towards multi-views. Compared with existing methods, CRISPR-M demonstrates significant performance advantages running on real-world datasets. Furthermore, experimental analysis of CRISPR-M under multiple metrics reveals its capability to extract features and validates its superiority on sgRNA off-target effect predictions.
-
CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts.
Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.
-
Unlocking loxP to Track Genome Editing In Vivo.
The development of CRISPR-associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly quantify and monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses. Here we report a new CRISPR "fingerprinting" approach to activating luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior cre recombinase (cre)-detector system and is designed for Cas editors able to target loxP including gRNAs for SaCas9 and ErCas12a. These CRISPRs cut specifically within loxP, an approach that is a departure from previous gene editing in vivo activity detection techniques that targeted adjacent stop sequences. In this sensor paradigm, CRISPR activity was monitored non-invasively in living cre reporter mice (FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, which will be referred to as LSL-luciferase and mT/mG throughout the paper) after intramuscular or intravenous hydrodynamic plasmid injections, demonstrating utility in two diverse organ systems. The same genome-editing event was examined at the cellular level in specific tissues by confocal microscopy to determine the identity and frequency of successfully genome-edited cells. Further, SaCas9 induced targeted editing at efficiencies that were comparable to cre, demonstrating high effective delivery and activity in a whole animal. This work establishes genome editing tools and models to track CRISPR editing in vivo non-invasively and to fingerprint the identity of targeted cells. This approach also enables similar utility for any of the thousands of previously generated loxP animal models.
PubMed returns only the first 10,000 results — refine the query to see the rest.