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: 26 182
Page 942 of 1000-
An efficient hairy root system for validation of CRISPR/Cas system activities in cotton.
The hairy root induction system has been widely applied in studying gene expression and function in plant species due to its rapidity and efficiency. The hairy root system is an efficient tool for evaluating the activities of CRISPR/Cas systems. Cotton hairy roots were primarily induced through cotton tissue culture under aseptic conditions and by injecting cotton stem tips under non-aseptic conditions. However, both methods are lab-intensive and time-consuming. In this study, an efficient cotton hairy root induction procedure was established via infecting cotton hypocotyls with Agrobacterium rhizogenes under non-sterile conditions. Cotton seedlings with expanded cotyledons were decapitated with a slanted cut, and the residual hypocotyl (maintained 1 cm apical portion) was inoculated with A. rhizogenes. Over 90% of the infected explants from all three tested varieties could produce hairy roots after 8 days of inoculation. The effictiveness of the method was tested by overexpressing two reporter genes (eGFP and GUS). The transformation efficiency of the GUS and eGFP were ranged from50-68.18% and 40.9-68.18%. In addition, the editing efficiency of target sites in different CRISPR/Cas systems were also tested in hairy root. This method provided technical support for screening suitable target sites for cotton gene editing.
-
Creating Hypoallergenic and Low Antinutritional Soybeans through CRISPR/Cas12a-Mediated Multiplex Gene Editing.
Soybean protein is a valuable plant-based nutrition source for food. However, allergens and antinutritional factors like Gly Bd 30k (P34), Kunitz trypsin inhibitor (KTI), and soybean agglutinin (LE) pose risks. In this study, we employed the CRISPR/Cas12a system to generate hypoallergenic and low antinutritional soybean lines without agronomic traits penalties. The edited lines exhibited markedly reduced levels and enzymatic activity of trypsin inhibitors, as well as soybean agglutinin content. Feeding mice with these edited soybeans, deficient in P34, KTI, and LE proteins, significantly alleviated allergenic responses. This research provides a promising resource of hypoallergenic and low antinutritional soybeans for applications in food production, animal feed, and immunotherapy.
-
Generation of Knockout Human Primary Keratinocytes by CRISPR/Cas9.
The culture of epidermal human primary keratinocytes (HPKs) represents a well-established model in biological and dermatological research. In addition, HPKs are used in three-dimensional organotypic cultures (OTCs), and gene therapeutic approaches have been reported for the treatment of patients suffering from epidermolysis bullosa, a severe blistering disease that can result in postnatal lethality. Therefore, there is a strong need for the development of techniques for the stable and specific genetic manipulation of HPKs, for example, by genome editing via the CRISPR/Cas9 approach. However, the main disadvantage of working with HPKs is the fact that these cells are prone to terminal differentiation and proliferate only for few passages in monoculture. As it is well known that the co-culture of HPKs with fibroblasts strongly increases the lifetime of the epidermal cells, we developed a protocol for the stable modification of HPKs by CRISPR/Cas9 via lentiviral transduction in the presence of 3T3-J2 fibroblasts as feeder cells. Selection of transduced HPKs is achieved with antibiotics in co-culture with antibiotic-resistant feeder cells. Modified HPKs generated by our protocol have the potential to generate epidermis-like structures in OTCs.
-
[Metabolic syndrome: Towards cell therapy by using CRISPR/Cas9-engineered human adipocytes].
-
Oligo-Modified Poly(β-Amino Ester)-Mediated mRNA Delivery: A Protocol for Gene Editing Applications.
Oligopeptide-modified poly(β-amino ester)s (OM-pBAEs) are polymer-based delivery systems that enable the encapsulation and intracellular transport of nucleic acids, including messenger RNA (mRNA) and mixtures of mRNA with DNA. OM-pBAEs form cationic nanoparticles (NPs) through electrostatic interactions with nucleic acids, protecting them from degradation and enhancing cellular uptake. Terminal oligopeptides composed of lysine, arginine, or histidine enhance polyplex stability, nucleic acid binding, and endosomal escape. This chapter describes a protocol for preparing OM-pBAE nanoparticles, including polymer synthesis, peptide modification, controlled mixing with nucleic acids to form polyplexes, precipitation in RNase-free water, stabilization with excipients, and optional freeze-drying for long-term storage. Nanoparticle formation occurs at a constant temperature of 25°C, with incubation to ensure reproducible self-assembly. Nanoparticles are characterized by using dynamic light scattering (DLS) to determine particle size, polydispersity, and surface charge, while nucleic acid encapsulation efficiency is assessed to evaluate loading capacity. This protocol provides a versatile, nonviral platform for efficient nucleic acid delivery, supporting applications such as gene-editing experiments.
-
Targeting LncRNAs with CRISPR/Cas9 for Kidney Therapeutics: A Review.
Long noncoding RNAs (lncRNAs) have emerged as key players in the pathogenesis of kidney diseases, including acute kidney injury (AKI), AKI-to-chronic kidney disease (CKD) transition, CKD, diabetic kidney disease (DKD), renal cell carcinoma (RCC), polycystic kidney diseases (PKD), and lupus nephritis (LN). Although the roles of lncRNAs in disease progression have been investigated in preclinical models, their underlying mechanisms remain poorly understood. The therapeutic potential of lncRNA-based therapies remains largely unexplored in clinical settings. Recently, an advancement in clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology offers a novel strategy for treating sickle cell anemia and β-thalassemia. Additionally, CRISPR/Cas9 is currently being evaluated in clinical trials for various diseases, including kidney diseases like RCC. However, the application of CRISPR/Cas9 to target lncRNAs is still in the early stages. Preclinical experiments have revealed that CRISPR/Cas9 could effectively target lncRNAs in kidney disorders. However, its clinical translation in AKI and CKD conditions remains unclear, and various biological challenges remain to be addressed. This review aims to investigate advancements in CRISPR/Cas9 that target lncRNAs in the kidney, highlighting the limitations and future directions for advancing CRISPR/Cas9-based lncRNA therapy and translating these findings into clinical applications.
-
Insights Gained from RNA Editing Targeted by the CRISPR-Cas13 Family.
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, especially type II (Cas9) systems, have been widely developed for DNA targeting and formed a set of mature precision gene-editing systems. However, the basic research and application of the CRISPR-Cas system in RNA is still in its early stages. Recently, the discovery of the CRISPR-Cas13 type VI system has provided the possibility for the expansion of RNA targeting technology, which has broad application prospects. Most type VI Cas13 effectors have dinuclease activity that catalyzes pre-crRNA into mature crRNA and produces strong RNA cleavage activity. Cas13 can specifically recognize targeted RNA fragments to activate the Cas13/crRNA complex for collateral cleavage activity. To date, the Cas13X protein is the smallest effector of the Cas13 family, with 775 amino acids, which is a promising platform for RNA targeting due to its lack of protospacer flanking sequence (PFS) restrictions, ease of packaging, and absence of permanent damage. This study highlighted the latest progress in RNA editing targeted by the CRISPR-Cas13 family, and discussed the application of Cas13 in basic research, nucleic acid diagnosis, nucleic acid tracking, and genetic disease treatment. Furthermore, we clarified the structure of the Cas13 protein family and their molecular mechanism, and proposed a future vision of RNA editing targeted by the CRISPR-Cas13 family.
-
Advances toward Curing HIV-1 Infection in Tissue Reservoirs.
A disease of more than 39.6 million people worldwide, HIV-1 infection has no curative therapy. To date, one man has achieved a sterile cure, with millions more hoping to avoid the potential pitfalls of lifelong antiretroviral therapy and other HIV-related disorders, including neurocognitive decline. Recent developments in immunotherapies and gene therapies provide renewed hope in advancing efforts toward a sterilizing or functional cure. On the horizon is research concentrated in multiple separate but potentially complementary domains: vaccine research, viral transcript editing, T-cell effector response targeting including checkpoint inhibitors, and gene editing. Here, we review the concept of targeting the HIV-1 tissue reservoirs, with an emphasis on the central nervous system, and describe relevant new work in functional cure research and strategies for HIV-1 eradication.
-
A CRISPR-Cas9 system for multiple genome editing and pathway assembly in Candida tropicalis.
Genetic manipulation is among the most important tools for synthetic biology; however, modifying multiple genes is extremely time-consuming and can sometimes be impossible when dealing with gene families. Here, we present a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system for use in the diploid yeast Candida tropicalis that is vastly superior to traditional techniques. This system enables the rapid and reliable introduction of multiple genetic deletions or mutations, as well as a stable expression using an integrated CRISPR-Cas9 cassette or a transient CRISPR-Cas9 cassette, together with a short donor DNA. We further show that the system can be used to promote the in vivo assembly of multiple DNA fragments and their stable integration into a target locus (or loci) in C. tropicalis. Based on this system, we present a platform for the biosynthesis of β-carotene and its derivatives. These results enable the practical application of C. tropicalis and the application of the system to other organisms.
-
High-efficiency prime editing with optimized, paired pegRNAs in plants.
Prime editing (PE) applications are limited by low editing efficiency. Here we show that designing prime binding sites with a melting temperature of 30 °C leads to optimal performance in rice and that using two prime editing guide (peg) RNAs in trans encoding the same edits substantially enhances PE efficiency. Together, these approaches boost PE efficiency from 2.9-fold to 17.4-fold. Optimal pegRNAs or pegRNA pairs can be designed with our web application, PlantPegDesigner.
PubMed returns only the first 10,000 results — refine the query to see the rest.