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: 26 147
Страница 516 из 1000-
Application of new breeding techniques in fruit trees.
Climate change and rapid adaption of invasive pathogens pose a constant pressure on the fruit industry to develop improved varieties. Aiming to accelerate the development of better-adapted cultivars, new breeding techniques have emerged as a promising alternative to meet the demand of a growing global population. Accelerated breeding, cisgenesis, and CRISPR/Cas genome editing hold significant potential for crop trait improvement and have proven to be useful in several plant species. This review focuses on the successful application of these technologies in fruit trees to confer pathogen resistance and tolerance to abiotic stress and improve quality traits. In addition, we review the optimization and diversification of CRISPR/Cas genome editing tools applied to fruit trees, such as multiplexing, CRISPR/Cas-mediated base editing and site-specific recombination systems. Advances in protoplast regeneration and delivery techniques, including the use of nanoparticles and viral-derived replicons, are described for the obtention of exogenous DNA-free fruit tree species. The regulatory landscape and broader social acceptability for cisgenesis and CRISPR/Cas genome editing are also discussed. Altogether, this review provides an overview of the versatility of applications for fruit crop improvement, as well as current challenges that deserve attention for further optimization and potential implementation of new breeding techniques.
-
Combating aggressive weeds: Reinforcing herbicide resistance in pigeonpea (Cajanus cajan L.) through genome editing.
Pigeonpea (Cajanus cajan L.) is a drought-tolerant, tropical grain legume, rich in dietary proteins, vitamins, and micronutrients. However, the longstanding problem of weed infestation in the fields is a major constraint that significantly hampers the productivity of pigeonpea. Glyphosate, a widely used post-emergent, broad-spectrum, systemic herbicide, has emerged as an effective weed management strategy at the field level. It inhibits the chloroplastic enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in the shikimate pathway by competitively inhibiting its substrate, phosphoenol pyruvate (PEP), thus curtailing the biosynthesis of essential aromatic amino acids (phenylalanine, tyrosine, and tryptophan). This makes glyphosate lethal to weeds and the main crop as well. To address this susceptibility towards glyphosate, we developed glyphosate-resistant pigeonpea plants by modifying the PEP-binding site within the native CcEPSPS enzyme at positions 182G-to-A, 183T-to-I, and 187P-to-S, for reducing glyphosate binding affinity. The targeted base-editing was achieved using the CRISPR-Cas9-based homology-directed repair (HDR) technique. T2-edited plants harbouring mCcEPSPS exhibited stable inheritance of the these GATIPS mutations, reduced glyphosate binding affinity, and maintained optimal photosynthetic and agronomic parameters post-glyphosate application. The mCcEPSPS enzyme efficiently catalysed the transformation of PEP and S3P to EPSP and demonstrated in vitro resistance to glyphosate. In contrast to treated control (TC) plants, the edited plants possessed excellent photosynthetic, agronomic, and physiological metrics following a post-foliar Roundup (6 ml/L) spray. This work offers the first efficient and precise gene-editing report in pigeonpea, offering an effective, sustainable strategy for broad-spectrum weed management to mitigate both quantitative and qualitative crop losses.
-
Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA.
DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G-editing TALEDs but not C-to-T-editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by >99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.
-
Repairing a deleterious domestication variant in a floral regulator gene of tomato by base editing.
Crop genomes accumulate deleterious mutations-a phenomenon known as the cost of domestication. Precision genome editing has been proposed to eliminate such potentially harmful mutations; however, experimental demonstration is lacking. Here we identified a deleterious mutation in the tomato transcription factor SUPPRESSOR OF SP2 (SSP2), which became prevalent in the domesticated germplasm and diminished DNA binding to genome-wide targets. We found that the action of SSP2 is partially redundant with that of its paralog SSP in regulating shoot and inflorescence architecture. However, redundancy was compromised during tomato domestication and lost completely in the closely related species Physalis grisea, in which a single ortholog regulates shoot branching. We applied base editing to directly repair the deleterious mutation in cultivated tomato and obtained plants with compact growth that provide an early fruit yield. Our work shows how deleterious variants have sensitized modern genotypes for phenotypic tuning and illustrates how repairing deleterious mutations with genome editing may allow predictable crop improvement.
-
Recent advances in therapeutic gene-editing technologies.
The advent of gene-editing technologies, particularly CRISPR-based systems, has revolutionized the landscape of biomedical research and gene therapy. Ongoing research in gene editing has led to the rapid iteration of CRISPR technologies, such as base and prime editors, enabling precise nucleotide changes without the need for generating harmful double-strand breaks (DSBs). Furthermore, innovations such as CRISPR fusion systems with DNA recombinases, DNA polymerases, and DNA ligases have expanded the size limitations for edited sequences, opening new avenues for therapeutic development. Beyond the CRISPR system, mobile genetic elements (MGEs) and epigenetic editors are emerging as efficient alternatives for precise large insertions or stable gene manipulation in mammalian cells. These advances collectively set the stage for next-generation gene therapy development. This review highlights recent developments of genetic and epigenetic editing tools and explores preclinical innovations poised to advance the field.
-
On-target and off-target activities of CRISPR therapeutics across scales.
Recent FDA-approved gene-editing therapies illustrate not only the transformative potential of biotechnologies using CRISPR (clustered regularly interspaced short palindromic repeats)-derived ribonucleoproteins in treating a broad range of diseases but also the spectrum of possible molecular variations CRISPR therapeutics can adopt. These include exagamglogene autotemcel, an ex vivo therapy for hemoglobinopathies using CRISPR nuclease Cas9, and kayjayguran abengcemeran, an in vivo therapy using a protospacer adjacent motif-altered base-editing Cas9 variant for an ultra-rare metabolic disorder. Together, these therapies underscore how far CRISPR has advanced beyond its original use as a tool in biological/biomedical research. In this opinion article, we argue that as CRISPR biotechnologies advance beyond the relative simplicity of in vitro applications, our understanding must also evolve to address the challenges of optimizing 'on-target' and 'off-target' mutational activities across the diverse contexts in which they occur.
-
Production of Cell Models with Differential Zygosity Using CRISPR Base Editors.
CRISPR base editors have revolutionized the ease with which single-nucleotide gene editing can be performed in cell and organismal models. Unlike conventional CRISPR/Cas9 gene editing, which induces a double-strand DNA break and relies on endogenous homology-directed repair (HDR) or non-homologous end joining (NHEJ) repair, base editors only induce a single-strand nick and exploit the mismatch repair (MMR) system for repair. In many instances, this results in fewer off-target effects and less cytotoxicity. Moreover, because base editors use an alternative repair mechanism, the zygosity of repair is often distinct from that of traditional HDR-based editing. Here, we provide a detailed protocol for performing gene editing using an adenine base editor (ABE) to induce a single A-G transition mutation in a gene of therapeutic relevance. We also illustrate how to sort and isolate cells with differential zygosity state, enabling the production of cell models suited to the study of both dominant and recessive mutations. This workflow can easily be adapted to the use of other variants of base editors (e.g., cytosine base editors) and can be employed in diverse cell lines.
-
In vivo base editing rescues ADPKD in a humanized mouse model.
Autosomal dominant polycystic kidney disease (ADPKD) is a genetic kidney disease, caused by mutations of the PKD1 and PKD2 genes, characterized by the development of renal cysts and extrarenal complications, such as cardiac hypertrophy. Recently, a revolutionary approach, adeno-associated virus (AAV) delivered CRISPR-Cas9 gene editing, has been developed to treat inherited diseases. However, the use of this technology in kidney diseases in vivo is challenged. In this study, we adapt one of the gene editing systems, adenine base editor (ABE9), to develop a broadly expressed and a kidney-specific promoter mediated base editors, and test the effects of these two systems delivered by AAV9 on preventing disease in humanized Pkd1RC/RC mice carrying an arginine (R) to cystine (C) mutation that mimics a mutation in ADPKD patients. We show that one dose of the broadly expressed dual ABE9-AAV9 treatment corrects the pathogenic variant in kidneys, hearts and livers, and result in delaying cyst growth, decrease heart hypertrophy and improve liver function. To confirm the specificity of the base editor system in kidneys, we show that one dose of the kidney specific promoter mediated dual-ABE9-AAV9 treatment corrects the Pkd1 gene mutation in the kidney, and not in the heart, resulting in delaying cyst growth in Pkd1RC/RC kidneys, supporting a promising strategy of using base editor to target specific organs. Treatment with ABE9 base editors mediated by either the broadly expressed or kidney specific promoter increased the survival rate of Pkd1RC/null mice. These preclinical studies support a potential that single-dose genetic therapies may be through the correction of pathogenic variants to prevent ADPKD development in the clinic.
-
Massively parallel assessment of human variants with base editor screens.
Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.
-
nCas9 Engineering for Improved Target Interaction Presents an Effective Strategy to Enhance Base Editing.
Base editors (BEs) are a recent generation of genome editing tools that couple a cytidine or adenosine deaminase activity to a catalytically impaired Cas9 moiety (nCas9) to enable specific base conversions at the targeted genomic loci. Given their strong application potential, BEs are under active developments toward greater levels of efficiency and safety. Here, a previously overlooked nCas9-centric strategy is explored for enhancement of BE. Based on a cytosine BE (CBE), 20 point mutations associated with nCas9-target interaction are tested. Subsequently, from the initial positive X-to-arginine hits, combinatorial modifications are applied to establish further enhanced CBE variants (1.1-1.3). Parallel nCas9 modifications in other versions of CBEs including A3A-Y130F-BE4max, YEE-BE4max, CGBE, and split-AncBE4max, as well as in the context of two adenine BEs (ABE), likewise enhance their respective activities. The same strategy also substantially improves the efficiencies of high-fidelity nCas9/BEs. Further evidence confirms that the stabilization of nCas9-substrate interactions underlies the enhanced BE activities. In support of their translational potential, the engineered CBE and ABE variants respectively enable 82% and 25% higher rates of editing than the controls in primary human T-cells. This study thus demonstrates a highly adaptable strategy for enhancing BE, and for optimizing other forms of Cas9-derived tools.
PubMed отдаёт не больше 10 000 первых результатов — уточните запрос, чтобы увидеть остальные.