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: 26 147
Page 502 of 1000-
Advances in gene editing for legume improvement: technologies, progress, and prospects.
Legumes are among the most important protein-rich crops in global agri-food systems. To meet the rising protein demand of a growing population, significant efforts are underway to enhance legume yield, nutritional quality, and resilience to environmental stresses through the manipulation of key genetic traits. Advanced technologies-including genetic engineering, gene editing, genomic selection, next-generation sequencing, single-cell genomics, and multi-omics-are accelerating legume improvement due to their high precision and efficiency. This review focuses on major gene-editing technologies, namely, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), TALENs (Transcription Activator-Like Effector Nucleases), ZFNs (Zinc Finger Nucleases), base editing (BE), and prime editing (PE), and their applications in key legume crops such as soybean (Glycine max), cowpea (Vigna unguiculata), chickpea (Cicer arietinum), groundnut (Arachis hypogaea), pea (Pisum sativum), barrel clover (Medicago truncatula), alfalfa (Medicago sativa), and Lotus japonicus. Among these platforms, CRISPR/Cas9 is the most widely adopted in legumes, largely due to its simplicity, versatility, and dependence on accurate genome sequence information and guide RNA (gRNA) design. Advances in next-generation sequencing and the growing availability of intuitive online gRNA design tools have streamlined CRISPR workflows, improving accessibility and precision. The present review indicates that CRISPR-P is the most used gRNA design tool in legume research, likely due to its early development for plant systems and integrated off-target prediction features. Therefore, alongside reviewing gene-editing applications, we emphasized the critical role of robust gRNA design tools as a foundation for successful genome editing. Future integration of artificial intelligence and large language models is expected to further enhance target prediction accuracy, minimize off-target effects, and enable more precise genome-editing strategies in legumes.
-
Having a Same Type IIS Enzyme's Restriction Site on Guide RNA Sequence Does Not Affect Golden Gate (GG) Cloning and Subsequent CRISPR/Cas Mutagenesis.
Golden gate/modular cloning facilitates faster and more efficient cloning by utilizing the unique features of the type IIS restriction enzymes. However, it is known that targeted insertion of DNA fragment(s) must not include internal type IIS restriction recognition sites. In the case of cloning CRISPR constructs by using golden gate (GG) cloning, this narrows down the scope of guide RNA (gRNA) picks because the selection of a good gRNA for successful genome editing requires some obligation of fulfillment, and it is unwanted if a good gRNA candidate cannot be picked only because it has an internal type IIS restriction recognition site. In this article, we have shown that the presence of a type IIS restriction recognition site in a gRNA does not affect cloning and subsequent genome editing. After each step of GG reactions, correct insertions of gRNAs were verified by colony color and restriction digestion and were further confirmed by sequencing. Finally, the final vector containing a Cas12a nuclease and four gRNAs was used for Agrobacterium-mediated citrus cell transformation. Sequencing of PCR amplicons flanking gRNA-2 showed a substitution (C to T) mutation in transgenic plants. The knowledge derived from this study could widen the scope of GG cloning, particularly of gRNAs selection for GG-mediated cloning into CRISPR vectors.
-
CRISPR base editor-based targeted random mutagenesis (BE-TRM) toolbox for directed evolution.
Directed evolution (DE) of desired locus by targeted random mutagenesis (TRM) tools is a powerful approach for generating genetic variations with novel or improved functions, particularly in complex genomes. TRM-based DE involves developing a mutant library of targeted DNA sequences and screening the variants for the desired properties. However, DE methods have for a long time been confined to bacteria and yeasts. Lately, CRISPR/Cas and DNA deaminase-based tools that circumvent enduring barriers such as longer life cycle, small library sizes, and low mutation rates have been developed to facilitate DE in native genetic environments of multicellular organisms. Notably, deaminase-based base editing-TRM (BE-TRM) tools have greatly expanded the scope and efficiency of DE schemes by enabling base substitutions and randomization of targeted DNA sequences. BE-TRM tools provide a robust platform for the continuous molecular evolution of desired proteins, metabolic pathway engineering, creation of a mutant library of desired locus to evolve novel functions, and other applications, such as predicting mutants conferring antibiotic resistance. This review provides timely updates on the recent advances in BE-TRM tools for DE, their applications in biology, and future directions for further improvements. [BMB Reports 2024; 57(1): 30-39].
-
Rescue of hearing by adenine base editing in a humanized mouse model of Usher syndrome type 1F.
Usher syndrome type 1F (USH1F), characterized by congenital lack of hearing and balance and progressive loss of vision, is caused by mutations in the PCDH15 gene. In the Ashkenazi population, a recessive truncation mutation accounts for a large proportion of USH1F cases. The truncation is caused by a single C→T mutation, which converts an arginine codon to a stop (R245X). To test the potential for base editors to revert this mutation, we developed a humanized Pcdh15R245X mouse model for USH1F. Mice homozygous for the R245X mutation were deaf and exhibited profound balance deficits, while heterozygous mice were unaffected. Here we show that an adenine base editor (ABE) is capable of reversing the R245X mutation to restore the PCDH15 sequence and function. We packaged a split-intein ABE into dual adeno-associated virus (AAV) vectors and delivered them into cochleas of neonatal USH1F mice. Hearing was not restored in a Pcdh15 constitutive null mouse despite base editing, perhaps because of early disorganization of cochlear hair cells. However, injection of vectors encoding the split ABE into a late-deletion conditional Pcdh15 knockout rescued hearing. This study demonstrates the ability of an ABE to correct the PCDH15 R245X mutation in the cochlea and restore hearing.
-
Eliminating base-editor-induced genome-wide and transcriptome-wide off-target mutations.
The fusion of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) capable of programmable C-to-T editing, which has potential in clinical applications but suffers from off-target (OT) mutations. Here, we used a cleavable deoxycytidine deaminase inhibitor (dCDI) domain to construct a transformer BE (tBE) system that induces efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, the tBE remains inactive at OT sites with the fusion of a cleavable dCDI, therefore eliminating unintended mutations. When binding at on-target sites, the tBE is transformed to cleave off the dCDI domain and catalyses targeted deamination for precise base editing. After delivery into mice through a dual-adeno-associated virus (AAV) system, the tBE system created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9, resulting in a ~30-40% decrease in total cholesterol. The development of tBE establishes a highly specific base editing system and its in vivo efficacy has potential for therapeutic applications.
-
Base editing: advances and therapeutic opportunities.
Base editing - the introduction of single-nucleotide variants (SNVs) into DNA or RNA in living cells - is one of the most recent advances in the field of genome editing. As around half of known pathogenic genetic variants are due to SNVs, base editing holds great potential for the treatment of numerous genetic diseases, through either temporary RNA or permanent DNA base alterations. Recent advances in the specificity, efficiency, precision and delivery of DNA and RNA base editors are revealing exciting therapeutic opportunities for these technologies. We expect the correction of single point mutations will be a major focus of future precision medicine.
-
Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli.
While there are several genome editing techniques available, few are suitable for dynamic and simultaneous mutagenesis of arbitrary targeted sequences in prokaryotes. Here, to address these limitations, we present a versatile and multiplex retron-mediated genome editing system (REGES). First, through systematic optimization of REGES, we achieve efficiency of ∼100%, 85 ± 3%, 69 ± 14% and 25 ± 14% for single-, double-, triple- and quadruple-locus genome editing, respectively. In addition, we employ REGES to generate pooled and barcoded variant libraries with degenerate RBS sequences to fine-tune the expression level of endogenous and exogenous genes, such as transcriptional factors to improve ethanol tolerance and biotin biosynthesis. Finally, we demonstrate REGES-mediated continuous in vivo protein evolution, by combining retron, polymerase-mediated base editing and error-prone transcription. By these case studies, we demonstrate REGES as a powerful multiplex genome editing and continuous evolution tool with broad applications in synthetic biology and metabolic engineering.
-
Strategies to develop climate-resilient chili peppers: transcription factor optimization through genome editing.
Chili peppers (Capsicum spp.), a globally significant crop revered for their nutritional, economic, and cultural importance, are increasingly imperiled by the converging burdens of climate-induced abiotic stresses, including drought, heat, and salinity, and relentless biotic assaults from pathogens and insect herbivores. These overlapping stressors not only destabilize yield but also compromise the metabolic intricacy responsible for the accumulation of health-promoting secondary metabolites. Although Capsicum exhibits remarkable genetic and phytochemical diversity, the integrated transcriptional, metabolic, and epigenetic frameworks that underpin its stress resilience remain poorly delineated. This review synthesizes recent advances in decoding core transcription factor families, such as CaNAC, CaWRKY, and CaMYB, that serve as pivotal regulators of osmotic adjustment, reactive oxygen species detoxification, hormonal crosstalk, and secondary metabolite biosynthesis under stress conditions. We further highlight how multi-omics-guided gene discovery, when paired with CRISPR/Cas-mediated genome editing, enables precise reprogramming of key regulatory loci to enhance adaptive responses. Emerging innovations, including base editing, prime editing, and novel nucleases like Cas12a and Cas13d, are expanding the functional genome-editing landscape, while the integration of morphogenic regulators and genotype-independent transformation platforms is beginning to circumvent long-standing obstacles in Capsicum genetic engineering. Lastly, we propose a transformative framework that converges transcription factor modulation, multi-omics strategies, precision phenotyping, and next-generation genome editing to accelerate the development of climate-resilient Capsicum cultivars with optimized metabolic traits. This strategic convergence of molecular insight and biotechnological innovation offers a robust foundation for building next-generation chili pepper varieties capable of withstanding intensifying environmental and pathogenic pressures, ultimately safeguarding yield, nutritional quality, and agricultural sustainability in the face of global climate change.
-
Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs.
Canonical CRISPR-knockout (KO) screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate targeted gene KOs. These methodologies may yield distorted results because DSB-associated effects are often falsely assumed to be consequences of gene perturbation itself, especially when high copy-number sites are targeted. In the present study, we report a DSB-independent, genome-wide CRISPR screening method, termed iBARed cytosine base editing-mediated gene KO (BARBEKO). This method leverages CRISPR cytosine base editors for genome-scale KO screens by perturbing gene start codons or splice sites, or by introducing premature termination codons. Furthermore, it is integrated with iBAR, a strategy we devised for improving screening quality and efficiency. By constructing such a cell library through lentiviral infection at a high multiplicity of infection (up to 10), we achieved efficient and accurate screening results with substantially reduced starting cells. More importantly, in comparison with Cas9-mediated fitness screens, BARBEKO screens are no longer affected by DNA cleavage-induced cytotoxicity in HeLa-, K562- or DSB-sensitive retinal pigmented epithelial 1 cells. We anticipate that BARBEKO offers a valuable tool to complement the current CRISPR-KO screens in various settings.
-
Engineering miniature IscB nickase for robust base editing with broad targeting range.
IscB has a similar domain organization to Cas9, but the small size of IscB is better suited for delivery by adeno-associated virus. To improve the low editing efficiency of OgeuIscB (IscB from human gut metagenome) in mammalian cells, we developed high-efficiency miniature base editors by engineering OgeuIscB nickase and its cognate ωRNA, termed IminiBEs. We demonstrated the robust editing efficiency of IminiCBE (67% on average) or IminiABE (52% on average). Fusing non-specific DNA-binding protein Sso7d to IminiBEs increased the editing efficiency of low-efficiency sites by around two- to threefold, and we termed it SIminiBEs. In addition, IminiCBE and SIminiCBE recognize NNRR, NNRY and NNYR target-adjacent motifs, which broaden the canonical NWRRNA target-adjacent motif sites for the wild-type IscB nickase. Overall, IminiBEs and SIminiBEs are efficient miniature base editors for site-specific genomic mutations.
PubMed returns only the first 10,000 results — refine the query to see the rest.