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The role of CRISPR in modern plant breeding: overcoming breeding barriers and legislative challenges through transgene-free genome editing.
Traditional plant breeding techniques-such as crossbreeding, mutation breeding, and marker-assisted selection-have significantly contributed to crop improvement over the past century. However, these methods are often limited by long breeding cycles, low precision, and the unintended transfer of undesirable traits. To address these challenges, transgenic breeding emerged as a powerful tool, enabling the introduction of specific foreign genes to confer desirable traits such as pest resistance or herbicide tolerance. While highly effective and precise, transgenic approaches face considerable regulatory and public acceptance barriers, particularly in regions with strict GMO legislation. The advent of CRISPR/Cas genome editing has revolutionized plant breeding by enabling precise, efficient, and targeted modification of native genes, significantly accelerating the development of improved crops. Among CRISPR-based methods, transgene-free genome editing has gained prominence for producing enhanced plant varieties without integrating foreign DNA, thus avoiding many regulatory constraints associated with GMOs. This review aims to provide a comprehensive overview of CRISPR-mediated genome editing technologies in crop innovation, emphasizing especially the use of transgene-free approaches in modern plant breeding and their transformative potential to overcome the limitations of conventional methods while providing a regulation-friendly pathway for crop improvement.
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The CRISPR Revolution: Unraveling the mysteries of Life's genetic code.
A biotechnological revolution is triggered by CRISPR-Cas systems' variety, measured quality, and proficiency. Identifying nucleic acid biomarkers, one of the methods that use CRISPR for diagnosis, is an extremely sensitive diagnostic method.A broad range of infectious and noninfecting diseases, mutations, and CRISPR deletions associated with genetic disorders have been detected using diagnostics. Furthermore, this technology is used to test proteins and micromolecules. We focus on how Cas proteins can be used to detect diseases in genes, agriculture, and cancer therapy. Furthermore, CRISPR technology has many negative impacts on the health of living organisms, environmental and population structures in spite of its numerous contributions to biomedical science. Therefore, an investigation into the impact of genome editing on nontargeted species is important for these reasons. CRISPR in the future is briefly discussed towards the end of this review.
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Prime Editing of Mouse Primary Neurons.
Prime editing is a hybrid genome editing technology that introduces small edits on the genome with high precision. It combines nickase Cas9 with reverse transcriptase to prime and synthesizes edited DNA from RNA, reducing unintended insertions and deletions on the genome. This protocol describes the design of prime editing guide RNAs (pegRNAs), cloning of plasmids, nucleofection of mouse primary neurons, and preparation for next-generation sequencing. Directions are given for pegRNA and PE3b gRNA design and construction using PegAssist, a publicly available webtool and plasmid set. Prime editing in neurons allows genome manipulation while maintaining endogenous gene expression, making it ideal for studying protein structure/function relationships and pathogenic variants in a native neuronal context.
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Detection of CRISPR/Cas9-Generated Off-Target Effect by Integration-Defective Lentiviral Vector.
Clustered regularly interspaced short palindromic repeat (CRISPR) and other gene editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) show great promises for research and therapeutic applications. One major concern is the off-target effects generated by these nucleases at unintended genomic sequences. In silico methods are usually used for off-target site prediction. However, based on currently available algorithms, the predicted cleavage activity at these potential off-target sites does not always reflect the true cleavage in vivo. Here we present an unbiased screening protocol using integration-defective lentiviral vector (IDLV) and deep sequencing to map the off-target sites generated by gene editing tools.
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Mitochondrial genome editing: another win for curiosity-driven research.
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Disgraced CRISPR-baby scientist's 'publicity stunt' frustrates researchers.
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Editing Candida: Origins and Advances of CRISPR Tools.
Pathogens causing candidiasis encompass a diverse group of ascomycetous yeasts that have become essential models for studying fungal adaptability, pathogenicity, and host-pathogen interactions. Although many candidiasis-promoting species exist as commensals within host microbiota, several have acquired virulence traits that enable opportunistic infections, positioning them as a leading cause of invasive fungal disease in humans. Deciphering the molecular and genetic determinants that underpin the biology of organisms responsible for candidiasis has long been a central objective in medical and molecular mycology. However, research progress has been constrained by intrinsic biological challenges, including noncanonical codon usage and the absence of a complete sexual cycle in diploid species, which have complicated traditional genetic manipulation. CRISPR-Cas9 genome editing has overcome many of these limitations, providing a precise, efficient, and versatile framework for targeted genomic modification. This system has facilitated functional genomic studies ranging from single-gene deletions to high-throughput mutagenesis, yielding new insights into the mechanisms governing virulence, antifungal resistance, and stress adaptation. Since its initial application in Candida albicans, CRISPR-Cas9 technology has been refined and adapted for other clinically and industrially relevant species, including Nakaseomyces glabratus (formerly referred to as Candida glabrata), Candida parapsilosis, and Candida auris. The present work provides an overview of the evolution of genetic approaches employed in research directed against candidiasis-associated species, with a particular focus on the development and optimization of CRISPR-based systems. It highlights how recent advancements have improved the genetic tractability of these pathogens and outlines emerging opportunities for both fundamental and applied studies in fungal biology.
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CRISPR/Cas9-Mediated Targeting of the Integrin Beta-2 Gene as a Gene-Editing Strategy for Leukocyte Adhesion Deficiency Type I.
BACKGROUND: Leukocyte adhesion deficiency (LAD) is a genetic immunodeficiency that severely impairs the leukocyte adhesion cascade and migration to the sites of infection and inflammation. As a result, the main consequence is recurrent and severe infections with bacteria and fungi, which can lead to death. The most common variant, LAD-I, is due to mutations in the integrin beta 2 (ITGB2) gene responsible for the β2 integrin (CD18) subunit. Corrective strategies based on gene therapy and genome editing are the most promising methods for treating genetic defects. METHODS: In the present experimental in vitro study conducted at Tarbiat Modares University, Tehran, Iran, in 2024, we designed a single-guide RNA (sgRNA) for the CRISPR/Cas9 system to target exon 6 of ITGB2 and performed validation experiments. We also investigated peptide nanocarriers as a potential strategy for delivering the CRISPR components. The PX458 plasmid encoding Cas9 and the designed sgRNA was delivered into HEK293T cells, followed by genome-editing analysis. RESULTS: Genome-editing analysis confirmed a measurable level of indel formation at the target locus. Furthermore, the results of MPG nanopeptide production and purification demonstrate the potential of peptide nanocarriers for genome-editing systems. CONCLUSION: Overall, this work establishes a foundation for subsequent knock-in and gene-correction studies in clinically relevant immune cell models, paving the way for potential LAD-I gene therapy applications.
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Challenges and progress related to gene editing in rare skin diseases.
Genodermatoses represent a large group of inherited skin disorders encompassing clinically-heterogeneous conditions that manifest in the skin and other organs. Depending on disease variant, associated clinical manifestations and secondary complications can severely impact patients' quality of life and currently available treatments are transient and not curative. Multiple emerging approaches using CRISPR-based technologies offer promising prospects for therapy. Here, we explore current advances and challenges related to gene editing in rare skin diseases, including different strategies tailored to mutation type and structural organization of the affected gene, considerations for in vivo and ex vivo applications, the critical issue of delivery into the skin, and immune aspects of therapy. Against the backdrop of a landmark FDA approval for the first re-dosable gene replacement therapy for a rare genetic skin disorder, gene editing approaches are inching closer to the clinics and the possibility of a local permanent cure for patients affected by these disorders.
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Bar-seq strategies for the LeishGEdit toolbox.
The number of fully sequenced genomes increases steadily but the function of many genes remains unstudied. To accelerate dissection of gene function in Leishmania spp. and other kinetoplastids we previously developed a streamlined pipeline for CRISPR-Cas9 gene editing, which we termed LeishGEdit. To facilitate high-throughput mutant screens we have adapted this pipeline by barcoding mutants with unique 17-nucleotide barcodes, allowing loss-of-function screens in mixed populations. Here we present primer design and analysis tools that facilitate these bar-seq strategies. We have developed a standalone easy-to-use pipeline to design CRISPR primers suitable for the LeishGEdit toolbox for any given genome and have generated a list of 14,995 barcodes. Barcodes and oligo sequences are now accessible through our website www.leishgedit.net allowing researchers to pursue bar-seq experiments in all currently available TriTrypDB genomes (release 41). This will streamline CRISPR bar-seq assays in kinetoplastids, enabling pooled mutant screens across the community.
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