Frontiers in genome editing最新文献

The advent of genetics, molecular biology, and genome sequencing has rapidly accelerated the development of elite genetic lines across various species, including poultry. It is now possible to introduce intra- or inter-species single nucleotide polymorphisms into chicken lines to enhance productivity. This advancement may mark the beginning of a new agricultural revolution, dramatically reducing the time required to improve poultry lines for commercial production environments. Transgenic technologies, including lentiviral vectors and piggyBac transposition, have enabled the generation of animals expressing exogenous genes. The emergence of new genome-editing tools is transforming avian biotechnology, allowing the creation of customized organisms for specific traits. Genome editing has become the most efficient method for studying gene function. First and second generation tools, such as zinc finger nucleases and transcription activator-like effector nucleases (TALENs), are limited by complex design and off-target effects. In contrast, the third generation Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9), represents a significant breakthrough. It encompasses guided RNA (gRNA) and the Cas9 endonuclease which together target specific DNA sequences and induces double-strand breaks that are repaired via error-prone non-homologous end joining, frequently causing insertions or deletions that disrupt gene function. Targeting specificity is achieved through gRNA-DNA base pairing and recognition of a protospacer adjacent motif by Cas9. Beyond gene knockout, CRISPR/Cas9 enables functional analysis of non-coding elements such as enhancers and insulators. Delivered via plasmid systems, Cas9 and gRNA are transiently expressed and degrade within 48-72 h, leaving no permanent genetic footprint. Since no exogenous DNA is integrated, this approach is generally considered less contentious than traditional transgenic methods in the context of genetically modified organism regulation. CRISPR/Cas9 has diverse applications in poultry, including enhancing disease resistance to avian influenza and Marek's disease, improving productivity traits such as growth, feed efficiency, and egg-laying, and enabling early in-ovo sexing to address ethical concerns around male chick culling. It also allows control of reproductive traits for breeding management, supports bio-pharming by producing therapeutic proteins or vaccines in eggs, and facilitates functional genomics through precise gene knockouts to study development, immunity, and metabolism.

CRISPR-Cas9 is a gene editing tool used extensively in biological research that is now making its way into clinical therapies. With the first CRISPR therapy obtaining approval by the United States' Food and Drug Administration (FDA) in late 2023, we look at clinical trials of emerging therapies involving CRISPR-Cas9, currently the most prevalent CRISPR-based tool in these trials. A CRISPR-based therapy is currently approved for treatment of both sickle-cell anemia and transfusion-dependent β-thalassemia but clinical trials for CRISPR-based therapeutics include a much broader range of targets. CRISPR-Cas9 is being explored to treat cancer, infectious disease, and more. This review highlights CRISPR-Cas9 clinical trials registered at clinicaltrials.gov as of 12/31/2024.

Targeted insertion (TIN) of transgenic trait cassettes has the potential to reduce timeline and cost for GM product development and commercialization. Precise genome engineering has made remarkable progress over the last several decades, particularly with the use of site-directed nucleases as core editing machinery. However, there are still many critical factors that can impact TIN efficiency including insertion site selection, nuclease optimization and expression, donor vector design, gene delivery, and stable event regeneration. Here, we develop workflows for target site sequence identification and gRNA screening for CRISPR-Cas12a system and demonstrate its successful application for TIN in maize with donor sequences up to 10 kilobase pairs (kb) in size. We first prioritize genomic regions for inserting transgenes in silico using bioinformatics tools and then test gRNA performance using a leaf protoplast transient assay. Despite its known low efficiency, we identify homology-directed repair (HDR) as the preferential pathway for directing targeted insertions of large sequences in immature embryos and demonstrate double-junction integrations at a rate of up to 4%. We further apply a molecular analysis workflow using large amplicon TaqMan assays and nanopore sequencing for streamlined identification and characterization of high-quality insertion events with intact large inserts. Analysis of TIN events across generations suggests that efficiency bottlenecks are not limited to donor targeted integration; attrition in efficiency also results from partial or additional donor insertion, chimerism, and close linkage with undesired sequence insertions such as those encoding the editing machinery. This work represents a major step forward in realizing the potential of precise genome engineering in maize for basic research and biotech trait development applications.

Plant genome editing has undergone a transformative shift with the advent of advanced molecular tools, offering unprecedented levels of precision, flexibility and efficiency in modifying genetic material. While classical site-directed nucleases such as ZFNs, TALENs and CRISPR-Cas9 have revolutionized genome engineering by enabling targeted mutagenesis and gene knockouts, the landscape is now rapidly evolving with the emergence of novel systems that go beyond the conventional double strand break (DSB)-mediated approaches. Advanced and recent tools include LEAPER, SATI, RESTORE, RESCUE, ARCUT, SPARDA, helicase-based approaches like HACE and Type IV-A CRISPR system, and transposon-based techniques like TATSI and piggyBac. These tools unlock previously inaccessible avenues of genome and transcriptome modulation. Some of these technologies allow DSB-free editing of DNA, precise base substitutions and RNA editing without altering the genomic DNA, a significant advancement for regulatory approval and for species with complex genomes or limited regeneration capacity. While LEAPER, RESCUE and RESTORE are the new advents in the RNA editing tool, SATI allows DSB-free approach for DNA editing, ARCUT offers less off-target and cleaner DNA repairs and Type IV-A CRISPR system induces gene silencing rather than editing. The transposon-based approaches include TATSI, piggyBac and TnpB, and helicases are used in HACE and Type IV-A CRISPR system. The prokaryotic Argonaute protein is used in SPARDA tool as an endonuclease to edit DNA. The transient and reversible nature of RNA editing tools such as RESTORE and LEAPER introduces a new layer of epigenetics-like control in plant systems, which could be harnessed for tissue-specific and environmentally-responsive trait expression. Simultaneously, innovations like ARCUT and SPARDA utilize chemically-guided editing, minimizing reliance on biological nucleases and reducing off-target risks. Their modularity and programmability are enabling gene function studies, synthetic pathway designs and targeted trait stacking. These advances represent a novel synthesis of genome engineering and systems biology, positioning plant genome editing not just as a tool of modification but as a platform for designing adaptive and intelligent crops, tailored to future environmental and nutritional challenges. Although, many of these recent tools remain to be applied on plant systems, they are proven to be effective elsewhere and hold a great potential to be effective in creating climate-resilient crops.

Potato is an important vegetatively propagated, starch-rich tuber crop. High amylose potatoes containing more resistant starch offer healthier food alternatives. However, the resistant starch content is low in most cultivated potato varieties. In this study, targeted mutation of the starch branching enzyme2 (SBE2.1 & SBE2.2 isoforms) had been done in the commercially significant potato cultivar, Kufri Chipsona-I using Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9 system) to develop high-amylose potato lines. SBE2 is one of the key enzymes involved in amylopectin biosynthesis, a starch component. Two isoforms, SBE2.1 & SBE2.2, were mutated using CRISPR-Cas9-mediated genome editing. After Agrobacterium-mediated genetic transformation, fifty transformed lines were generated on herbicide Basta selection medium, out of which 70% were found positive for bar and Cas9 genes. Overall, six mutant lines, viz. K301, K302, K303, K304, K305, K306, derived from distinct events, exhibited deletions and substitutions in the target exons. The CRISPR-Cas9 edited K304 potato line exhibited both insertion-deletion (indel) and substitution mutations in three out of the four selected targets across both genes, and was therefore identified as the most efficiently edited line. The harvested tubers from SBE2.1 & SBE2.2 mutant K304 line showed the highest amylose (95.91%) and resistant starch content (8.69 g/100 g). Evaluation of starch using X-ray crystallography (XRD) illustrated an altered crystallinity index (CI%) in all six mutant events in comparison to the wild study. Furthermore, ¹H-NMR study demonstrated a substantial decline in branch chain elongation in amylopectin, and thus a low degree of branching in a range of 1.15%-3.66% was reported in mutant lines, relative to the wild type (5.46%). The present study demonstrated the efficacy of CRISPR-Cas9-mediated mutagenesis of starch biosynthetic genes to develop high-amylose potato lines with elevated resistant starch content for improved health benefits.

Background: The functional characterization of Epithelial Cell Adhesion Molecule (EpCAM) in colorectal cancer (CRC) progression has been constrained by methodological limitations, particularly the potential for truncated protein isoforms to confound traditional genetic knockout approaches. This study aimed to develop a novel CRISPR/Cas9 strategy to overcome this challenge and systematically elucidate the context-dependent oncogenic roles of EpCAM across diverse CRC models.

Methods: We engineered EpCAM overexpression (pCDH-EpCAM) and CRISPR/Cas9 knockdown (pGMC-KO-EpCAM) vectors using restriction digestion and T4 DNA ligation. A strategic dual-exon targeting approach (exons 1 and 3) was employed to minimize the risk of functional escape variants. Selected CRC cell lines (HT-29, HT-115, HRT-18) were genetically modified using optimized Lipofectamine 2000 transfection. Functional impacts were quantitatively assessed through: (i) flow cytometry for EpCAM surface expression (CD326-PE); (ii) daily cell counting over 8 days for proliferation kinetics; and (iii) scratch wound healing (0/24/48 h) and Transwell migration assays (8-μm pores, 18 h) to evaluate metastatic potential.

Results: Successful genetic modulation was achieved and validated: HT-29-OE-EpCAM-2 exhibited an 89% EpCAM-positive rate versus 12% in wild-type (WT) (*p*<0.001), while HRT-18-KD-EpCAM-3 showed a significant reduction to 4% EpCAM-positive cells (vs. 15% in WT, *p*<0.001). EpCAM overexpression accelerated proliferation, with HT-29-OE cells showing a 20.1% increase in peak density on day 5 (30.76 ± 0.15 × 10⁴ vs. WT 25.62 ± 0.25 × 10⁴; *p*<0.001). Conversely, EpCAM knockdown in HRT-18 cells prolonged the doubling time by 8.8% (30.8 h vs. WT 28.3 h; *p*<0.05). Migration capacity was profoundly altered: HT-115-OE cells achieved complete scratch closure (100% vs. 74.05% in WT, *p*<0.001), whereas HRT-18-KD cells showed an 80.5% reduction (*p*<0.001). Transwell migration hierarchy confirmed the pro-metastatic role of EpCAM (HT-29-OE > HT-115-OE > HRT-18-KD; ANOVA *p* = 0.0024).

Conclusion: This study establishes a robust dual-vector toolkit for reliable EpCAM manipulation, highlighting a novel exon-targeting strategy that mitigates the limitations of previous approaches. Our findings demonstrate that EpCAM is a master regulator of CRC aggressiveness, dictating proliferative and metastatic phenotypes in a cell context-dependent manner. The genetically defined models provide a validated platform for therapeutic screening and safety assessment, forming a foundational resource for advancing EpCAM-targeted therapies and diagnostic applications.

Advancements in genome editing technologies, notably CRISPR/Cas9, base editing (BE), and prime editing (PE), have revolutionized plant biotechnology, offering unprecedented precision in crop improvement to address the ongoing global warming challenge. This review provides a critical analysis of recent developments in SpCas9-based editing tools, emphasizing enhancements in editing efficiency and specificity and follow the chronological development of editing tools. We explore methodological innovations, including dual pegRNA strategies and site-specific integrases, that have expanded the potential of PE for precise gene insertions. By integrating insights into DNA repair mechanisms and leveraging SpCas9 enhancements, we outline future directions for the application of genome editing in plant breeding.

[This corrects the article DOI: 10.3389/fgeed.2025.1620438.].

Genome editing for the treatment of human disease (GenE-HumDi) is an EU-funded COST Action for the development and consolidation of academic, industrial and healthcare feedback networks aiming to accelerate, foster and harmonize the approval of genome-editing (GE) therapies. GenE-HumDi offers mobility grants, supports educational courses, and hosts conferences and meetings to promote synergistic interactions among and across partners active in the discovery, validation, optimization, manufacturing and clinical application of genomic medicines. Furthermore, it provides young and early career scientists with a supportive and world-class environment to foster networking and international collaborations within the GE field. We compiled the proceedings of the second Annual GenE-HumDi Meeting held in Limassol, Cyprus, in 2024. Over three days, renowned experts from the field updated an audience of over 70 GenE-HumDi members and non-member scientists on the latest discoveries and ongoing projects, discussed the status of the field, and identified GenE-HumDi action priorities to advance research and development for GE medicines. Seven focused discussion groups identified gaps in knowledge, standardization and dissemination for new GE tools, delivery methods, safety monitoring, validation for clinical use, and progress in industrial manufacturing and regulatory issues. Simultaneously, publicity about the event itself contributed to outreach and dissemination of GE for human diseases. Therefore, the conclusions of that meeting, summarized here, serve as a compass toward GE application in Europe through coordination, enhanced collaboration and focus on critical developments.