Frontiers in genome editing最新文献

Since 2009, seven people living with human immunodeficiency virus (PLHIV) have been declared cured of HIV after receiving allogeneic hematopoietic stem cell transplants (alloHSCTs) to treat hematologic malignancies. In this sense, cure signifies the absence of viral DNA/RNA and undetectable viral loads without the use of antiretroviral therapy (ART). Five of these transplants utilized mutated C-C motif chemokine receptor type 5 (CCR5^Δ32/Δ32) stem cells. Much has been learned from these and past cases, and although effective, bone marrow transplants cannot be easily or safely translated to cure the millions of PLHIV across the globe. A successful eradicating cure includes both the prevention of HIV from entering new cells and the elimination of tissue reservoirs. Protecting hematopoietic stem and progenitor cells (HSPCs) from infection is a key consideration since there is evidence that HSPCs themselves, not only their descendants, are susceptible to infection. Gene therapy approaches have the potential to bring about an eradicating HIV cure that could be highly effective, broadly applicable, less expensive, and practical to implement. Current strategies are tackling this problem by removing the integrated proviral DNA from infected cells and/or eliminating the co-receptor(s) necessary for HIV viral entry into target cells. Both approaches hold promise, but they require overcoming key challenges (i.e., vector toxicity, transduction efficacy, elimination of reservoir cells, etc.). This review summarizes and examines the lessons learned about curing HIV through bone marrow transplants, the current gene therapy methodologies, pitfalls of eradication strategies as well as future directions of the field.

The main aim of present study was to analyze the consumers' preferences about genome/gene-edited food products in Iran. For this purpose, an extended version of the theory of planned behavior was used as a social intervention tool. The theory of planned behavior was firstly extended using the introduction and new variable of trust in gene-edited products and perceived benefits of gene-edited food products, but in the next step, it was also analyzed statistically. To achieve the main objective of the research, a representative sample was selected from the population of purchasers of gene-edited products, and data were collected using a cross-sectional survey. The validity and reliability of the data collection tool was evaluated and confirmed using different quantitative and qualitative methods in the pilot stages and after the main survey. The results of structural equation modeling showed that the attitude towards gene-edited food products, perceived behavioral control, and the subjective norms of gene-edited products had positive and significant effects on the intention to purchase these products. The results of the study indicated that two newly introduced variables to the theory of planned behavior, namely, trust in gene-edited products and the perceived benefits of gene-edited products also had positive and significant effects on the intention to purchase these products. Based on the results, the framework employed and extended in this study can provide the basis for effective interventions to improve consumers' preference for gene-edited food products. Also, some practical suggestions were provided for policymakers, managers, and producers of these products.

Selectable marker genes (SMGs) are essential for identifying transgenic plants but raise concerns regarding biosafety, regulatory compliance, and public acceptance. In this study, we used a CRISPR/Cas9-based strategy to eliminate the SMG from transgenic tobacco plants. Leaf discs from plants carrying DsRED (SMG) and aminoglycoside phosphotransferase (gene of interest, GOI) were re-transformed with a CRISPR vector containing four gRNAs designed to target both flanking regions of the SMG cassette. Approximately 20% of the regenerated shoots exhibited loss of red fluorescence, and PCR and sequencing analyses confirmed that about half of these carried a smaller amplicon, indicating a successful SMG excision efficiency of around 10%. Mutation analysis further revealed the presence of small indels at gRNA target sites, in addition to the deletion of SMG cassette. Quantitative real-time PCR (qPCR) analysis confirmed the absence of DsRED expression in SMG-deleted lines, while the Cas9 and GOI remained actively expressed. The SMG-free plants displayed normal growth, flowering, and seed production, indicating CRISPR marker excision had no adverse effects on plant development and fertility. In addition, Cas9-free, marker-free transgenic plants were recovered through segregation in T1 generation. This approach is adaptable to various transgenic plant species and provides a practical solution for generating marker-free transgenic crops, thereby enhancing their acceptance and commercialization.

Introduction: Accurately predicting the on-target activity of sgRNAs remains a challenge in CRISPR-Cas9 applications, due to the limited generalization of existing models across datasets, small-sample settings, and complex sequence contexts. Current methods often rely on shallow architectures or unimodal encodings, limiting their ability to capture the intricate dependencies underlying Cas9-mediated cleavage.

Methods: We present CRISPR-FMC, a dual-branch hybrid neural network that integrates One-hot encoding with contextual embeddings from a pre-trained RNA-FM model. Multi-scale convolution (MSC), BiGRU, and Transformer blocks are employed to extract hierarchical sequence features, while a bidirectional cross-attention mechanism with a residual feedforward network enhances multimodal fusion and generalization.

Results: Across nine public CRISPR-Cas9 datasets, CRISPR-FMC consistently outperforms existing baselines in both Spearman and Pearson correlation metrics, showing particularly strong performance under low-resource and cross-dataset conditions. Ablation experiments confirm the contribution of each module, and base substitution analysis reveals a pronounced sensitivity to the PAM-proximal region.

Discussion: The PAM-proximal sensitivity aligns with established biological evidence, indicating the model's capacity to capture biologically relevant sequence determinants. These results demonstrate that CRISPR-FMC offers a robust and interpretable framework for sgRNA activity prediction across heterogeneous genomic contexts.

Polyploidy, or whole-genome duplication (WGD), is a significant evolutionary force. Following allopolyploidy, duplicate gene copies (homeologs) have divergent evolutionary trajectories: some genes are preferentially retained in duplicate, while others tend to revert to single-copy status. Examining the effect of homeolog loss (i.e., changes in gene dosage) on associated phenotypes is essential for unraveling the genetic mechanisms underlying polyploid genome evolution. However, homeolog-specific editing has been demonstrated in only a few crop species and remains unexplored beyond agricultural applications. Tragopogon (Asteraceae) includes an evolutionary model system for studying the immediate consequences of polyploidy in nature. In this study, we developed a CRISPR-mediated homeolog-specific editing platform in allotetraploid T. mirus. Using the MYB10 and DFR genes as examples, we successfully knocked out the targeted homeolog in T. mirus (4x) without editing the other homeolog (i.e., no off-target events). The editing efficiencies, defined as the percentage of plants with at least one allele of the targeted homeolog modified, were 35.7% and 45.5% for MYB10 and DFR, respectively. Biallelic modification of the targeted homeolog occurred in the T₀ generation. These results demonstrate the robustness of homeolog-specific editing in polyploid Tragopogon, laying the foundation for future studies of genome evolution following WGD in nature.

Prime editing (PE) enables precise genome modification, i.e., all 12 types of base substitution, as well as designed insertion and deletion. Previously, we developed an efficient PE system using a pair of engineered pegRNAs (epegRNAs), appending an RNA pseudoknot sequence to the 3'ends of pegRNAs to enhance stability and prevent degradation of the 3'extension. Using a wild-type nSpCas9-based PE system (PE-wt) recognizing an NGG-protospacer adjacent motif (PAM) in this approach, two NGG-PAMs (NGG and CCN) adjacent to the target site are required for targeting by paired pegRNAs; however, this is not the PAM configuration available at most target sites. Using an nSpCas9-NG variant recognizing NG-PAM in PE (PE-NG) can expand applicability. Here, we compare the PE efficiency of PE-wt with paired epegRNAs targeting a distal NGG-PAM versus PE-NG with paired epegRNAs targeting NG-PAMs adjacent to the target site. By introducing substitution and designated deletion mutations into target genes via PE-wt and PE-NG with paired epegRNAs, we demonstrated that PE-wt could edit the target site efficiently despite targeting the distal PAM site when either of the paired epegRNAs for PE-NG targets PGC-PAM. If epegRNAs for PE-NG are designed to recognize NGA and NGT-PAM, there is no significant difference in frequency between PE-NG and PE-wt. These findings indicate that PE efficiency via PE-NG is particularly low at the NGC-PAM in rice.

Alzheimer's disease (AD), a progressive neurodegenerative disorder characterized by Aβ plaques, tau protein neuronal fiber tangles, and neuroinflammation, poses a significant global health problem, and current therapies focus on the symptoms rather than the cause. This paper gives a new multidimensional therapeutic form to AD treatment by exploring the integrated application of stem cell therapy and CRISPR/Cas9 gene editing technology. The study comprehensively dissected the roles of neural stem cells (NSCs), induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) in neural replacement, neuroinflammation modulation and neuroplasticity enhancement, and also explored the application of CRISPR/Cas9 in modifying the pathogenic variants of AD-related genes (APP, PSEN1 and PSEN2). The key findings suggest that gene-edited iPSCs can reduce abnormal Aβ and tau protein accumulation in AD models, improve cognitive function, and provide a platform for disease modeling and drug screening. Stem cell transplantation promotes neurogenesis and synaptic plasticity by secreting neurotrophic factors to improve the brain microenvironment. Despite the challenges of off-target effects, immune rejection, and long-term safety, the synergistic application of these two technologies offers a breakthrough solution for AD treatment. This paper highlights the translational potential of combining stem cells with gene editing technology, which is expected to drive clinical applications in the next 5-10 years. The integration of these advanced technologies not only addresses the limitations of current AD treatments, but also paves the way for a personalized medical approach that is expected to revolutionize the AD treatment landscape and bring new hope to patients worldwide.

CRISPR-Cas9 is a breakthrough genome-editing platform that can cut chosen DNA sequences with unprecedented speed, accuracy, and affordability. By reprogramming a single guide RNA, researchers now alter gene function, correct pathogenic variants, or introduce novel traits. Earlier tools such as zinc-finger nucleases and TALENs performed similar tasks but were significantly more complex and costly. Yet CRISPR's very power raises urgent ethical concerns: Who controls its use, and how can society prevent germ-line enhancement, eugenic selection, or unequal access that favors wealthy nations and patients? A well-publicized case of embryo editing already showed how premature, unregulated experiments can erode public trust. This perspective therefore frames CRISPR's scientific promise alongside its social responsibilities, arguing that proactive, globally coordinated governance is essential to unlock benefits while preventing new forms of genetic inequality.

Introduction: With the growing number of Cas9 nucleases available to genetic engineers, selecting the most suitable one for a given application can be challenging. A major complication arises from the differing protospacer adjacent motif (PAM) sequence requirements of each Cas9 variant, which makes direct comparisons difficult. To ensure a fair comparison, it is essential to identify common target sites that are not biased by the natural genetic landscape of the chosen target.

Methods: To address this challenge, we developed CATS (Comparing Cas9 Activities by Target Superimposition), a novel bioinformatic tool. CATS automates the detection of overlapping PAM sequences across different Cas9 nucleases and identifies allele-specific targets, particularly those arising from pathogenic mutations. One of the key parameters in CATS is the proximity of PAM sites, which helps minimize sequence composition bias. The tool integrates data from continuously updated sources and includes ClinVar information to facilitate the targeting of disease-causing mutations.

Results: CATS significantly reduces the time and effort required for CRISPR/Cas9 experimental design. It streamlines the comparison of Cas9 nucleases with different PAM requirements, enabling researchers to select the most appropriate nuclease for their specific target. The tool's automation, speed, and user-friendly interface make it accessible to researchers regardless of their computational expertise.

Discussion: By enabling the identification of overlapping PAMs and allele-specific targets, CATS supports the implementation of Cas9-based applications in both research and clinical settings. Its ability to incorporate genetic variants makes it particularly useful for designing therapeutic approaches that selectively target mutated alleles while sparing healthy ones. Ultimately, CATS contributes to the development of more effective and precise genetic therapies.