Editing Methods Research Articles

The E3 ligase TaE3V-B1 ubiquitinates proteins encoded by the vernalization gene TaVRN1 and regulates developmental processes in wheat

Abstract In wheat (Triticum aestivum), early maturity is desired to avoid the hot and dry summer season, especially in view of climate change. Here, we report that TaE3V1, a C3H2C3 RING-type E3 ligase that interacts with TaVRN1, is associated with early development. Aside from its RING domain, TaE3V1 does not harbor any domains that are conserved in other RING-type or other E3 ligase proteins. TaE3V-B1b, encoded by the functional TaE3V1 allele, interacts with and ubiquitinates TaVRN1. In contrast, TaE3V-B1a, encoded by a natural nonfunctional TaE3V1 allele, neither interacts with TaVRN1 nor has E3 ligase activity. TaE3V-B1b activity decreases with plant age under warmer temperatures, but not under the low temperatures required for vernalization. We employed a gene editing method to simultaneously inactivate the three homoeologous TaE3V1 genes to validate their functions. Overall, our results suggest that the naturally mutated and edited TaE3V1 alleles can accelerate wheat development and aid adaptation to warming climates.

Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells.

The safety of CRISPR-based gene editing methods is of the utmost priority in clinical applications. Previous studies have reported that Cas9 cleavage induced frequent aneuploidy in primary human T cells, but whether cleavage-mediated editing of base editors would generate off-target structure variations remains unknown. Here, we investigate the potential off-target structural variations associated with CRISPR/Cas9, ABE, and CBE editing in mouse embryos and primary human T cells by whole-genome sequencing and single-cell RNA-seq analyses. The results show that both Cas9 and ABE generate off-target structural variations (SVs) in mouse embryos, while CBE induces rare SVs. In addition, off-target large deletions are detected in 32.74% of primary human T cells transfected with Cas9 and 9.17% of cells transfected with ABE. Moreover, Cas9-induced aneuploid cells activate the P53 and apoptosis pathways, whereas ABE-associated aneuploid cells significantly upregulate cell cycle-related genes and are arrested in the G0 phase. A percentage of 16.59% and 4.29% aneuploid cells are still observable at 3 weeks post transfection of Cas9 or ABE. These off-target phenomena in ABE are universal as observed in other cell types such as B cells and Huh7. Furthermore, the off-target SVs are significantly reduced in cells treated with high-fidelity ABE (ABE-V106W). This study shows both CRISPR/Cas9 and ABE induce off-target SVs in mouse embryos and primary human T cells, raising an urgent need for the development of high-fidelity gene editing tools.

An improved face attributes editing method based on DDIM

The main advantage of DDIM is that it guarantees the quality of the generated images while increasing the efficiency of the generation by modifying the sampling strategy in the diffusion process. DiffusionRig, which addresses the problem of maintaining identity consistency by learning the person-specific facial prior in a tiny personalized dataset, is a successful representation of the DDIM strategy. Based on DiffusionRig, in this article, we propose an improved face attributes editing method based on DDIM to improve naturalness and accuracy of editing results in complex face attribute editing tasks and the generalization ability. Our method combines DDIM and DECA together and use two-stage training strategy. To reduce DiffusionRig’s limitations in handling face attribute editing tasks that require nonlinear understanding and fine-tuning, our method also introduces a channel attention mechanism and a depth-separable convolution technique in the training model. In the first stage we trained our model on the open FFHQ dataset, which consists of 30,000 high-resolution face image. In the second stage, the model was refined by using a tiny personalized dataset. Face attribute editing experiments,comparative experiment with DiffusionRig, and a series of ablation experiments have been performed. Then, the validity of our improved method is verified by qualitative and quantitative analysis.

GSEditPro: 3D Gaussian Splatting Editing with Attention‐based Progressive Localization

AbstractWith the emergence of large‐scale Text‐to‐Image(T2I) models and implicit 3D representations like Neural Radiance Fields (NeRF), many text‐driven generative editing methods based on NeRF have appeared. However, the implicit encoding of geometric and textural information poses challenges in accurately locating and controlling objects during editing. Recently, significant advancements have been made in the editing methods of 3D Gaussian Splatting, a real‐time rendering technology that relies on explicit representation. However, these methods still suffer from issues including inaccurate localization and limited manipulation over editing. To tackle these challenges, we propose GSEditPro, a novel 3D scene editing framework which allows users to perform various creative and precise editing using text prompts only. Leveraging the explicit nature of the 3D Gaussian distribution, we introduce an attention‐based progressive localization module to add semantic labels to each Gaussian during rendering. This enables precise localization on editing areas by classifying Gaussians based on their relevance to the editing prompts derived from cross‐attention layers of the T2I model. Furthermore, we present an innovative editing optimization method based on 3D Gaussian Splatting, obtaining stable and refined editing results through the guidance of Score Distillation Sampling and pseudo ground truth. We prove the efficacy of our method through extensive experiments.

Balanced Training Sets Improve Deep Learning-Based Prediction of CRISPR sgRNA Activity.

CRISPR-Cas systems have transformed the field of synthetic biology by providing a versatile method for genome editing. The efficiency of CRISPR systems is largely dependent on the sequence of the constituent sgRNA, necessitating the development of computational methods for designing active sgRNAs. While deep learning-based models have shown promise in predicting sgRNA activity, the accuracy of prediction is primarily governed by the data set used in model training. Here, we trained a convolutional neural network (CNN) model and a large language model (LLM) on balanced and imbalanced data sets generated from CRISPR-Cas12a screening data for the yeast Yarrowia lipolytica and evaluated their ability to predict high- and low-activity sgRNAs. We further tested whether prediction performance can be improved by training on imbalanced data sets augmented with synthetic sgRNAs. Lastly, we demonstrated that adding synthetic sgRNAs to inherently imbalanced CRISPR-Cas9 data sets from Y. lipolytica and Komagataella phaffii leads to improved performance in predicting sgRNA activity, thus underscoring the importance of employing balanced training sets for accurate sgRNA activity prediction.

Advances in text-guided 3D editing: a survey

In 3D Artificial Intelligence Generated Content (AIGC), compared with generating 3D assets from scratch, editing extant 3D assets satisfies user prompts, allowing the creation of diverse and high-quality 3D assets in a time and labor-saving manner. More recently, text-guided 3D editing that modifies 3D assets guided by text prompts is user-friendly and practical, which evokes a surge in research within this field. In this survey, we comprehensively investigate recent literature on text-guided 3D editing in an attempt to answer two questions: What are the methodologies of existing text-guided 3D editing? How has current progress in text-guided 3D editing gone so far? Specifically, we focus on text-guided 3D editing methods published in the past 4 years, delving deeply into their frameworks and principles. We then present a fundamental taxonomy in terms of the editing strategy, optimization scheme, and 3D representation. Based on the taxonomy, we review recent advances in this field, considering factors such as editing scale, type, granularity, and perspective. In addition, we highlight four applications of text-guided 3D editing, including texturing, style transfer, local editing of scenes, and insertion editing, to exploit further the 3D editing capacities with in-depth comparisons and discussions. Depending on the insights achieved by this survey, we discuss open challenges and future research directions. We hope this survey will help readers gain a deeper understanding of this exciting field and foster further advancements in text-guided 3D editing.

Establishment of a Genetic Transformation and Gene Editing Method by Floral Dipping in Descurainia sophia

Establishment of a Genetic Transformation and Gene Editing Method by Floral Dipping in Descurainia sophia

PFB-Diff: Progressive Feature Blending diffusion for text-driven image editing

Diffusion models have demonstrated their ability to generate diverse and high-quality images, sparking considerable interest in their potential for real image editing applications. However, existing diffusion-based approaches for local image editing often suffer from undesired artifacts due to the latent-level blending of the noised target images and diffusion latent variables, which lack the necessary semantics for maintaining image consistency. To address these issues, we propose PFB-Diff, a Progressive Feature Blending method for Diffusion-based image editing. Unlike previous methods, PFB-Diff seamlessly integrates text-guided generated content into the target image through multi-level feature blending. The rich semantics encoded in deep features and the progressive blending scheme from high to low levels ensure semantic coherence and high quality in edited images. Additionally, we introduce an attention masking mechanism in the cross-attention layers to confine the impact of specific words to desired regions, further improving the performance of background editing and multi-object replacement. PFB-Diff can effectively address various editing tasks, including object/background replacement and object attribute editing. Our method demonstrates its superior performance in terms of editing accuracy and image quality without the need for fine-tuning or training. Our implementation is available at https://github.com/CMACH508/PFB-Diff.

CRISPR/Cas12a-based genome editing for cyanophage of Anabeana sp.

Efforts have been conducted on cyanobacterial genome editing, yet achieving genome editing in cyanophages remains challenging. Editing cyanophage genomes is crucial for understanding and manipulating their interactions with cyanobacterial hosts, offering potential solutions for controlling cyanobacterial blooms. In this study, we developed a streamlined CRISPR-Cas12a-based method for efficient cyanophage genome editing and then applied this method to the cyanophages A-1(L) and A-4(L) of Anabeana sp. PCC.7120. Multiple hypothetical genes were edited and knocked out from these two cyanophage genomes, generating viable mutants with varying capabilities to inhibit cyanobacterial growth. All these mutants displayed significant inhibitory effects on the host, indicating that these genes were non-essential for phage life cycle and the deletion led to little impairment of the cyanophages in infectious efficiency to their host. By iterative and simultaneous gene knockouts in cyanophage A-4(L), we achieved the minimal genome mutant with a 2400 bp reduction in genome size, representing a 5.75 % decrease compared to the wild type (WT). In conclusion, these cyanophage mutants can facilitate the identification of nonessential genes for cyanophages biology and the insertion of foreign genes for synthetic biology research. This advancement holds promise in addressing the widespread issue of water blooms and the associated environmental hazards.

독립운동가 박용만의 출판 활동 연구 : 1910년대 미주와 하와이, 1920년대 중국 북경을 중심으로

This study aims to analyze the meaning of the six books published by independence activist Park Yong-man from 1911 to 1927 after his arrival in the United States in 1905, as well as the publishing process and activities. To this end, the publication circumstances of the six books published were investigated based on the covers, copyrights, and prefaces, and the editing and production methods of the books were analyzed. In addition, related materials were analyzed to identify the context and meaning surrounding Park Yong-man’s publishing activities. The results of the analysis are as follows. First, Park Yong-man made efforts to find the optimal method that suited the purpose and characteristics of the books in the time, space, and technological environments of the United States and China in the 1910s and 1920s. As a result, the characteristics of his publishing activities became differentiated depending on the time and place. Second, Park Yong-man’s publishing activities expanded beyond the role of an “author” to that of a “publisher,” and his social network with independence movement organizations and figures played an important role. Third, Park Yong-man’s publishing activities were a ‘socio-cultural activity and independence movement’ that overcame the many difficulties experienced by overseas Koreans during the Japanese colonial period in a political, social, and technological environment with high complexity and changing characteristics.

Genome Editing Technologies towards Tomato Improvement: Recent Advances and Future Perspectives

Tomato (Solanum lycopersicon L.) is the world's second major vegetable crop and a superior model plant for studies on fruit biology. However, the changing climatic conditions are hugely impacting the yield and quality of tomato. CRISPR/Cas9 technology has been widely used in tomato breeding for enhanced disease resistance, herbicide tolerance, domestication and urban farming of wild tomato, and improved fruit yield and quality. Furthermore, new and advanced editing systems like Cas12a, Cas12b, base editing, and prime editing have been recently applied for high-precision tomato improvement. CRISPR variants, PAM-less genome editing, advanced transformation protocols, and gene delivery systems have played a critical role in fast breeding. This review offers an informative summary of recent progress in various genome editing methods and applications for improving tomatoes. It also focuses on critical issues, regulatory concerns, and prospects of genome editing platforms to improve tomato and allied crops.

First Confirmed Description of Acremonium egyptiacum from Greece and Molecular Identification of Acremonium and Acremonium-like Clinical Isolates.

Acremonium and the recently separated acremonium-like genera, such as Sarocladium, are emerging causes of opportunistic disease in humans, mainly post-traumatic infections in immunocompetent hosts, but also invasive infections in immunocompromised patients, such as those undergoing transplantation. Acremonium egyptiacum has emerged as the major pathogenic Acremonium species in humans, implicated mainly in nail but also in disseminated and organ specific infections. In this first study of acremonium-like clinical isolates in Greece, 34 isolates were identified and typed by sequencing the internal transcribed spacer, and their antifungal susceptibility was determined by a modified CLSI standard M38 3rd Edition method for filamentous fungi. A. egyptiacum was the primary species (18 isolates) followed by Sarocladium kiliense (8), Acremonium charticola, Gliomastix polychroma, Proxiovicillium blochii, Sarocladium terricola, Sarocladium zeae, and Stanjemonium dichromosporum (all with one isolate). Two isolates, each with a novel ITS sequence, possibly represent undescribed species with an affinity to Emericellopsis. All three A. egyptiacum ITS barcode types described to date were identified, with 3 being the major type. Flutrimazole, lanoconazole, and luliconazole presented the lower minimum inhibitory concentration (MIC) values against A. egyptiacum, with a geometric mean (GM) MIC of 2.50, 1.92, and 1.57 μg/mL, respectively. Amphotericin B, itraconazole, posaconazole, voriconazole, terbinafine, amorolfine, and griseofulvin MICs were overall high (GM 12.79-29.49 μg/mL). An analysis of variance performed on absolute values showed that flutrimazole, lanoconazole, and luliconazole were equivalent and notably lower than those of all the other drugs tested against A. egyptiacum. Antifungal susceptibility of the three different A. egyptiacum genotypes was homogeneous. Overall, the high MICs recorded for all systemically administered drugs, and for some topical antifungals against the tested A. egyptiacum and other acremonium-like clinical isolates, justify the routine susceptibility testing of clinical isolates.

Cell-Penetrating Peptides and CRISPR-Cas9: A Combined Strategy for Human Genetic Disease Therapy.

The advent of Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated nuclease 9 (Cas9) technology has revolutionized the field of genetic engineering, offering unprecedented potential for the targeted manipulation of DNA sequences. Advances in the mechanism of action of the CRISPR-Cas9 system allowed potential applicability for the treatment of genetic diseases. CRISPR-Cas9's mechanism of action involves the use of an RNA guide molecule to target specific DNA sequences and the Cas9 enzyme to induce precise DNA cleavage. In the context of the CRISPR-Cas9 system, this review covers non-viral delivery methods for gene editing based on peptide internalization. Here we describe critical areas of discussion such as immunogenicity, emphasizing the importance of safety, efficiency, and cost-effectiveness, particularly in the context of treating single-mutation genetic diseases using advanced editing techniques genetics as prime editor and base editor. The text discusses the versatility of Cell-Penetrating Peptides (CPPs) in forming complexes for delivering biomolecules, particularly Ribonucleoprotein (RNP) for genome editing with CRISPR-Cas9 in human cells. In addition, it emphasizes the promise of combining CPPs with DNA base editing and prime editing systems. These systems, known for their simplicity and precision, hold great potential for correcting point mutations in human genetic diseases. In summary, the text provides a clear overview of the advantages of using CPPs for genome editing with CRISPR-Cas9, particularly in conjunction with advanced editing systems, highlighting their potential impact on clinical applications in the treatment of single-mutation genetic diseases.

Genome editing in angiosperm chloroplasts: targeted DNA double-strand break and base editing.

Chloroplasts are organelles that are derived from a photosynthetic bacterium and have their own genome. Genome editing is a recently developing technology that allows for specific modifications of target sequences. The first successful application of genome editing in chloroplasts was reported in 2021, and since then, this research field has been expanding. Although the chloroplast genome of several dicot species can be stably modified by a conventional method, which involves inserting foreign DNAs into the chloroplast genome via homologous recombination, genome editing offers several advantages over this method. In this review, we introduce genome editing methods targeting the chloroplast genome and describe their advantages and limitations. So far, CRISPR/Cas systems are inapplicable for editing the chloroplast genome because guide RNAs, unlike proteins, cannot be efficiently delivered into chloroplasts. Therefore, protein-based enzymes are used to edit the chloroplast genome. These enzymes contain a chloroplast-transit peptide, the DNA-binding domain of transcription activator-like effector nuclease (TALEN), or a catalytic domain that induces DNA modifications. To date, genome editing methods can cause DNA double-strand break or introduce C:G-to-T:A and A:T-to-G:C base edits at or near the target sequence. These methods are expected to contribute to basic research on the chloroplast genome in many species and to be fundamental methods of plant breeding utilizing the chloroplast genome.

Kinesin-7 CENP-E mediates centrosome organization and spindle assembly to regulate chromosome alignment and genome stability.

Chromosome congression and alignment are essential for cell cycle progression and genomic stability. Kinesin-7 CENP-E, a plus-end-directed kinesin motor, is required for chromosome biorientation, congression and alignment in cell division. However, it remains unclear how chromosomes are aligned and segregated in the absence of CENP-E in mitosis. In this study, we utilize the CRISPR-Cas9 gene editing method and high-throughput screening to establish CENP-E knockout cell lines and reveal that CENP-E deletion results in defects in chromosome congression, alignment and segregation, which further promotes aneuploidy and genomic instability in mitosis. Both CENP-E inhibition and deletion lead to the dispersion of spindle poles, the formation of the multipolar spindle and spindle disorganization, which indicates that CENP-E is necessary for the organization and maintenance of spindle poles. In addition, CENP-E heterozygous deletion in spleen tissues also leads to the accumulation of dividing lymphocytes and cell cycle arrest in vivo. Furthermore, CENP-E deletion also disrupts the localization of key kinetochore proteins and triggers the activation of the spindle assembly checkpoint. In summary, our findings demonstrate that CENP-E promotes kinetochore-microtubule attachment and spindle pole organization to regulate chromosome alignment and spindle assembly checkpoint during cell division.

A Simple and Efficient Procedure for Developing Mouse Germline Stem Cell Lines with Gene Knock-in via CRISPR/Cas9 Technology.

Cultured mammalian spermatogonial stem cells (SSCs), also known as germline stem cells (GSCs), hold great promise for applications such as fertility preservation, gene therapy, and animal breeding, particularly in conjunction with accurate gene editing. Although the in vitro development of mouse GSC (mGSC) lines, and gene-targeting procedures for such lines, were initially established about two decades ago, it remains challenging for beginners to efficiently accomplish these tasks, partly because mGSCs proliferate more slowly and are more resistant to lipid-mediated gene transfection than pluripotent stem cells (PSCs). Meanwhile, methods for mGSC culture and gene editing have been evolving constantly to become simpler and more efficient. Here, we describe how to develop mGSC lines from small mouse testis samples and how to carry out gene knock-in in these cells using CRISPR/Cas9 technology, detailing three basic protocols that constitute a streamlined procedure. Using these simple and efficient procedures, site-specific knock-in mGSC lines can be obtained in 3 months. We hope that these protocols will help researchers use genetically modified GSCs to explore scientific questions of interest and to accumulate experience for application to GSC research in other mammalian species. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Establishment of mouse GSCs lines from small testicular samples Basic Protocol 2: Preparation of plasmids for gene knock-in using the CRISPR/Cas9 system Basic Protocol 3: Establishment of gene knock-in mGSC lines by electroporation gene delivery.

Edit Temporal-Consistent Videos with Image Diffusion Model

Large-scale text-to-image (T2I) diffusion models have been extended for text-guided video editing, yielding impressive zero-shot video editing performance. Nonetheless, the generated videos usually show spatial irregularities and temporal inconsistencies as the temporal characteristics of videos have not been faithfully modeled. In this paper, we propose an elegant yet effective Temporal-Consistent Video Editing (TCVE) method to mitigate the temporal inconsistency challenge for robust text-guided video editing. In addition to the utilization of a pretrained T2I 2D Unet for spatial content manipulation, we establish a dedicated temporal Unet architecture to faithfully capture the temporal coherence of the input video sequences. Furthermore, to establish coherence and interrelation between the spatial-focused and temporal-focused components, a cohesive spatial-temporal modeling unit is formulated. This unit effectively interconnects the temporal Unet with the pretrained 2D Unet, thereby enhancing the temporal consistency of the generated videos while preserving the capacity for video content manipulation. Quantitative experimental results and visualization results demonstrate that TCVE achieves state-of-the-art performance in both video temporal consistency and video editing capability, surpassing existing benchmarks in the field. Codes are released at https://github.com/mdswyz/TCVE .

Heritable Tissue-Culture-Free Gene Editing in Nicotiana benthamiana through Viral Delivery of SpCas9 and sgRNA.

Conventional plant gene editing requires laborious tissue-culture-mediated transformation, which restricts the range of applicable plant species. In this study, we developed a heritable and tissue-culture-free gene editing method in Nicotiana benthamiana using tobacco ringspot virus (TRSV) as a vector for in planta delivery of Cas9 and single-guide RNA (sgRNA) to shoot apical meristems. Agrobacterium-mediated inoculation of the TRSV vector induced systemic and heritable gene editing in NbPDS. Transient downregulation of RNA silencing enhanced gene editing efficiency, resulting in an order of magnitude increase (0.8% to 13.2%) in the frequency of transgenerational gene editing. While the TRSV system had a preference for certain sgRNA sequences, co-inoculation of a TRSV vector carrying only Cas9 and a tobacco rattle virus vector carrying sgRNA successfully introduced systemic mutations with all five tested sgRNAs. Extensively gene-edited lateral shoots occasionally grew from plants inoculated with the virus vectors, of which the transgenerational gene editing frequency ranged up to 100%. This virus-mediated heritable gene editing method makes plant gene editing easy, requiring only the inoculation of non-transgenic plants with a virus vector(s) to obtain gene-edited individuals.

A Humanized Yeast Model for Studying TRAPP Complex Mutations; Proof-of-Concept Using Variants from an Individual with a TRAPPC1-Associated Neurodevelopmental Syndrome.

Variants in membrane trafficking proteins are known to cause rare disorders with severe symptoms. The highly conserved transport protein particle (TRAPP) complexes are key membrane trafficking regulators that are also involved in autophagy. Pathogenic genetic variants in specific TRAPP subunits are linked to neurological disorders, muscular dystrophies, and skeletal dysplasias. Characterizing these variants and their phenotypes is important for understanding the general and specialized roles of TRAPP subunits as well as for patient diagnosis. Patient-derived cells are not always available, which poses a limitation for the study of these diseases. Therefore, other systems, like the yeast Saccharomyces cerevisiae, can be used to dissect the mechanisms at the intracellular level underlying these disorders. The development of CRISPR/Cas9 technology in yeast has enabled a scar-less editing method that creates an efficient humanized yeast model. In this study, core yeast subunits were humanized by replacing them with their human orthologs, and TRAPPC1, TRAPPC2, TRAPPC2L, TRAPPC6A, and TRAPPC6B were found to successfully replace their yeast counterparts. This system was used for studying the first reported individual with an autosomal recessive disorder caused by biallelic TRAPPC1 variants, a girl with a severe neurodevelopmental disorder and myopathy. We show that the maternal variant (TRAPPC1 p.(Val121Alafs*3)) is non-functional while the paternal variant (TRAPPC1 p.(His22_Lys24del)) is conditional-lethal and affects secretion and non-selective autophagy in yeast. This parallels defects seen in fibroblasts derived from this individual which also showed membrane trafficking defects and altered Golgi morphology, all of which were rescued in the human system by wild-type TRAPPC1. This study suggests that humanized yeast can be an efficient means to study TRAPP subunit variants in the absence of human cells and can assign significance to variants of unknown significance (VUS). This study lays the foundation for characterizing further TRAPP variants through this system, rapidly contributing to disease diagnosis.

Conquering Limitations: Exploring the Factors that Drive Successful Agrobacterium-Mediated Genetic Transformation of Recalcitrant Plant Species.

Agrobacterium-mediated transformation is a preferred method for genetic engineering and genome editing of plants due to its numerous advantages, although not all species exhibit transformability. Genetic engineering and plant genome editing methods are technically challenging in recalcitrant crop plants. Factors affecting the poor rate of transformation in such species include host genotype, Agrobacterium genotype, type of explant, physiological condition of the explant, vector, selectable marker, inoculation method, chemical additives, antioxidative compounds, transformation-enhancing compounds, medium formulation, optimization of culture conditions, and pre-treatments. This review provides novel insights into the key factors involved in gene transfer facilitated by Agrobacterium and proposes potential solutions to overcome existing barriers to transformation in recalcitrant species, thereby contributing to improvement programs for these species. This review introduces the key factors that impact the effectiveness of a molecular breeding program using Agrobacterium-mediated transformation, specifically focusing on recalcitrant plant species.