Editing Applications Research Articles

Biology and applications of CRISPR-Cas12 and transposon-associated homologs.

CRISPR-associated Cas12 proteins are a highly variable collection of nucleic acid-targeting proteins. All Cas12 variants use RNA guides and a single nuclease domain to target complementary DNA or, in rare cases, RNA. The high variability of Cas12 effectors can be explained by a series of independent evolution events from different transposon-associated TnpB-like ancestors. Despite basic structural and functional similarities, this has resulted in unprecedented variation of the Cas12 effector proteins in terms of size, domain composition, guide structure, target identity and interference strategy. In this Review, we compare the unique molecular features of natural and engineered Cas12 and TnpB variants. Furthermore, we provide an overview of established genome editing and diagnostic applications and discuss potential future directions.

Structure-Guided Image Completion With Image-Level and Object-Level Semantic Discriminators.

Structure-guided image completion aims to inpaint a local region of an image according to an input guidance map from users. While such a task enables many practical applications for interactive editing, existing methods often struggle to hallucinate realistic object instances in complex natural scenes. Such a limitation is partially due to the lack of semantic-level constraints inside the hole region as well as the lack of a mechanism to enforce realistic object generation. In this work, we propose a learning paradigm that consists of semantic discriminators and object-level discriminators for improving the generation of complex semantics and objects. Specifically, the semantic discriminators leverage pretrained visual features to improve the realism of the generated visual concepts. Moreover, the object-level discriminators take aligned instances as inputs to enforce the realism of individual objects. Our proposed scheme significantly improves the generation quality and achieves state-of-the-art results on various tasks, including segmentation-guided completion, edge-guided manipulation and panoptically-guided manipulation on Places2 datasets. Furthermore, our trained model is flexible and can support multiple editing use cases, such as object insertion, replacement, removal and standard inpainting. In particular, our trained model combined with a novel automatic image completion pipeline achieves state-of-the-art results on the standard inpainting task.

Comparison of CRISPR/Cas9 and Cas12a for gene editing in Chlamydomonas reinhardtii

CRISPR/Cas-based technologies have revolutionized biology, offering a wide range of gene editing and engineering applications due to their diverse enzyme characteristics. Among the CRISPR/Cas nucleases, Cas9, and more recently, Cas12a (formerly known as Cpf1), have been employed in various gene editing applications in many eukaryotes, including the model green alga Chlamydomonas reinhardtii. To provide a comprehensive picture of their applicability in single-strand templated DNA repair and gene editing, we first mapped their target space by analysing the corresponding PAM frequencies, and then compared Cas9 and Cas12a activities by targeting overlapping regions at three independent loci in the Chlamydomonas genome. We identified 8 and 32 times more target sites for Cas9 compared to Cas12a within promoter regions and coding sequences, respectively. We found that Cas9 and Cas12a RNPs- co-delivered with ssODN repair templates- induced similar levels of total editing, achieving as much as 20–30 % in all viably recovered cells. Importantly, the level of precision editing was slightly higher for Cas12a. In contrast, Cas9 alone was able to induce more edits at the FKB12 locus than its Cas12a counterpart, overall making Cas9 the preferable enzyme for genome engineering among the currently available nucleases in C. reinhardtii.

Adaptation of bacterial natural single guide RNA (tracr-L) for efficient plant genome editing.

A long tracrRNA (tracr-L),which naturally act as single guide RNA,and its truncated version, Δtracr-L, from S. pyogenes, efficiently induce Cas9-mediated double-strand breaks (DSBs) in plant genomic loci, as demonstrated by in vitro cleavage assay and protoplast transfection. CRISPR-Cas system provides a form of immune memory in prokaryotes and archaea, protecting them against viruses and foreign genetic elements. In Streptococcus pyogenes, this system includes the pre-crRNA along with another non-coding RNA, tracrRNA, which aids in CRISPR-based immunity. In S. pyogenes, two distinct tracrRNAs are produced: a long form (tracr-L) and a short form (tracr-S). The tracr-S regulates crRNA biogenesis and Cas9 cleavage, while tracr-L suppresses CRISPR-Cas expression by targeting the Cas9 promoter to prevent autoimmunity. Deleting 79 nucleotides from tracr-L results in Δtracr-L, which retains similar functionality in gene repression. This study investigates, for thefirst time, the effectiveness of tracr-L, and Δtracr-L in genome editing within plant systems. In vitro cleavage assays using purified Cas9 and synthesized sgRNAs targeting the Cas9 gene, OsPDS, and the OsSWEET11 promoter revealed that across all target sites, tracr-S demonstrated the highest cleavage efficiency compared to tracr-L and Δtracr-L. For in vivo genome editing, we transfected rice protoplasts with tracr-L, Δtracr-L, and tracr-S, targeting three rice genes: OsPDS, OsSPL14, and the promoter of OsSWEET14. Amplicon deep sequencing revealed various types of indels at the target regions across all three tracrRNA versions, indicating comparable levels of efficiency. This study establishes the utility of both the long-form tracrRNA (tracr-L) and its truncated variant (Δtracr-L) in eukaryote genome editing. These two new forms of tracrRNA provide proof of concept and expand the CRISPR-Cas toolkit for plant genome editing applications, and for eukaryotes more broadly.

Rapid in silico directed evolution by a protein language model with EVOLVEpro.

Directed protein evolution is central to biomedical applications but faces challenges like experimental complexity, inefficient multi-property optimization, and local maxima traps. While in silico methods using protein language models (PLMs) can provide modeled fitness landscape guidance, they struggle to generalize across diverse protein families and map to protein activity. We present EVOLVEpro, a few-shot active learning framework that combines PLMs and regression models to rapidly improve protein activity. EVOLVEpro surpasses current methods, yielding up to 100-fold improvements in desired properties. We demonstrate its effectiveness across six proteins in RNA production, genome editing, and antibody binding applications. These results highlight the advantages of few-shot active learning with minimal experimental data over zero-shot predictions. EVOLVEpro opens new possibilities for AI-guided protein engineering in biology and medicine.

Deciphering Cas9 specificity: Role of domain dynamics and RNA:DNA hybrid interactions revealed through machine learning and accelerated molecular simulations

CRISPR/Cas9 technology is widely used for gene editing, but off-targeting still remains a major concern in therapeutic applications. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. This study compares the dynamics of the highly specific Cas9 from Francisella novicida (FnCas9) to the commonly used SpCas9. Using long-scale atomistic Gaussian accelerated molecular dynamic simulations and machine learning techniques, we deciphered the structural factors behind FnCas9's higher specificity in native and off-target forms. Our analysis revealed that Cas9's cleavage specificity relies more on its domain rearrangement than on RNA:DNA heteroduplex shape, with significant conformational variations in Cas9 domains among off-target forms, while the RNA:DNA hybrid showed minimal changes, especially in FnCas9 compared to SpCas9. REC1-REC3 domains contacts with the RNA:DNA hybrid in FnCas9 acted as critical discriminator of off-target effects playing a pivotal role in influencing specificity. In FnCas9, allosteric signal transmission involves the REC3 and HNH domain, bypassing REC2, leading to a superior efficiency in information transmission. This study offers a quantitative framework for understanding the structural basis of elevated specificity, paving the way for the rational design of Cas9 variants with improved precision and specificity in genome editing applications.

Colorful Diffuse Intrinsic Image Decomposition in the Wild

Intrinsic image decomposition aims to separate the surface reflectance and the effects from the illumination given a single photograph. Due to the complexity of the problem, most prior works assume a single-color illumination and a Lambertian world, which limits their use in illumination-aware image editing applications. In this work, we separate an input image into its diffuse albedo, colorful diffuse shading, and specular residual components. We arrive at our result by gradually removing first the single-color illumination and then the Lambertian-world assumptions. We show that by dividing the problem into easier sub-problems, in-the-wild colorful diffuse shading estimation can be achieved despite the limited ground-truth datasets. Our extended intrinsic model enables illumination-aware analysis of photographs and can be used for image editing applications such as specularity removal and per-pixel white balancing.

A deep mutational scanning platform to characterize the fitness landscape of anti-CRISPR proteins.

Deep mutational scanning is a powerful method for exploring the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, facilitates their characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli that combines synthetic gene circuits based on CRISPR interference with flow cytometry coupled sequencing and mathematical modeling. Using this pipeline, we characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of CRISPR-Cas9. The resulting mutational fitness landscapes revealed considerable mutational tolerance for both Acrs, suggesting an intrinsic redundancy with respect to Cas9 inhibitory features, and - for AcrIIA5 - indicated mutations that boost Cas9 inhibition. Subsequent in vitro characterization suggested that the observed differences in inhibitory potency between mutant inhibitors were mostly due to changes in binding affinity rather than protein expression levels. Finally, to demonstrate that our pipeline can inform Acrs-based genome editing applications, we employed a selected subset of mutant inhibitors to increase CRISPR-Cas9 target specificity by modulating Cas9 activity. Taken together, our work establishes deep mutational scanning as a powerful method for anti-CRISPR protein characterization and optimization.

Instrumentation and application of the CRISPR-Cas system

Abstract. The CRISPR-Cas gene editing system has revolutionized molecular biology by providing a versatile and precise method for genetic manipulation. We explore the basic mechanisms of CRISPR-Cas technology, focusing on the role of Cas proteins and guide RNAs in targeting and cleaving specific DNA sequences. Various types of CRISPR-Cas systems are also discussed, focusing on the widely used CRISPR-Cas9, and their applications in genome editing, including gene knockout, gene knock-in, and gene regulation. Additionally, we present advances in CRISPR-Cas technologies, such as base editing and plasmid editing, demonstrating their potential for therapeutic applications. This paper aims to provide a comprehensive overview of the status and applications of CRISPR-Cas technology and its future directions in gene editing.

Characterizing the collateral activity of CRISPR/Cas13 in mammalian cells: Implications for RNA editing and therapeutic applications

The CRISPR/Cas13 system has garnered attention as a potential tool for RNA editing. However, the degree of collateral activity among various Cas13 orthologs and their cytotoxic effects in mammalian cells remain contentious, potentially impacting their applications. In this study, we observed differential collateral activities for LwaCas13a and RfxCas13d in 293T and U87 cells by applying both sensitive dual-fluorescence (mRuby/GFP) reporter and quantifiable dual-luciferase (Fluc/Rluc) reporter, with LwaCas13a displaying notable activity contrary to previous reports. However, significant collateral RNA cleavage exerted only a modest impact on cell viability. Furthermore, collateral activity of LwaCas13a mildly impeded, but did not arrest, porcine embryo development. Our findings reveal that distinct collateral RNA cleavage by Cas13 slightly suppresses mammalian cell proliferation and embryo development. This could account for the lack of reported collateral effects in numerous prior studies and offers new insights into the implications of the collateral activity of Cas13 for clinical application.

Nanoparticle Carriers: A New Era of Precise CRISPR/Cas9 Gene Editing.

The revolutionary CRISPR/Cas9 gene editing technology holds immense potential for treating genetic diseases and tackling conditions like cancer. However, efficient delivery remains a significant challenge. This is where nanoparticles come into play, emerging as powerful allies in the realm of drug delivery. Nanoparticles can accommodate larger insertion sizes, enabling the incorporation of larger Cas9 enzymes and complex guide RNAs, thus opening up the possibility of editing previously inaccessible genetic regions. Their relatively straightforward and scalable production processes make them cost-effective options for wider applications. Notably, nanoparticles excel in vivo, demonstrating efficient tissue penetration and targeted delivery, which are crucial for maximizing therapeutic impact while minimizing side effects. This review aims to explore the potential of nanoparticle-based delivery systems for CRISPR/Cas9, highlighting their advantages and challenges in gene editing applications. The diverse range of nanoparticles further bolsters their potential. Polymeric nanoparticles, for instance, offer tunable properties for customization and controlled release of the CRISPR cargo. Lipid-based nanoparticles facilitate efficient cellular uptake and endosomal escape, ensuring the CRISPR components reach the target DNA. Even gold nanoparticles, known for their unique biocompatibility and photothermal properties, hold promise in light-activated editing strategies. Non-viral delivery systems, particularly those based on nanoparticles, stand out due to their inherent advantages. Collectively, the evidence paints a promising picture: nanoparticles are not merely passive carriers but active participants in the CRISPR/Cas9 delivery landscape. Their versatility, efficiency, and safety position them as key enablers of a future where gene editing can revolutionize drug development, offering personalized and targeted therapies for a wide range of diseases.

The Influence of Video Editing Applications in Improving Student Learning Achievement in Fiqh Subjects at MTS MTI Pariangan

This research aims to determine how video editing applications improve learning achievement in Fiqh subjects at MTs MTI Pariangan. This type of research is quantitative and uses experimental methods. The population in this study were all class VIII students. Meanwhile, the sample for this research consisted of 46 students, namely class VIII. 1 (as the control class) and class VIII. 2 (as the experimental class). The sampling technique uses cluster random sampling. The results of this study show that the average score between the experimental and control classes is different. Namely, the experimental class is 92.3, and the control class is 69.42. Therefore, it can be seen that the experimental class score is higher than the score of the control class students, and after testing, The prerequisite for normality and homogeneity is that the two samples are declared to have a standard or homogeneous distribution so that research can be carried out in the control class and experimental class. Learning using learning videos edited through video editing applications on prostration of gratitude, prostration of recitations and prostration of sahwi is significantly superior to learning using conventional models. Therefore, video editing applications influence improving student learning achievement.

A small-molecule carrier for the intracellular delivery of a membrane-impermeable protein with retained bioactivity.

Intracellular protein delivery has the potential to revolutionize cell-biological research and medicinal therapy, with broad applications in bioimaging, disease treatment, and genome editing. Herein, we demonstrate successful delivery of a functional protein, cytochrome c (CYC), by using a boron cluster anion as molecular carrier of the superchaotropic anion type (B12Br11OPr2-). CYC was delivered into lipid bilayer vesicles as well as living cells, with a cellular uptake ratio approaching 90%. Mechanistic studies showed that CYC was internalized into cells through a permeation pathway directly into the cytoplasm, bypassing endosomal entrapment. Upon carrier-assisted internalization, CYC retained its bioactivity, as reflected by an induced cell apoptosis rate of 25% at low dose (1 µM). This study furbishes a direct protein delivery method by a molecular carrier with high efficiency, confirming the potential of inorganic cluster ions as protein transport vehicles with an extensive range of future cell-biological or biomedical applications.

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.

Characterization of argonaute nucleases from mesophilic bacteria Pseudobutyrivibrio ruminis

Mesophilic Argonautes (Agos) from microbial resources have received significant attention due to their potential applications in genome editing and molecular diagnostics. This study characterizes a novel Ago from Pseudobutyrivibrio ruminis (PrAgo), which can cleave single-stranded DNA using guide DNA (gDNA). PrAgo, functioning as a multi-turnover enzyme, effectively cleaves DNA using 5′-phosphate gDNA, 14–30 nucleotides in length, in the presence of both Mn2+ and Mg2+ ions. PrAgo demonstrates DNA cleavage activity over a broad pH range (pH 4–12), with optimal activity at pH 11. As a mesophilic enzyme, PrAgo cleaves efficiently DNA at temperatures ranging from 25 to 65 °C, particularly at 65 °C. PrAgo does not show strong preferences for the 5′-nucleotide in gDNA. It shows high tolerance for single-base mismatches, except at positions 13 and 15 of gDNA. Continuous double-nucleotide mismatches at positions 10–16 of gDNA significantly reduce cleavage activity. Furthermore, PrAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at 65 °C. Additionally, molecular dynamic simulations suggest that interactions between the PAZ domain and different nucleic acids strongly influence cleavage efficiency. These findings expand our understanding of Protokaryotic Agos and their potential applications in biotechnology.Graphical

Current Updates of CRISPR/Cas System and Anti-CRISPR Proteins: Innovative Applications to Improve the Genome Editing Strategies.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated sequence (CRISPR/Cas) system is a cutting-edge genome-editing tool employed to explore the functions of normal and disease-related genes. The CRISPR/Cas system has a remarkable diversity in the composition and architecture of genomic loci and Cas protein sequences. Owing to its excellent efficiency and specificity, this system adds an outstanding dimension to biomedical research on genetic manipulation of eukaryotic cells. However, safe, efficient, and specific delivery of this system to target cells and tissues and their off-target effects are considered critical bottlenecks for the therapeutic applications. Recently discovered anti-CRISPR proteins (Acr) play a significant role in limiting the effects of this system. Acrs are relatively small proteins that are highly specific to CRISPR variants and exhibit remarkable structural diversity. The in silico approaches, crystallography, and cryo-electron microscopy play significant roles in elucidating the mechanisms of action of Acrs. Acrs block the CRISPR/Cas system mainly by employing four mechanisms: CRISPR/Cas complex assembly interruption, target-binding interference, target cleavage prevention, and degradation of cyclic oligonucleotide signaling molecules. Engineered CRISPR/Cas systems are frequently used in gene therapy, diagnostics, and functional genomics. Understanding the molecular mechanisms underlying Acr action may help in the safe and effective use of CRISPR/Cas tools for genetic modification, particularly in the context of medicine. Thus, attempts to regulate prokaryotic CRISPR/Cas surveillance complexes will advance the development of antimicrobial drugs and treatment of human diseases. In this review, recent updates on CRISPR/Cas systems, especially CRISPR/Cas9 and Acrs, and their novel mechanistic insights are elaborated. In addition, the role of Acrs in the novel applications of CRISPP/Cas biotechnology for precise genome editing and other applications is discussed.

Organization, density, and content of collagen in the body wall of sea cucumbers Acaudina rosettis and Phyllophorus sp.

Marine collagen has been known to have lower immunogenicity compared to bovine collagen. The primary component in the body wall of sea cucumbers is collagen. This study aimed to investigate the density, bundle organization, and yield of collagen in the two sea cucumber species, Acaudina rosettis and Phyllophorus sp. collected from the Madura Strait, Indonesia. The body wall parts, including the dorsal anterior, dorsal median, dorsal posterior, ventral anterior, ventral median, and ventral posterior, from five samples of each species, were fixed and decalcified. They were histologically processed using the paraffin method before staining them with Van Gieson to visualize collagen in the cross-sections of body wall tissue. Collagen density was determined using the ImageJ software, while collagen bundle organization was examined negatively by utilizing the image editing application. Collagen density data were analyzed using repeated-measures ANOVA (α = 0.05). Collagen content in the body wall of each species was examined using acid solubilization-based collagen extraction, which did not significantly differ between different parts of the body wall in each species (p = 0.161 for A. rosettis, p = 0.095 for Phyllophorus sp.). However, Phyllophorus sp. exhibited a higher collagen density and collagen bundles, which were larger and more organized compared to A. rosettis. The collagen content was higher in A. rosettis (10.07%) compared to Phyllophorus sp. (8.07%). Further study can be performed to explore the potential of collagen from these sea cucumber species for commercial use.

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.

Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells.

Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in a heterogeneous mixture of undesirable editing outcomes. We recently leveraged a type I-F CRISPR-associated transposase (CAST) from the Pseudoalteromonas Tn7016 transposon (PseCAST) for DSB-free, RNA-guided DNA integration in human cells, taking advantage of its programmability and large payload capacity. PseCAST is the only characterized CAST system that has achieved human genomic DNA insertions, but multiple lines of evidence suggest that DNA binding may be a critical bottleneck that limits high-efficiency activity. Here we report structural determinants of target DNA recognition by the PseCAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), which revealed novel subtype-specific interactions and RNA-DNA heteroduplex features. By combining our structural data with target DNA library screens and rationally engineered protein mutations, we uncovered CAST variants that exhibit increased integration efficiency and modified PAM stringency. Structure predictions of key interfaces in the transpososome holoenzyme also revealed opportunities for the design of hybrid CASTs, which we leveraged to build chimeric systems that combine high-activity DNA binding and DNA integration modules. Collectively, our work provides unique structural insights into type I-F CAST systems while showcasing multiple diverse strategies to investigate and engineer new RNA-guided transposase architectures for human genome editing applications.

Molecular approaches for the management of papaya ringspot virus infecting papaya: a comprehensive review.

Papaya ringspot virus (PRSV) is a catastrophic disease that causes huge yield losses in papaya cultivation around the world. Yield losses in severely infected plants can be upto 100%. Because of this disease, papaya cultivation has been shifted to other crops in some areas of the world. Many conventional methods and breeding approaches are used against this disease, which turns out to be less effective. Considering the yield loss caused by PRSV in papaya, it is high time to focus on alternative control methods. To implement effective management strategies, molecular approaches such as Marker Assisted Breeding (MAS) or transgenic methods involving post-transcriptional gene silencing targeting the genome viz., coat protein, replicase gene, or HC Pro can be pursued. However, the public's reluctance to widely accept the transgenic approach due to health and environmental concerns necessitates a consideration of non-transgenic alternatives. Prioritizing safety and ensuring efficient virus control, non-transgenic approaches which encompass cross-protection, genome editing, and topical applications of dsRNA to induce gene silencing within the host, can be adopted. This review aims to provide comprehensive insights of various molecular tools used in managing PRSV which in turn will help in sustainable agriculture.