Genome Editing Technology Research Articles

Highly Efficient Homozygous CRISPR/Cas9 Gene Editing Based on Single-Cell-Originated Somatic Embryogenesis in Liriodendron tulipifera

The clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system is the most widely used gene-editing tool to date. However, its application in the genetic improvement of forestry trees has been largely limited. Here, we first established a highly efficient multi-target editing system in the magnoliid woody plant Liriodendron tulipifera. Using phytoene desaturase gene (PDS) as an example, we systematically compared CRISPR/Cas9 and CRSPR/Cpf1 expression systems for loss-of-function analysis and conducted genetic transformations using transient and stable transformation. Ultimately, our findings indicated that the CRISPR/Cas9 system, when applied to transformation based on single-cell-originated somatic embryogenesis, yielded the highest gene-editing efficiency, with mutation rates of nearly 100%. Furthermore, we obtained a total of 137 regeneration plantlets via somatic embryogenesis, of which 82.48% exhibited an albino phenotype. The Illumina sequencing results of albino seedlings and the callus tissue obtained from dedifferentiation of mutant plants revealed that the mutation at the T1 target site was homozygous. These results indicate that CRISPR/Cas9-based multiplex genome-editing technology can not only accelerate the identification of gene function but also be incorporated into the genetic improvement and breeding of tulip trees, supporting the scale propagation of genome-edited plantlets via somatic embryogenesis.

Advancements in genome editing tools for genetic studies and crop improvement

The rapid increase in global population poses a significant challenge to food security, compounded by the adverse effects of climate change, which limit crop productivity through both biotic and abiotic stressors. Despite decades of progress in plant breeding and genetic engineering, the development of new crop varieties with desirable agronomic traits remains a time-consuming process. Traditional breeding methods often fall short of addressing the urgent need for improved crop varieties. Genome editing technologies, which enable precise modifications at specific genomic loci, have emerged as powerful tools for enhancing crop traits. These technologies, including RNA interference, Meganucleases, ZFNs, TALENs, and CRISPR/Cas systems, allow for the targeted insertion, deletion, or alteration of DNA fragments, facilitating improvements in traits such as herbicide and insect resistance, nutritional quality, and stress tolerance. Among these, CRISPR/Cas9 stands out for its simplicity, efficiency, and ability to reduce off-target effects, making it a valuable tool in both agricultural biotechnology and plant functional genomics. This review examines the functional mechanisms and applications of various genome editing technologies for crop improvement, highlighting their advantages and limitations. It also explores the ethical considerations associated with genome editing in agriculture and discusses the potential of these technologies to contribute to sustainable food production in the face of growing global challenges.

Nature Inspired Delivery Vehicles for CRISPR-Based Genome Editing.

The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genome editing technologies has opened up groundbreaking possibilities for treating a wide spectrum of genetic disorders and diseases. However, the success of these technologies relies heavily on the development of efficient and safe delivery systems. Among the most promising approaches are natural and synthetic nanocarrier-mediated delivery systems, including viral vectors, extracellular vesicles (EVs), engineered cellular membrane particles, liposomes, and various nanoparticles. These carriers enhance the efficacy of the CRISPR system by providing a unique combination of efficiency, specificity, and reduced immunogenicity. Synthetic carriers such as liposomes and nanoparticles facilitate CRISPR delivery with high reproducibility and customizable functions. Viral vectors, renowned for their high transduction efficiency and broad tropism, serve as powerful vehicles for delivering CRISPR components to various cell types. EVs, as natural carriers of RNA and proteins, offer a stealth mechanism to evade immune detection, allowing for the targeted delivery of genome editors with minimal off-target effects. Engineered cellular membrane particles further improve delivery by simulating the cellular environment, enhancing uptake, and minimizing immune response. This review explores the innovative integration of CRISPR genome editors with various nanocarrier systems, focusing on recent advancements, applications, and future directions in therapeutic genome editing.

A CRISPR view on genetic screens inToxoplasma gondii.

Genome editing technologies, such as CRISPR-Cas9, have revolutionised the study of genes in a variety of organisms, including unicellular parasites. Today, the CRISPR-Cas9 technology is vastly applied in high-throughput screens to investigate interactions between the Apicomplexan parasite Toxoplasma gondii and its hosts. In vitro and in vivo T. gondii screens performed in naive and restrictive conditions have led to the discovery of essential and fitness-conferring T. gondii genes, as well as factors important for virulence and dissemination. Recent studies have adapted the CRISPR-Cas9 screening technology to study T. gondii genes based on phenotypes unrelated to parasite survival. These advances were achieved by using conditional systems coupled with imaging, as well as single-cell RNA sequencing and phenotypic selection. Here, we review the state-of-the-art of CRISPR-Cas9 screening technologies with a focus on T. gondii, highlighting strengths, current limitations and future avenues for its development, including its application to other Apicomplexan species.

Perspectives of Genome Editing Mediated Haploid Inducer Systems in Legumes

Genome editing-mediated haploid inducer systems (HISs) present a promising strategy for enhancing breeding efficiency in legume crops, which are vital for sustainable agriculture due to their nutritional benefits and ability to fix nitrogen. Traditional legume breeding is often slow and complicated by the complexity of legumes’ genomes and the challenges associated with tissue culture. Recent advancements have broadened the applicability of HISs in legume crops, facilitating a reduction in the duration of the breeding cycle. By integrating genome editing technology with haploid breeding systems, researchers can achieve precise genetic modifications and rapidly produce homozygous lines, thereby significantly accelerating the development of desired traits. This review explores the current status and future prospects of genome editing-mediated HISs in legumes, emphasizing the mechanisms of haploid induction; recent breakthroughs; and existing technical challenges. Furthermore, we highlight the necessity for additional research to optimize these systems across various legume species, which has the potential to greatly enhance breeding efficiency and contribute to the sustainability of legume production.

Генно-модифицированные организмы: угроза или путь к решению проблемы обеспечения продовольственной безопасности страны?

The article analyzes the concept of "genetically modified organisms" and provides a scheme for their creation. The current state of production of genetically modified crops in the world is considered. The issue of ambiguous attitudes towards GMOs among scientists and the public in the world and in Russia is analyzed. The prospects for the development of new areas of biotechnology in our country, including genomic editing technology, are shown. The analysis of a number of government documents adopted in Russia in recent years aimed at the development and state support of the latest areas of biotechnology is given

EVALUATION OF CRISPR/CAS9 GENOME-EDITING SYSTEM IN HUMAN STEM CELLS HSCS: THERAPEUTICS AND DIAGNOSTICS PROSPECTS

Background: CRISPR/Cas9 genome-editing technology has revolutionized human stem cell (HSC) research, offering novel therapeutic and diagnostic applications. HSCs play a crucial role in regenerative medicine and genetic therapies due to their ability to self-renew and differentiate into various blood cell lineages. The precise genome-editing capability of CRISPR/Cas9 allows for targeted gene modifications, enabling the correction of inherited disorders, disease modeling and the discovery of novel biomarkers. Despite significant advancements, challenges such as off-target effects, delivery efficiency, and ethical concerns persist, requiring further research and optimization. Objective: This systematic review evaluates the therapeutic and diagnostic potential of CRISPR/Cas9 genome editing in HSCs, focusing on its efficacy in gene correction for hematologic disorders, disease modeling and biomarker discovery. Methods: A systematic review was conducted following PRISMA guidelines, analyzing studies published between 2015 and 2024. Literature searches were performed in PubMed, Web of Science, and Scopus using MeSH-aligned keywords. The inclusion criteria encompassed peer-reviewed studies utilizing CRISPR/Cas9 for gene modification in HSCs for therapeutic and diagnostic applications. Exclusion criteria included studies that lacked experimental validation, did not focus on HSCs, or were non-English publications. Out of 85 initially retrieved studies, 40 met the inclusion criteria, and 15 were selected for final synthesis. Results: CRISPR/Cas9 gene-editing strategies in HSCs were categorized as gene knockout (53%), gene activation (40%), and dual knockout/activation (7%). Hematological disorders, including sickle cell anemia and beta-thalassemia, accounted for 35% of studies, demonstrating up to 90% correction in β-globin mutations. Neurodegenerative diseases constituted 20% of studies, where knockout of amyloid precursor protein (APP) in Alzheimer’s models resulted in a 60% reduction in plaque accumulation. Muscular dystrophy studies (10%) showed 75% improvement in dystrophin expression through gene activation. High-throughput CRISPR screening was employed in 15% of studies for biomarker identification. Despite promising outcomes, off-target mutations were observed in 28% of studies, and viral vector-based delivery methods were used in 65%, raising safety concerns. Conclusion: CRISPR/Cas9 genome editing in HSCs presents a ground-breaking approach for treating genetic disorders and enhancing precision medicine. Its potential to correct disease-causing mutations, model complex disorders, and identify novel therapeutic targets is substantial. However, challenges in delivery methods, long-term safety, and ethical considerations remain barriers to clinical translation. Future research should focus on optimizing high-fidelity Cas9 variants, improving non-viral delivery methods, and addressing ethical concerns to ensure the safe and effective application of CRISPR/Cas9 in regenerative medicine.

Treating genetic blood disorders in the era of CRISPR-mediated genome editing.

In the setting of monogenic disease, advances made in genome editing technologies can, in principle, be deployed as a therapeutic strategy to precisely correct a specific gene mutation in an affected cell type and restore functionality. Using the β-hemoglobinopathies and hemophilia as exemplars, we review recent experimental breakthroughs utilizing CRISPR-derived genome editing technology that have translated to significant improvements in the management of inherited hematologic disorders. Yet there are also challenges facing the use of CRISPR mediated genome editing in these patients and we discuss possible ways to obviate those issues for furtherance of clinical benefit.

Establishment of a CRISPR-Cas9-Mediated Genome Editing System in Flax.

Flax is an important crop used for oil and fiber production. Although genetic engineering has been possible in flax, it is not commonly used to produce cultivars. However, the use of genome editing technology, which can produce site-specific mutations without introducing foreign genes, may be a valuable tool for creating elite cultivars that can be easily cultivated. The purpose of this study was to investigate the potential of genome editing in flax by establishing the clustered regularly interspaced short palindromic repeats (CR ISPR)-CRISPR-associated protein 9 (CRISPR-Cas9) genome editing system using the phytoene desaturase (PDS) gene, which produces albino mutants that are easily identifiable. Four sgRNAs were designed from two PDS genes of Flax (LuPDS1 and LuPDS2), and CRISPR-Cas9 genome editing vectors were constructed. After gene transformation, albino phenotypes were observed in transformed callus and regenerated plantlets on selection media. Polymerase chain reaction (PCR) amplification and sequencing of the PDS genes revealed deletions and insertions in the albino tissues, indicating successful editing of the PDS genes. Potential off-target sites were analyzed, but no off-target mutations were found, indicating the specificity of the CRISPR-Cas9 system. The establishment of a flax genome editing system using the CRISPR-Cas9 technology opens up new possibilities for the genetic engineering of flax. This study demonstrates the potential of genome editing in creating elite cultivars that can be easily cultivated, which can have significant implications for the flax industry.

The OsGATA gene family as a promising candidate for applying the CRISPR/Cas genome editing technology to improve the nutritional and yield qualities of rice (Oryza sativa L.)

Molecular breeding of rice (Oryza sativa L.) for yield is of great importance for ensuring food security of the population. Living organisms manifest genetically determined responses to environmental factors, including stressors. Photosynthetic activity affects all metabolic processes in plant cells. The genes involved in photosynthesis, in their turn, are regulated by differentially expressed genes associated with circadian rhythms. Plants, as sedentary organisms, require more efficient regulation of gene expression. GATA factors are transcription factors (TFs) that affect the production of phytohormones and mediate the stress response. GATA factors are divided into four main classes (A to D), based on the difference in the structure of the zinc finger domain, and into seven subfamilies, depending on the availability of additional domains. GATA TFs incorporate domain structures that may be involved in the regulation of circadian rhythms. Effects on the circadian rhythms influence other regulatory metabolic pathways in plants, which makes the study of genes associated with circadian rhythms relevant and significant. The most well-known and popular method of gene editing at the moment is the CRISPR/Cas technology. More than 30 rice genes were successfully genomically edited using the CRISPR/Cas technology in the period from 2018 through 2023. This helped to improve their valuable agronomic traits.This review summarizes all information about the classification and known functions of OsGATA genes and OsGATA TFs and provides evidence for the possibility of influencing the regulation of rice photoperiodicity by editing these genes.

PcoCas12a: A novel CRISPR enzyme from Prevotella copri enhancing TCR-T-cell tumor suppression.

Genome editing technologies have been widely utilized in cell engineering, demonstrating immense potential in cell and gene therapy. However, an optimal gene-editing enzyme for immune cell editing remains unidentified. In this study, we identified a novel gene editing enzyme, termed CRISPR/PcoCas12a, derived from Prevotella copri, which recognizes a 5'-YYN PAM sequence. We demonstrated that CRISPR/PcoCas12a offers a broader range of editing sites and superior editing efficiency at specific loci compared to AsCas12a. Furthermore, we illustrated its capability to enhance tumor suppression by targeting DGKα in TCR-T cells. DGKα functions as a negative regulator of T cell function, and its knockout significantly boosts the antitumor efficacy of TCR-T cells. The knockout efficiency and tumor suppressor ability of PcoCas12a targeting DGKα were markedly higher than those achieved with AsCas12a. Single-cell sequencing data confirmed that PcoCas12a-mediated DGKα gene knockout improves the tumor suppressive capabilities of T cells by promoting T-cell activation and strengthening immune regulatory responses. These findings establish PcoCas12a as a highly efficient enzyme for T cell editing, indicating its potential application in T-cell therapy.

Immunoinformatics: A Veritable Toolbox for Livestock Omics and Veterinomics.

Immunoinformatics, an integrative field consisting of bioinformatics and immunology, has showcased its potential in addressing zoonotic diseases, as evidenced during the Coronavirus disease 2019 (COVID-19) pandemic. However, its application in livestock health remains largely untapped. This opinion commentary explores how immunoinformatics, combined with advancements in genomics, multi-omics integration, and genome editing technologies, can revolutionize livestock management by enhancing disease resistance, vaccine development, and productivity. We examine the current and critical challenges, such as limited breed-specific data, computational barriers, and economic constraints, while highlighting the transformative potential of immunoinformatics in addressing these issues. We underscore the importance of leveraging immunoinformatics to bridge the phenotype-genotype gap, develop effective diagnostics and vaccines, and tackle emerging pathogens with zoonotic potential. By emphasizing interdisciplinary collaboration and the need for accessible veterinomics solutions, particularly in low- and middle-income countries, we outline herein the actionable steps to harness immunoinformatics for sustainable livestock management and global food security. In all, this opinion commentary aims to inspire a renewed focus on and veritable innovations in planetary health by unpacking and recognizing immunoinformatics' key role in shaping the future of veterinomics, the convergence of veterinary medicine with omics systems science, in the pursuit of agricultural sustainability.

Breeding by Design for Functional Rice with Genome Editing Technologies.

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.

The ift140 -Deficient Zebrafish: A Model for Renal Cystogenesis and an F0-Based Screen to Identify Genetic Modifiers of Kidney Cysts.

Genetic modifiers are believed to play an important role in the onset and severity of polycystic kidney disease (PKD), but identifying these modifiers has been challenging due to the lack of effective methodologies. In this study, we investigated zebrafish mutants of IFT140 , a newly identified ADPKD gene, and observed phenotypes similar to those seen in mammalian models, including kidney cysts and bone defects. Using efficient microhomology-mediated end joining (MMEJ)-based genome editing technology, we demonstrated that ift140 CRISPRants recapitulate mutant phenotypes while bypassing the early lethality of the mutants to allow for renal cyst analysis in adult fish. In addition to cilia defects, non-cilia phenotypes, such as disrupted cell polarity and aberrant microtubule stabilization in kidney epithelial cells, may also contribute to ift140 -associated cystogenesis. Importantly, the ability to detect ift140 -associated renal cysts with ease allowed us to develop an F0-based genetic screen to identify potential protective modifiers. A pilot screen of sixteen genes previously linked to dysregulated signaling pathways in ADPKD revealed both known and novel modifiers, including mtor and ulk1a . Inhibition of mtor and ulk1a reversed both cilia-related and non-cilia-related abnormalities. In summary, our study underscores the potential of using zebrafish as a model for the efficient discovery of genetic modifiers of kidney cysts.

CRISPR challenges in clinical developments.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated proteins) is a novel genome editing technology with potential applications in treating diseases. Currently, its use in humans is restricted to clinical trials, although its growth rate is significant, and some have received initial FDA approval. It is crucial to examine and address the challenges for this technology to be implemented in clinical settings. This review aims to identify and explore new research ideas to increase of CRISPR's efficiency in treating genetic diseases and cancer, as well as its future prospects. Given that a substantial amount of previous research has focused on CRISPR-Cas delivery strategies and materials, this overview introduces specific conditions and strategies. It also discusses some of the challenges and opportunities in this field, offering a unique perspective.

Ovomucoid-null eggs produced via genome-editing technology: Protein composition and physicochemical properties

Ovomucoid-null eggs produced via genome-editing technology: Protein composition and physicochemical properties

Current approaches in CRISPR-Cas systems for diabetes.

In the face of advancements in health care and a shift towards healthy lifestyle, diabetes mellitus (DM) still presents as a global health challenge. This chapter explores recent advancements in the areas of genetic and molecular underpinnings of DM, addressing the revolutionary potential of CRISPR-based genome editing technologies. We delve into the multifaceted relationship between genes and molecular pathways contributing to both type1 and type 2 diabetes. We highlight the importance of how improved genetic screening and the identification of susceptibility genes are aiding in early diagnosis and risk stratification. The spotlight then shifts to CRISPR-Cas9, a robust genome editing tool capable of various applications including correcting mutations in type 1 diabetes, enhancing insulin production in T2D, modulating genes associated with metabolism of glucose and insulin sensitivity. Delivery methods for CRISPR to targeted tissues and cells are explored, including viral and non-viral vectors, alongside the exciting possibilities offered by nanocarriers. We conclude by discussing the challenges and ethical considerations surrounding CRISPR-based therapies for DM. These include potential off-target effects, ensuring long-term efficacy and safety, and navigating the ethical implications of human genome modification. This chapter offers a comprehensive perspective on how genetic and molecular insights, coupled with the transformative power of CRISPR, are paving the way for potential cures and novel therapeutic approaches for DM.

Diverse Cre recombinase expression pattern in Albumin-Cre driver rats

Rats (Rattus norvegicus) have been widely utilized as model animals due to their physiological characteristics, making them suitable for surgical and long-term studies. They have played a crucial role in biomedical research, complementing studies conducted in mice. The advent of genome editing technologies has facilitated the generation of genetically modified rat strains, advancing studies in experimental animals. Among these innovations, Cre-driver rat models have emerged as powerful tools for spatiotemporal control of gene expression. However, their development and characterization remain less advanced compared to mouse models. In this study, we developed liver-targeting Cre knock-in rats and reporter knock-in rats to evaluate Cre recombinase expression profiles in different genetic contexts. Our results revealed that insertion orientation and promoter origin significantly influence Cre expression patterns. Notably, forward insertion of the Albumin (Alb) promoter-driven Cre sequence at the ROSA26 locus resulted in ubiquitous Cre expression, while reverse insertion confined Cre expression predominantly to the liver. Interestingly, Cre expression under an endogenous Alb promoter unexpectedly induced expression in non-liver tissues, which may suggest a potential link to the in vivo dynamics of albumin. These findings underscore the importance of rigorous characterization in Cre-based transgenic systems. By elucidating the roles of promoter origin, insertion site, and orientation, our study provides valuable insights for optimizing Cre-driver rat models. These findings pave the way for refining genetic strategies to enhance tissue specificity and reliability in functional genomics and disease modeling.

Targeted C-to-T Base Editing in the Arabidopsis Plastid Genome.

Arabidopsis thaliana, particularly the ecotype Columbia-0 (Col-0), has been extensively employed in the study of genetics of the nuclear genome. However, the difficulty of modifying the plastid genome of Col-0, the most widely used ecotype, has hindered investigation of the functional interactions between nuclear-encoded and plastid-encoded genes in this ecotype. Recently, we achieved targeted base editing, substituting a specific C:G pair with a T:A pair in the plastid genome of Col-0 through the application of genome-editing technology. This article introduces the method employed to accomplish this targeted base editing. The process involves four steps: (i) designing and constructing a binary vector encoding the genome-editing enzyme, (ii) introducing the binary vector into the nuclear genome of Col-0 via floral dipping, (iii) identifying base-edited plants, and (iv) verifying inheritance of the edited base(s) by the next generation. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design and construction of a binary vector encoding ptpTALECD or ptpTALECD_v2 Basic Protocol 2: Agrobacterium-mediated introduction of a binary vector into the Arabidopsis nuclear genome Basic Protocol 3: Selection of plants harboring T-DNA in the nucleus and detection of base editing in the plastid genome.

Increasing the Level of Knock-in of a Construct Encoding the HIV-1 Fusion Inhibitor, MT-C34 Peptide, into the <i>CXCR4</i> Locus in the CEM/R5 T Cell Line

The low efficiency of knock-in, especially in primary human cells, limits the use of genome editing technology for therapeutic purposes, which makes it important to develop approaches for increasing knock-in levels. In this work, using a knock-in model of the peptide fusion inhibitor of HIV MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line, we analyzed the effectiveness of several approaches to increasing knock-in levels. First, donor DNA modification aimed at improving the efficiency of plasmid transport into the nucleus was evaluated, namely the introduction into the donor plasmid of the SV40 DNA transport sequence (DTS) or the binding sites for the transcription factor NF-κB, whose effects on knock-in levels have not been described. In the MT-C34 knock-in model into the CXCR4 locus, this modification was ineffective. The second approach, modifying the Cas9 nuclease by introducing two additional nuclear localization signals (NLS), increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining pathway using DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. The combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the nonhomologous end joining pathway significantly increased the level of knock-in of the HIV-1 peptide fusion inhibitor into the clinically relevant locus CXCR4, which can be used to develop effective gene therapy approaches for the treatment of HIV infection.