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CRISPR-Cas9 technology has revolutionized genetic engineering, offering precise and efficient genome editing capabilities. This review explores the application of CRISPR-Cas9 for cystic fibrosis (CF), particularly targeting mutations in the CFTR gene. CF is a multiorgan disease primarily affecting the lungs, gastrointestinal system (e.g., CF-related diabetes (CFRD), CF-associated liver disease (CFLD)), bones (CF-bone disease), and the reproductive system. CF, a genetic disorder characterized by defective ion transport leading to thick mucus accumulation, is often caused by mutations like ΔF508 in the CFTR gene. This review employs a systematic methodology, incorporating an extensive literature search across multiple academic databases, including PubMed, Web of Science, and ScienceDirect, to identify 40 high-quality studies focused on CRISPR-Cas9 applications for CFTR gene editing. The data collection process involved predefined inclusion criteria targeting experimental approaches, gene-editing outcomes, delivery methods, and verification techniques. Data analysis synthesized findings on editing efficiency, off-target effects, and delivery system optimization to present a comprehensive overview of the field. The review highlights the historical development of CRISPR-Cas9, its mechanism, and its transformative role in genetic engineering and medicine. A detailed examination of CRISPR-Cas9's application in CFTR gene correction emphasizes the potential for therapeutic interventions while addressing challenges such as off-target effects, delivery efficiency, and ethical considerations. Future directions include optimizing delivery systems, integrating advanced editing tools like prime and base editing, and expanding personalized medicine approaches to improve treatment outcomes. By systematically analyzing the current landscape, this review provides a foundation for advancing CRISPR-Cas9 technologies for cystic fibrosis treatment and related disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Background: Livestock, particularly cattle, are crucial for biotechnology fields, such as genetic breeding, infectious diseases, bioreactors, and specific disease models. However, genetic engineering in cattle has lagged due to long gestation periods, single embryo pregnancies, and high rearing costs. Additionally, the slow validation of germline transmission and the absence of germline-competent embryonic stem cells hinder progress. With the development of genome editing technologies like ZFN, TALEN, and CRISPR-Cas9, recent advancements have shown that Cas9-expressing pigs and chickens have been successfully produced. We hypothesize that generating CRISPR/Cas9-expressing cattle and their resources will provide a powerful resource for bovine genome editing, advancing our understanding of bovine genetics and disease resistance., Results: In this study, two types of Cas9-expressing cattle were successfully produced: Cas9-RFP-fatty acid dehydrogenase I (FatI), Cas9-GFP-sgRNA for the prion protein (sgPRNP). Somatic cells from these cattle were induced to mutate multiple target genes when single-guide RNAs (sgRNAs) were transfected into the somatic cells. Additionally, semen from Cas9 expressing male cattle was frozen and used to fertilize wild-type oocytes, successfully transmitting the transgene (Cas9, reporter genes, FatI), and sgPRNP) to the next generation. Furthermore, the gene editing capabilities of Cas9, including knockout and high-efficiency knock-in, were confirmed in embryos derived from F1 semen through in vitro production., Conclusion: These data demonstrate, for the first time, that Cas9-expressing cattle were successfully born, and this transgene was transmitted to the next-generation calves (F1) and F2 embryos. In addition, somatic and germ cells derived from F0 and F1generations were used to evaluate the potential for gene editing (knockout and knock-in) in multiple genes. PRNP-mutated F1 cattle are currently being raised as a resistance model for bovine spongiform encephalopathy. These transgenic bovine models and their derivatives will serve as a valuable resource for both in vitro and in vivo genome editing, advancing our genetic understanding of bovine genomics and diseases., Competing Interests: Declarations. Ethics approval and consent to participate: This study was carried out in accordance with the guidelines for Institutional Animal Care and Use Committee of Seoul National University (IACUC # SNU-180403–1-2). Additionally, approval for the animal experiments was obtained from the Institutional Animal Care and Use Committee (IACUC #106) of the Gyeongsangbuk-do Livestock Research Institute, and all relevant national laws and policies regarding animal care and use were followed throughout. All procedures, including embryo transfer for animal production, animal care and rearing, genotyping through tissue sampling, blood collection, and sperm collection, were conducted with approval from the IACUC #106. The heifers were housed in a stanchion barn with adequate space and were fed according to the Korean feeding standard program. Rice straw, mineral blocks, and water were provided without restriction. All animal procedures, including blood collection and embryo transfer, were performed by a veterinarian. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The gene editing field has advanced rapidly since the development of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system because of its applicability in precisely modifying the genome. Among its multiple applications, the correction of genetic diseases has emerged as a potential curative treatment for many disorders that have eluded a cure to date. Despite its efficiency in achieving therapeutic levels of correction, the unexpected adverse effects of editing due to CRISPR/Cas9 nuclease activity are a major concern when translating these new strategies to the clinic. Multiple in silico tools and empirical methods have been developed to evaluate these off-target edits as well as other adverse alterations of the genome, including rearrangements, not only in ex vivo experiments but also in in vivo experiments. In this review, we summarize the available methods for the assessment of off-target effects of CRISPR/Cas9 systems, highlighting their advantages and limitations. Special attention will be paid to their application in pre-clinical studies and clinical trials, both in the manufacturing product and in the long-term follow-up of patients., Competing Interests: Declaration of competing interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Aeromonas hydrophila and Edwardsiella piscicida are significant bacterial pathogens in aquaculture, causing severe diseases and tremendous economic losses worldwide. Additionally, both of them can act as opportunistic pathogens in humans, leading to severe infections. Efficient genome editing tools for these pathogens are essential for understanding their pathogenic mechanisms and physiological behaviors, enabling the development of targeted strategies to control and mitigate their effects. In this study, we adapted the CRISPR-Cas9 system for high-efficiency, marker-less genome editing in multiple naturally isolated strains of these two aquaculture pathogens. We developed a streamlined procedure that successfully generated deletion mutants of the aerA gene (encoding for aerolysin, a pore-forming toxin that plays a critical role in the pathogenicity) and the gfp insertion mutants in three naturally isolated A. hydrophila strains. Additionally, we deleted five putative hemolysin-encoding genes in both A. hydrophila ML10-51K and its ∆aerA derivative. The same system was also applied to the naturally isolated E. piscicida S11-285 strain, successfully deleting the ssaV gene (a component of the Type III Secretion System-a critical virulence mechanism in many pathogenic bacteria). The methodologies developed herein could be broadly applied to other pathogenic strains from natural environments, providing valuable tools for studying bacterial pathogenesis and aiding in the development of effective control strategies., (© 2024 Wiley Periodicals LLC.)

Ensuring sufficient gRNA transcript levels is critical for obtaining optimal CRISPR-Cas9 gene editing efficiency. The standard gRNA scaffold contains a sequence of four thymine nucleotides (4T), which is known to inhibit transcription from Pol III promoters such as the U6 promoter. Our study showed that using standard plasmid transfection protocols, the presence of these 4Ts did not significantly affect editing efficiency, as most of the gRNAs tested (55 gRNAs) achieved near-perfect editing outcomes. We observed that gRNAs with lower activity were T-rich and had reduced gRNA transcript levels. However, this issue can be effectively resolved by increasing transcript levels, which can be readily achieved by shortening the 4T sequences. In this study, we demonstrated this by modifying the sequences to 3TC. Although the 3TC scaffold modification did not improve editing efficiency for already efficient gRNAs when high vector quantities were available, it proved highly beneficial under conditions of limited vector availability, where the 3TC scaffold yielded higher editing efficiency. Additionally, we demonstrated that the 3TC scaffold is compatible with SpCas9 high-fidelity variants and ABEmax base editing, enhancing their editing efficiency. Another commonly used natural Cas9 variant, SaCas9, also benefited from the 3TC scaffold sequence modification, which increased gRNA transcription and subsequently improved editing activity. This modification was applied to the EDIT-101 therapeutic strategy, where it demonstrated marked improvements in performance. This study highlights the importance of shortening the 4T sequences in the gRNA scaffold to optimize gRNA transcript expression for enhanced CRISPR-Cas9 gene editing efficiency. This optimization is particularly important for therapeutic applications, where the quantity of vector is often limited, ensuring more effective and optimal outcomes., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Editing the +58 region of the BCL11A erythroid enhancer has shown promise in treating β-globin disorders. To address variations in fetal hemoglobin (HbF) response, we investigated editing both +58 and +55 enhancers. Rhesus macaques transplanted with edited hematopoietic stem/progenitor cells (HSPCs) following busulfan conditioning exhibited durable, high-level (∼90%) editing frequencies post transplantation with sustained HbF reactivation over 4 years, without hematological perturbations. HbF levels were further boosted by stress erythropoiesis or hydroxyurea. Bone marrow analysis revealed that gene edits were predominantly programmed deletions, programmed inversions, and short indels, each disrupting the enhancer core TGN 7-9 WGATAR half E-box/GATA binding motifs. Nonprogrammed long deletions were disfavored in engrafting cells. CD45 antibody-drug conjugate (ADC) conditioning achieved comparable engraftment and HbF reactivation, whereas lentiviral vector tracking showed polyclonal reconstitution with dynamics similar to animals conditioned with total body irradiation (TBI) or busulfan. Joining CD45-ADC conditioning with combined enhancer editing presents an effective strategy for β-hemoglobinopathies, enabling durable HbF reactivation without chemotherapy., Competing Interests: Declaration of interests R.P., L.L., A.B., K.H., T.T., and L.M.O. were employees of Magenta Therapeutics at the time of the initiation of the project., (Published by Elsevier Inc.)

Squamous cell carcinomas of the head and neck (HNSCC) originate in the upper aerodigestive tract, including the oral cavity, pharynx, and larynx. Current treatments of locally advanced HNSCC often lead to high treatment failure, and disease recurrence, resulting in poor survival rates. Advances in mRNA technologies and lipid nanoparticle (LNP) delivery systems led to several clinical trials involving LNP-CRISPR-Cas9 mRNA-based therapeutics. Despite these advances, achieving cell-type-specific extrahepatic mRNA delivery is still challenging. This study introduces a safe and effective intratumoral EGFR-targeted CRISPR-LNP delivery strategy for knocking out SOX2, which is a cancer-specific gene. To assess their therapeutic potential, it is shown that LNPs made from ionizable lipids with helper lipids co-encapsulating Cas9 mRNA and sgRNA targeting SOX2 (sgSOX2), lead to a ≈60% reduction in HNSCC cell viability in vitro. Next, using a xenograft HNSCC mouse model, targeted delivery of 𝜶EGFR- CRISPR-sgSOX2-LNPs to HNSCC cells resulted in a 90% inhibition of tumor growth and a 90% increase in survival for > 84 days, with tumor disappearance observed in 50% of the mice. These findings emphasize the potential of targeted mRNA-Cas9-LNPs in clinically accessible solid tumors, specifically in reaching tumor cells and inducing persistent therapeutic responses in tumors with high-recurrence rates like HNSCC., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

As global populations grow and climate change increasingly disrupts agricultural systems, ensuring food security and nutritional resilience has become a critical challenge. In addition to grains and legumes, vegetables are very important for both human and animals because they contain vitamins, minerals, and fibre. Enhancing the ability of vegetables to withstand climate change threats is essential; however, traditional breeding methods face challenges due to the complexity of the genomic clonal multiplication process. In the postgenomic era, gene editing (GE) has emerged as a powerful tool for improving vegetables. GE can help to increase traits such as abiotic stress tolerance, herbicide tolerance, and disease resistance; improve agricultural productivity; and improve nutritional content and shelf-life by fine-tuning key genes. GE technologies such as Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR-Cas9) have revolutionized vegetable breeding by enabling specific gene modifications in the genome. This review highlights recent advances in CRISPR-mediated editing across various vegetable species, highlighting successful modifications that increase their resilience to climatic stressors. Additionally, it explores the potential of GE to address malnutrition by increasing the nutrient content of vegetable crops, thereby contributing to public health and food system sustainability. Additionally, it addresses the implementation of GE-guided breeding strategies in agriculture, considering regulatory, ethical, and public acceptance issues. Enhancing vegetable genetics via GE may provide a reliable and nutritious food supply for an expanding global population under more unpredictable environmental circumstances., Competing Interests: Declarations. Ethics approval and consent to participate: Ethics approval and consent to participate are not applicable for this study. Consent for publication: All the authors provided their consent for its publication. Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The CRISPR-Cas9 technology, one of the groundbreaking genome editing methods for addressing genetic disorders, has emerged as a powerful, precise, and efficient tool. However, its clinical translation remains hindered by challenges in delivery efficiency and targeting specificity. This review provides a comprehensive analysis of the structural features, advantages, and potential applications of various non-viral and stimuli-responsive systems, examining recent progress to emphasize the potential to address these limitations and advance CRISPR-Cas9 therapeutics. We describe how recent reports emphasize that nonviral vectors, including lipid-based nanoparticles, extracellular vesicles, polymeric nanoparticles, gold nanoparticles, and mesoporous silica nanoparticles, can offer diverse advantages to enhance stability, cellular uptake, and biocompatibility, based on their structures and physio-chemical stability. We also summarize recent progress on stimuli-responsive nanoformulations, a type of non-viral vector, to introduce precision and control in CRISPR-Cas9 delivery. Stimuli-responsive nanoformulations are designed to respond to pH, redox states, and external triggers, facilitate controlled and targeted delivery, and minimize off-target effects. The insights in our review suggest future challenges for clinical applications of gene therapy technologies and highlight the potential of delivery systems to enhance CRISPR-Cas9's clinical efficacy, positioning them as pivotal tools for future gene-editing therapies., Competing Interests: The authors declare no conflict of interest.

Cardiovascular diseases (CVDs) remain a significant global health challenge, with many current treatments addressing symptoms rather than the genetic roots of these conditions. The advent of CRISPR-Cas9 technology has revolutionized genome editing, offering a transformative approach to targeting disease-causing mutations directly. This article examines the potential of CRISPR-Cas9 in the treatment of various CVDs, including atherosclerosis, arrhythmias, cardiomyopathies, hypertension, and Duchenne muscular dystrophy (DMD). The technology's ability to correct single-gene mutations with high precision and efficiency positions it as a groundbreaking tool in cardiovascular therapy. Recent developments have extended the capabilities of CRISPR-Cas9 to include mitochondrial genome editing, a critical advancement for addressing mitochondrial dysfunctions often linked to cardiovascular disorders. Despite its promise, significant challenges remain, including off-target effects, ethical concerns, and limitations in delivery methods, which hinder its translation into clinical practice. This article also explores the ethical and regulatory considerations surrounding gene editing technologies, emphasizing the implications of somatic versus germline modifications. Future research efforts should aim to enhance the accuracy of CRISPR-Cas9, improve delivery systems for targeted tissues, and ensure the safety and efficacy of treatments in the long term. Overcoming these obstacles could enable CRISPR-Cas9 to not only treat but also potentially cure genetically driven cardiovascular diseases, heralding a new era in precision medicine for cardiovascular health.

Optimized laboratory conditions for research models are crucial for the success of scientific projects. This includes controlling the entire life cycle, having access to all developmental stages and maintaining stable physiological conditions. Reducing the life cycle of a research model can also enhance the access to biological material and speed up genetic tool development. Thus, we optimized the rearing conditions for the sea anemone Nematostella vectensis, a cnidarian research model, to study embryonic and post-metamorphic processes, such as regeneration. We adopted a semi-automated aquaculture system for N. vectensis and developed a dietary protocol optimized for the different life stages. Thereby, we increased spawning efficiencies, juvenile growth and survival rates, and considerably reduced the overall life cycle down to 2 months. To further improve the obtention of CRISPR-Cas9 mutants, we optimized the design of sgRNAs leading to full knockout animals in F0 polyps using a single sgRNA. Finally, we show that NHEJ-mediated transgene insertion is possible in N. vectensis. In summary, our study provides additional resources for the scientific community that uses or plans to use N. vectensis as a research model., Competing Interests: Competing interests O.D. and E.R. own shares in, and O.D. is employee of Planktovie, the supplier of rotifers and RG complete used in this research., (© 2025. Published by The Company of Biologists.)

Key Message: Viral vector-mediated gene editing is enhanced for cultivated tomato under low temperature conditions, enabling higher mutation rates, heritable, and virus-free gene editing for efficient breeding. The CRISPR/Cas system, a versatile gene-editing tool, has revolutionized plant breeding by enabling precise genetic modifications. The development of robust and efficient genome-editing tools for crops is crucial for their application in plant breeding. In this study, we highly improved virus-induced genome-editing (VIGE) system for cultivated tomato. Vectors of tobacco rattle virus (TRV) and potato virus X (PVX) were used to deliver sgRNA targeting phytoene desaturase (SlPDS), along with mobile RNA sequences of tFT or tRNA Ileu , into Cas9-overexpressing cultivated tomato (S. lycopersicum cv. Moneymaker). Our results demonstrate that low temperature significantly enhanced viral vector-mediated gene editing efficiency in both cotyledons and systemic upper leaves. However, no mutant progeny was obtained from TRV- and PVX301-infected MM-Cas9 plants. To address this challenge, we employed tissue culture techniques and found that low-temperature incubations at the initiation stage of tissue culture lead to enhanced editing efficiency in both vectors, resulting in a higher mutation rate (> 70%) of SlPDS in regenerated plants. Heritable gene-edited and virus-free progenies were successfully identified. This study presents a straightforward approach to enhance VIGE efficiency and the expeditious production of gene-edited lines in tomato breeding., Competing Interests: Declarations. Conflict of interest: The authors declare no competing financial interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Granulocyte-colony-stimulating factor (G-CSF) is commonly used to accelerate recovery from neutropenia following chemotherapy and autologous transplantation of hematopoietic stem and progenitor cells (HSPCs) for malignant disorders. However, its utility after ex vivo gene therapy in human HSPCs remains unexplored. We show that administering G-CSF from day 1 to 14 post-transplant impedes engraftment of CRISPR-Cas9 gene-edited human HSPCs in murine xenograft models. G-CSF affects gene-edited HSPCs through a cell-intrinsic mechanism, causing proliferative stress and amplifying the early p53-mediated DNA damage response triggered by Cas9-mediated DNA double-strand breaks. This underscores a threshold mechanism where p53 activation must reach a critical level to impair cellular function. Transiently inhibiting p53 or delaying the initiation of G-CSF treatment to day 5 post-transplant attenuates its negative impact on gene-edited HSPCs. The potential for increased HSPC toxicity associated with post-transplant G-CSF administration in CRISPR-Cas9 autologous HSPC gene therapy warrants consideration in clinical trials., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Genetic modifications using CRISPR-Cas9 have revolutionized cancer research and other preclinical studies. Exceptionally, these efficient tools are inadequate in a few disease models and cell lines due to the aberrant differentiation states and the accumulation of excessive somatic mutations that compromise the robustness of viral gene delivery and stable transduction. A couple of B lymphoma cell lines fall into this category where lentiviral transfection becomes inefficient and exhibits variable efficiency. Additionally, lentiviral delivery requires high biosafety levels. To address this challenge, we have developed a two-step strategy that supports CRISPR-Cas9 through lentivirus and murine ecotropic γ-retrovirus. By engineering B lymphoma cell lines to express Cas9 and mCat1, a specific receptor for ecotropic retroviruses, we enable efficient and safe gene editing through ecotropic γ-retrovirus. We demonstrate the efficacy of this method by generating IgM-deficient B lymphoma cell lines. This innovative approach simplifies protocols, enhances accessibility, and paves the way for standardized gene manipulation of B cell lymphoma models for molecular cell biology research., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Genome-wide CRISPR screens are a powerful tool to interrogate and identify gene function in a wide variety of applications and cell types. CRISPR-Cas9 technology using pooled CRISPR single guide RNA (sgRNA) libraries enables genetic editing in bulk in a large population of cells of interest. After selection of gene-edited cells, phenotyping effects can be evaluated by quantifying abundance (over- or under-representation) of individual sgRNAs using DNA sequencing. In addition to cell survival, these assays can be applied to investigations of drug sensitivity, as well as almost any cellular process with a clear phenotypic read out, for example, receptor internalization, migration, autophagy, and differentiation.Here, taking as an example the identification of molecular components governing B-cell antigen uptake through the B-cell receptor, we describe whole-genome, small-scale, and in vivo validation methods to identify and validate genes regulating BCR-mediated antigen uptake., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The CRISPR/Cas system is a very powerful genome-editing tool that has been developed over the past decade to optimize genome editing for many organisms. Here, we describe a rapid genome-editing method for fission yeast using the CRISPR-Cas9 system. It allows rapid generation of desired auxotrophic and non-auxotrophic strains without perturbing the local genome content by avoiding the insertion of selection markers at target loci., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

With the rapid development of CRISPR-Cas9 technology, gene editing has become a powerful tool for studying gene function. Specifically, in the study of the mechanisms by which natural immune responses combat viral infections, gene knockout mouse models have provided an indispensable platform. This article describes a detailed protocol for constructing gene knockout mice using the CRISPR-Cas9 system. This field focuses on the design of single-guide RNAs (sgRNAs) targeting the antiviral immune gene cGAS, embryo microinjection, and screening and verification of gene editing outcomes. Furthermore, this study provides methods for using cGAS gene knockout mice to analyze the role of specific genes in natural immune responses. Through this protocol, researchers can efficiently generate specific gene knockout mouse models, which not only helps in understanding the functions of the immune system but also offers a powerful experimental tool for exploring the mechanisms of antiviral innate immunity., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Vibrio natriegens is a gram-negative bacterium, which has received increasing attention due to its very fast growth with a doubling time of under 10 min under optimal conditions. To enable a wide range of projects spanning from basic research to biotechnological applications, we developed NT-CRISPR as a new method for genome engineering. This book chapter provides a step-by-step protocol for the use of this previously published tool. NT-CRISPR combines natural transformation with counterselection through CRISPR-Cas9. Thereby, genomic regions can be deleted, foreign sequences can be integrated, and point mutations can be introduced. Furthermore, up to three simultaneous modifications are possible., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

CRISPR-Cas tools have recently been adapted for cell lineage tracing during development. Combined with single-cell RNA sequencing, these methods enable scalable lineage tracing with single-cell resolution. Here, I describe, scGESTALTv2, which combines cumulative CRISPR-Cas9 editing of a lineage barcode array with transcriptional profiling via droplet-based single-cell RNA sequencing (scRNA-seq). The technique is applied in developing zebrafish brains to generate mutations in the barcode array during development. The recorded lineages along with cellular transcriptomes are then extracted via scRNA-seq to define cell relationships among thousands of profiled brain cells and dozens of cell types., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

CRISPR-Cas9 technology is an established, powerful tool for genome editing through the ability to target specific DNA sequences of interest for introduction of desired genetic modifications. CRISPR-Cas9 is utilized for a variety of purposes, ranging from a research molecular biology tool to treatment for human diseases. Due to its prominence across a variety of applications, it is critical that undergraduates in the life sciences are educated on CRISPR-Cas9 technology. To this end, we created an intensive eight-week long course-based undergraduate research experience (CURE) designed for students to understand CRISPR-Cas9 genome editing and perform it in Saccharomyces cerevisiae. Students enrolled in the CURE participate in 2, 3-h sessions a week and are engaged in the entire process of CRISPR-Cas9 genome editing, from preparation of genome editing reagents to characterization of mutant yeast strains. During the process, students master fundamental techniques in the life sciences, including sterile technique, Polymerase Chain Reaction (PCR), primer design, sequencing requirements, and data analysis. The course is developed with flexibility in the schedule for repetition of techniques in the event of a failed experiment, providing an authentic research experience for the students. Additionally, we have developed the course to be easily modified for the editing of any yeast gene, offering the potential to expand the course in research-driven classroom or laboratory settings., (© 2024 The Author(s). Biochemistry and Molecular Biology Education published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Fetal hemoglobin (HbF) reactivation expression through CRISPR-Cas9 is a promising strategy for the treatment of sickle cell disease (SCD). Here, we describe a genome editing strategy leading to reactivation of HbF expression by targeting the binding sites (BSs) for the lymphoma-related factor (LRF) repressor in the γ-globin promoters. CRISPR-Cas9 treatment in healthy donor (HD) and patient-derived HSPCs resulted in a high frequency of LRF BS disruption and potent HbF synthesis in their erythroid progeny. LRF BS disruption did not impair HSPC engraftment and differentiation but was more efficient in SCD than in HD cells. However, SCD HSPCs showed a reduced engraftment and a myeloid bias compared with HD cells. We detected off-target activity and chromosomal rearrangements, particularly in SCD samples (likely because of the higher overall editing efficiency) but did not impact the target gene expression and HSPC engraftment and differentiation. Transcriptomic analyses showed that the editing procedure results in the up-regulation of genes involved in DNA damage and inflammatory responses, which was more evident in SCD HSPCs. This study provides evidence of efficacy and safety for an editing strategy based on HbF reactivation and highlights the need of performing safety studies in clinically relevant conditions, i.e., in patient-derived HSPCs., Competing Interests: Declaration of interests A.M. is named as inventor on a patent describing GE approaches for hemoglobinopathies (WO/2020/053224/PCT/EP2019/074131: Methods for increasing fetal hemoglobin content in eukaryotic cells and uses thereof for the treatment of hemoglobinopathies)., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In this work, we developed a plasmid-based CRISPR-Cas9 strategy for editing Lactococcus cremoris, which allows easy generation of plasmid-free strains with the desired modification. We constructed versatile shuttle vectors based on the theta-type pAMβ1 promiscuous replicon and p15A ori, expressing both the Cas9 nuclease gene (under pH-regulated promoters derived from P170) and a single-guide RNA for specific targeting (under a strong constitutive promoter). The vectors designed for plasmid targeting were very effective for low- and high-copy-number plasmid curing in L. cremoris, and their targeting efficiency was shown to be tunable by regulating cas9 expression. For chromosome editing, we implemented a host-independent method that enhances double-homologous recombination events using plasmids expressing the genes encoding λRed-phage Redβ recombinase and Escherichia coli single-stranded DNA binding protein (EcSSB). By coupling either the endogenous recombination machinery or the Redβ-EcSSB-assisted recombination system with our novel chromosome-targeting CRISPR-Cas9 plasmids, we efficiently generated and selected thousands of gene-edited cells. Examination of the impact of the constructed CRISPR-Cas9 vectors on host fitness revealed no Cas9-associated toxicity, and, remarkably, these vectors exhibited a very high loss rate when growing the bacterial host cells in the absence of selective pressure., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Gene editing technologies, particularly CRISPR-Cas9, have revolutionized agriculture by offering precise and efficient tools to enhance crop production. The vegetatively propagated crops, crucial for global food security, face challenges such as climate change, pests, and limited genetic diversity. CRISPR-Cas9 enables targeted modifications to improve traits like disease resistance, drought tolerance, and nutritional content, thereby boosting productivity and sustainability. Despite its transformative potential, the adoption of gene editing in vegetatively propagated crops is hampered by technical complexities and regulatory frameworks. This review explores recent advancements, challenges, and prospects of gene editing in vegetatively propagated crops, emphasizing strategies to overcome technical barriers and regulatory constraints. Addressing these issues is essential for realizing the full agricultural potential of gene editing and ensuring food security in a changing global climate., Competing Interests: Declaration of competing interest I am writing to declare that there are no conflicts of interest related to this submission. All authors listed have contributed significantly to the research and have approved the final version of the manuscript for submission., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Cystic fibrosis (CF) is a hereditary disorder characterized by mutations in the CFTR gene, leading to impaired chloride ion transport and subsequent thickening of mucus in various organs, particularly the lungs. Despite significant progress in CF management, current treatments focus mainly on symptom relief and do not address the underlying genetic defects. Stem cell and gene therapies present promising avenues for tackling CF at its root cause. Stem cells, including embryonic, induced pluripotent, mesenchymal, hematopoietic, and lung progenitor cells, offer regenerative potential by differentiating into specialized cells and modulating immune responses. Similarly, gene therapy aims to correct CFTR gene mutations by delivering functional copies of the gene into affected cells. Various approaches, such as viral and nonviral vectors, gene editing with CRISPR-Cas9, small interfering RNA (siRNA) therapy, and mRNA therapy, are being explored to achieve gene correction. Despite their potential, challenges such as safety concerns, ethical considerations, delivery system optimization, and long-term efficacy remain. This review provides a comprehensive overview of the current understanding of CF pathophysiology, the rationale for exploring stem cell and gene therapies, the types of therapies available, their mechanisms of action, and the challenges and future directions in the field. By addressing these challenges, stem cell and gene therapies hold promise for transforming CF management and improving the quality of life of affected individuals., Competing Interests: Conflict of Interest Statement. The authors declare that there is no conflict of interest with any financial organization or corporation or individual that can inappropriately influence this work., (© 2024. Huazhong University of Science and Technology.)

Main Conclusion: This paper highlights the need for innovative approaches to enhance cold tolerance. It underscores how genome-editing tools can deepen our understanding of genes involved in cold stress. Cold stress is a significant abiotic factor in high-altitude regions, adversely affecting plant growth and limiting crop productivity. Plants have evolved various mechanisms in response to low temperatures that enable resistance at both physiological and molecular levels during chilling and freezing stress. Several cold-inducible genes have been isolated and characterized, with most playing key roles in providing tolerance against low-temperature stress. However, many plants fail to survive at low temperatures due to the absence of cold acclimatization mechanisms. Conventional breeding techniques, such as inter-specific or inter-genic hybridization, have had limited effectiveness in enhancing the cold resistance of essential crops. Thus, it is crucial to develop crops with improved adaptability, high yields and resistance to cold stress using advanced genomic approaches. The current availability of gene editing tools offers the opportunity to introduce targeted modifications in plant genomes efficiently, thereby developing cold-tolerant varieties. This review discusses advancements in gene editing tools, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)/Cas12a(Cpf1), prime editing (PE) and retron library recombineering (RLR). We focus specifically on the CRISPR/Cas system, which has garnered significant attention in recent years as a groundbreaking tool for genome editing across various species. These techniques will enhance our understanding of molecular interactions under low-temperature stress response and highlight the progress of genome editing in designing future climate-resilient crops., Competing Interests: Declarations. Conflict of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here, we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks, enhancing the reconstitution capacity of edited HSPCs. However, this results in lower HDR efficiency, rendering ex vivo culture necessary yet detrimental. Mechanistically, ex vivo activation triggers a multi-step process initiated by p38 mitogen-activated protein kinase (MAPK) phosphorylation, which generates mitogenic reactive oxygen species (ROS), promoting fast cell-cycle progression and subsequent proliferation-induced DNA damage. Thus, p38 inhibition before gene editing delays G1/S transition and expands transcriptionally defined HSCs, ultimately endowing edited cells with superior multi-lineage differentiation, persistence throughout serial transplantation, enhanced polyclonal repertoire, and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications., Competing Interests: Declaration of interests R.D.M., L.d.V., F.M., A.C., L.N., and S.F. are inventors of patents on applications of gene editing in HSPCs owned and managed by the San Raffaele Scientific Institute and the Telethon Foundation. L.N. is a founder and quota holder of GeneSpire., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Conventional pre-genomics breeding methodologies have significantly improved crop yields since the mid-twentieth century. Genomics provides breeders with advanced tools for whole-genome study, enabling a direct genotype-phenotype analysis. This shift has led to precise and efficient crop development through genomics-based approaches, including molecular markers, genomic selection, and genome editing. Molecular markers, such as SNPs, are crucial for identifying genomic regions linked to important traits, enhancing breeding accuracy and efficiency. Genomic resources viz. genetic markers, reference genomes, sequence and protein databases, transcriptomes, and gene expression profiles, are vital in plant breeding and aid in the identification of key traits, understanding genetic diversity, assist in genomic mapping, support marker-assisted selection and speeding up breeding programs. Advanced techniques like CRISPR/Cas9 allow precise gene modification, accelerating breeding processes. Key techniques like Genome-Wide Association study (GWAS), Marker-Assisted Selection (MAS), and Genomic Selection (GS) enable precise trait selection and prediction of breeding outcomes, improving crop yield, disease resistance, and stress tolerance. These tools are handy for complex traits influenced by multiple genes and environmental factors. This paper explores new genomic technologies like molecular markers, genomic selection, and genome editing for plant breeding showcasing their impact on developing new plant varieties., (© 2024. The Author(s).)

Prostate cancer (PC) is a leading cause of cancer-related deaths in men worldwide. Interleukin-30 (IL-30) is a PC progression driver, and its suppression would be strategic for fighting metastatic disease. Biocompatible lipid nanoparticles (NPs) were loaded with CRISPR-Cas9gRNA to delete the human IL30 (hIL30) gene and functionalized with anti-PSCA-Abs (Cas9hIL30-PSCA NPs). Efficiency of the NPs in targeting IL-30 and the metastatic potential of PC cells was examined in vivo in xenograft models of lung metastasis, and in vitro by using two organ-on-chip (2-OC)-containing 3D spheroids of IL30 + PC-endothelial cell co-cultures in circuit with either lung-mimicking spheroids or bone marrow (BM)-niche-mimicking scaffolds. Cas9hIL30-PSCA NPs demonstrated circulation stability, genome editing efficiency, without off-target effects and organ toxicity. Intravenous injection of three doses/13 days, or five doses/20 days, of NPs in mice bearing circulating PC cells and tumor microemboli substantially hindered lung metastasization. Cas9hIL30-PSCA NPs inhibited PC cell proliferation and expression of IL-30 and metastasis drivers, such as CXCR2, CXCR4, IGF1, L1CAM, METAP2, MMP2, and TNFSF10, whereas CDH1 was upregulated. PC-Lung and PC-BM 2-OCs revealed that Cas9hIL30-PSCA NPs suppressed PC cell release of CXCL2/GROβ, which was associated with intra-metastatic myeloid cell infiltrates, and of DKK1, OPG, and IL-6, which boosted endothelial network formation and cancer cell migration. Development of a patient-tailored nanoplatform for selective CRISPR-mediated IL-30 gene deletion is a clinically valuable tool against PC progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

This narrative review examines the promising potential of integrating artificial intelligence (AI) with CRISPR-Cas9 genome editing to advance CAR T-cell therapy. AI algorithms offer unparalleled precision in identifying genetic targets, essential for enhancing the therapeutic efficacy of CAR T-cell treatments. This precision is critical for eliminating negative regulatory elements that undermine therapy effectiveness. Additionally, AI streamlines the manufacturing process, significantly reducing costs and increasing accessibility, thereby encouraging further research and development investment. A key benefit of AI integration is improved safety; by predicting and minimizing off-target effects, AI enhances the specificity of CRISPR-Cas9 edits, contributing to safer CAR T-cell therapy. This advancement is crucial for patient safety and broader clinical adoption. The convergence of AI and CRISPR-Cas9 has transformative potential, poised to revolutionize personalized immunotherapy. These innovations could expand the application of CAR T-cell therapy beyond hematologic malignancies to various solid tumors and other non-hematologic conditions, heralding a new era in cancer treatment that substantially improves patient outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

CRISPR-Cas9 has emerged as a powerful tool in livestock breeding, enabling precise genetic modifications to address genetic diseases, enhance productivity, and develop disease-resistant animal breeds. A thorough analysis of previous research highlights the potential of CRISPR-Cas9 in overcoming genetic disorders by targeting specific mutations in genes. Furthermore, its integration with reproductive biotechnologies and genomic selection facilitates the production of gene-edited animals with high genomic value, contributing to genetic enhancement and improved productivity. Additionally, CRISPR-Cas9 opens new avenues for developing disease-resistant livestock and creating innovative breeding models for high-quality production. A key trend in the field is the development of multi-sgRNA vectors to correct mutations in various genes linked to productivity traits or certain diseases within individual genomes, thereby increasing resistance in animals. However, despite the potential advantages of CRISPR-Cas9, public acceptance of genetically modified agricultural products remains uncertain. Would consumers be willing to purchase such products? It is essential to advocate for bold and innovative research into genetically edited animals, with a focus on safety, careful promotion, and strict regulatory oversight to align with long-term goals and public acceptance. Continued advancements in this technology and its underlying mechanisms promise to improve poultry products and genetically modified livestock. Overall, CRISPR-Cas9 technology offers a promising pathway for advancing livestock breeding practices, with opportunities for genetic improvement, enhanced disease resistance, and greater productivity., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Conditional regulation is a highly beneficial system for studying the function of essential genes in Plasmodium falciparum and dimerizable Cre recombinase (DiCre) is a recently adapted conditional regulation system suitable for this purpose. In the DiCre system, two inactive fragments of Cre are reconstituted to form a functionally active enzyme in the presence of rapamycin. Different loci have been targeted to generate parasite lines that express the DiCre enzyme. Here, we have used marker-free CRISPR-Cas9 gene editing to integrate the DiCre cassette in a redundant cg6 locus. We have shown the utility of the newly generated ∆cg6DC4 parasites in mediating robust, rapid, and highly specific excision of exogenously encoded gfp sequence. The ∆cg6DC4 parasites are also capable of conditional excision of an endogenous parasite gene, PF3D7_1246000. Conditional deletion of PF3D7_1246000 did not cause any inhibition in the asexual proliferation of the parasites. Furthermore, the health and morphology of the mutant parasites were comparable to that of the control parasites in Giemsa smears. The availability of another stable DiCre parasite strain competent for conditional excision of target genes will expedite functional characterization and validation of novel drug and vaccine targets against malaria., (© 2024. The Author(s).)

Hereditary tyrosinemia type 1 is an autosomal recessive disorder caused by mutations (pathogenic variants) in fumarylacetoacetate hydrolase, an enzyme involved in tyrosine degradation. Its loss results in the accumulation of toxic metabolites that mainly affect the liver and kidneys and can lead to severe liver disease and liver cancer. Tyrosinemia type 1 has a global prevalence of approximately 1 in 100,000 births but can reach up to 1 in 1,500 births in some regions of Québec, Canada. Mutating functionally related "modifier' genes (i.e. genes that, when mutated, affect the phenotypic impacts of mutations in other genes) is an emerging strategy for treating human genetic diseases. In vivo somatic genome editing in animal models of these diseases is a powerful means to identify modifier genes and fuel treatment development. In this study, we demonstrate that mutating additional enzymes in the tyrosine catabolic pathway through liver-specific genome editing can relieve or worsen the phenotypic severity of a murine model of tyrosinemia type 1. Neonatal gene delivery using recombinant adeno-associated viral vectors expressing Staphylococcus aureus Cas9 under the control of a liver-specific promoter led to efficient gene disruption and metabolic rewiring of the pathway, with systemic effects that were distinct from the phenotypes observed in whole-body knockout models. Our work illustrates the value of using in vivo genome editing in model organisms to study the direct effects of combining pathological mutations with modifier gene mutations in isogenic settings., Competing Interests: Conflicts of interest The authors declare that they have no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Sickle cell disease (SCD) is a common, severe genetic blood disorder. Current pharmacotherapies are partially effective and allogeneic hematopoietic stem cell transplantation is associated with immune toxicities. Genome editing of patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers an alternative potentially curative approach to treat SCD. Although the FDA released guidelines for evaluating genome editing risks, it remains unclear how best to approach pre-clinical assessment of genome-edited cell products. Here, we describe rigorous pre-clinical development of a therapeutic γ-globin gene promoter editing strategy that supported an investigational new drug application cleared by the FDA. We compared γ-globin promoter and BCL11A enhancer targets, identified a potent HbF-inducing lead candidate, and tested our approach in mobilized CD34 + hematopoietic stem progenitor cells (HSPCs) from SCD patients. We observed efficient editing, HbF induction to predicted therapeutic levels, and reduced sickling. With single-cell analyses, we defined the heterogeneity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased off-target discovery followed by targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new ex vivo HSC genome editing strategies toward clinical trials for treating SCD and other blood disorders., Competing Interests: Declaration of interests A.S. has received consultant fees from Spotlight Therapeutics, Medexus Inc., Vertex Pharmaceuticals, Sangamo Therapeutics, and Editas Medicine. He is a medical monitor for RCI BMT CSIDE clinical trials, for which he receives financial compensation. He has also received research funding from CRISPR Therapeutics and honoraria from Vindico Medical Education. A.S. is the St. Jude Children’s Research Hospital site principal investigator of clinical trials for genome editing of sickle cell disease sponsored by Vertex Pharmaceuticals/CRISPR Therapeutics (NCT03745287), Novartis Pharmaceuticals (NCT04443907), and Beam Therapeutics (NCT05456880). The industry sponsors provide funding for the clinical trial, which includes salary support paid to A.S.’s institution. A.S. has no direct financial interest in these therapies. J.S.Y. is an equity owner of Beam Therapeutics. M.J.W. is on advisory boards for Cellarity Inc., Novartis, and Forma Therapeutics. S.Q.T. is a co-inventor on licensed patents for CHANGE-seq and other genome engineering technologies. S.Q.T. is a member of the scientific advisory boards of Prime Medicine and Ensoma., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Kluyveromyces marxianus, a thermotolerant, fast-growing, Crabtree-negative yeast, is a promising chassis for the manufacture of various bioproducts. Although several genome editing tools are available for this yeast, these tools still require refinement to enable more convenient and efficient genetic modification. In this study, we engineered the K. marxianus NBRC 104275 strain by impairing the nonhomologous end joining and enhancing the homologous recombination machinery, which resulted in improved homology-directed repair effective on homology arms of up to 40 bp in length. Additionally, we simplified the CRISPR-Cas9 editing system by constructing a strain for integrative expression of Cas9 nuclease and plasmids bearing different selection markers for gRNA expression, thereby facilitating iterative genome editing without the need for plasmid curing. We demonstrated that tRNA was more effective than the hammerhead ribozyme for processing gRNA primary transcripts, and readily assembled tRNA-gRNA arrays were used for multiplexed editing of at least four targets. This editing tool was further employed for simultaneous scarless in vivo assembly of a 12-kb cassette from three fragments and marker-free integration for expressing a fusion variant of fatty acid synthase, as well as the integration of genes for starch hydrolysis. Together, the genome editing tool developed in this study makes K. marxianus more amenable to genetic modification and will facilitate more extensive engineering of this nonconventional yeast for chemical production., (© 2024 Wiley Periodicals LLC.)

The world population is steadily growing, exerting increasing pressure to feed in the future, which would need additional production of major crops. Challenges associated with changing and unpredicted climate (such as heat waves) are causing global food security threats. Cereal crops are a staple food for a large portion of the world's population. They are mostly affected by these environmentally generated abiotic stresses. Therefore, it is imperative to develop climate-resilient cultivars to support the sustainable production of main cereal crops (Rice, wheat, and maize). Among these stresses, heat stress causes significant losses to major cereals. These issues can be solved by comprehending the molecular mechanisms of heat stress and creating heat-tolerant varieties. Different breeding and biotechnology techniques in the last decade have been employed to develop heat-stress-tolerant varieties. However, these time-consuming techniques often lack the pace required for varietal improvement in climate change scenarios. Genome editing technologies offer precise alteration in the crop genome for developing stress-resistant cultivars. CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat/Cas9), one such genome editing platform, recently got scientists' attention due to its easy procedures. It is a powerful tool for functional genomics as well as crop breeding. This review will focus on the molecular mechanism of heat stress and different targets that can be altered using CRISPR/Cas genome editing tools to generate climate-smart cereal crops. Further, heat stress signaling and essential players have been highlighted to provide a comprehensive overview of the topic., Competing Interests: Declaration of competing interest On behalf of all authors, the corresponding author states that there is no conflict of interest., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Background: Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies., Methods: We introduced, as a proof-of-concept, using CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy (designing specific gRNAs), into a Duchenne patient's cell line. The edited cell line was characterized evaluating the dystrophin expression and the myogenic status., Results: Dystrophin expression was restored, and the myogenic defects were ameliorated in the edited myoblasts harbouring a specific del45-55. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations (despite the low cleavage efficiency of our gRNAs) as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55., Conclusions: Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology., (© 2024. The Author(s).)

Background: This study investigates the role of CXXC5 in the self-renewal and differentiation of hematopoietic stem cells (HSCs) within the bone marrow microenvironment, utilizing advanced methodologies such as single-cell RNA sequencing (scRNA-seq), CRISPR-Cas9, and proteomic analysis., Methods: We employed flow cytometry to isolate HSCs from bone marrow samples, followed by scRNA-seq analysis using the 10x Genomics platform to examine cell clustering and CXXC5 expression patterns. CRISPR-Cas9 and lentiviral vectors facilitated the knockout and overexpression of CXXC5 in HSCs. The impact on HSCs was assessed through qRT-PCR, Western blot, CCK-8, CFU, and LTC-IC assays, alongside flow cytometry to measure apoptosis and cell proportions. A mouse model was also used to evaluate the effects of CXXC5 manipulation on HSC engraftment and survival rates., Results: Our findings highlight the diversity of cell clustering and the significant role of CXXC5 in HSC regulation. Knockout experiments showed reduced proliferation and accelerated differentiation, whereas overexpression led to enhanced proliferation and delayed differentiation. Proteomic analysis identified key biological processes influenced by CXXC5, including cell proliferation, differentiation, and apoptosis. In vivo results demonstrated that CXXC5 silencing impaired HSC engraftment in a bone marrow transplantation model., Conclusion: CXXC5 is crucial for the regulation of HSC self-renewal and differentiation in the bone marrow microenvironment. Its manipulation presents a novel approach for enhancing HSC function and provides a potential therapeutic target for hematological diseases., (© 2024. The Author(s) under exclusive licence to The Genetics Society of Korea.)

Sugarcane (Saccharum spp. hybrid) is a prime feedstock for commercial production of biofuel and table sugar. Optimizing canopy architecture for improved light capture has great potential for elevating biomass yield. LIGULELESS1 (LG1) is involved in leaf ligule and auricle development in grasses. Here, we report CRISPR/Cas9-mediated co-mutagenesis of up to 40 copies/alleles of the putative LG1 in highly polyploid sugarcane (2n = 100-120, x = 10-12). Next generation sequencing revealed co-editing frequencies of 7.4%-100% of the LG1 reads in 16 of the 78 transgenic lines. LG1 mutations resulted in a tuneable leaf angle phenotype that became more upright as co-editing frequency increased. Three lines with loss of function frequencies of ~12%, ~53% and ~95% of lg1 were selected following a randomized greenhouse trial and grown in replicated, multi-row field plots. The co-edited LG1 mutations were stably maintained in vegetative progenies and the extent of co-editing remained constant in field tested lines L26 and L35. Next generation sequencing confirmed the absence of potential off targets. The leaf inclination angle corresponded to light transmission into the canopy and tiller number. Line L35 displaying loss of function in ~12% of the lg1 NGS reads exhibited an 18% increase in dry biomass yield supported by a 56% decrease in leaf inclination angle, a 31% increase in tiller number, and a 25% increase in internode number. The scalable co-editing of LG1 in highly polyploid sugarcane allows fine-tuning of leaf inclination angle, enabling the selection of the ideotype for biomass yield., (© 2024 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Although CRISPR-Cas9 gene therapies have proven to be a powerful tool across many applications, improvements are necessary to increase the specificity of this technology. Cas9 cutting in off-target sites remains an issue that limits CRISPR's application in human-based therapies. Treatment of autosomal dominant diseases also remains a challenge when mutant alleles differ from the wild-type sequence by only one base pair. Here, we utilize synthetic peptide nucleic acids (PNAs) that bind selected spacer sequences in the guide RNA (gRNA) to increase Cas9 specificity up to 10-fold. We interrogate variations in PNA length, binding position, and degree of homology with the gRNA. Our findings reveal that PNAs bound in the region distal to the protospacer adjacent motif (PAM) site effectively enhance specificity in both on-target/off-target and allele-specific scenarios. In addition, we demonstrate that introducing deliberate mismatches between PNAs bound in the PAM-proximal region of the gRNA can modulate Cas9 activity in an allele-specific manner. These advancements hold promise for addressing current limitations and expanding the therapeutic potential of CRISPR technology.

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 nonviral 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 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. [Figure: see text].

Soybean Glycine max L., paleopolyploid genome, poses challenges to its genetic improvement. However, the development of reference genome assemblies and genome sequencing has completely changed the field of soybean genomics, allowing for more accurate and successful breeding techniques as well as research. During the single-cell revolution, one of the most advanced sequencing tools for examining the transcriptome landscape is single-cell RNA sequencing (scRNA-seq). Comprehensive resources for genetic improvement of soybeans may be found in the SoyBase and other genomics databases. CRISPR-Cas9 genome editing technology provides promising prospects for precise genetic modifications in soybean. This method has enhanced several soybean traits, including as yield, nutritional value, and resistance to both biotic and abiotic stresses. With base editing techniques that allow for precise DNA modifications, the use of CRISPR-Cas9 is further increased. With the availability of the reference genome for soybeans and the following assembly of wild and cultivated soybeans, significant chromosomal rearrangements and gene duplication events have been identified, offering new perspectives on the complex genomic structure of soybeans. Furthermore, major single nucleotide polymorphisms (SNPs) linked to stachyose and sucrose content have been found through genome-wide association studies (GWAS), providing important tools for enhancing soybean carbohydrate profiles. In order to open up new avenues for soybean genetic improvement, future research approaches include investigating transcriptional divergence processes, enhancing genetic resources, and incorporating CRISPR-Cas9 technologies., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The budding yeast Saccharomyces cerevisiae is an excellent model organism for studying a variety of critical cellular processes. Traditional methods to knock in or -out at specific yeast loci utilize polymerase chain reaction-based techniques, in which marker cassettes with gene-specific homologies are integrated into the genome via homologous recombination. While simple and cost-effective, these methods are limited by marker availability when multiple edits are desired. More recently, CRISPR-Cas9 technology has introduced methods to edit the yeast genome without the need for selectable markers. Although efficient, this method is hindered by additional reagents and lengthy protocols to design and test unique guide RNAs and donor templates for each desired edit. In this study, we have combined these two approaches and have developed a highly efficient economical method to edit the yeast genome marker-free. We have designed two universal donor templates that efficiently repair commonly used selectable markers when targeted by a novel guideRNA-Cas9 designed to promoter regions in Ashbya gossypii found in most integration modules. Furthermore, we find our newly designed guideRNA-Cas9 successfully multiplexes when multiple markers are present. Using these new tools, we have significantly improved the cost and efficiency to generate single or multiple marker-free genetic modifications. In this study, we demonstrate the effectiveness of these new tools by marker-free ablating PRC1, PEP4, and PRB1 vacuolar proteases typically inactivated before many biochemical and membrane-trafficking studies using budding yeast., (© 2024 The Author(s). Yeast published by John Wiley & Sons Ltd.)

Phosphomannomutase 2 deficiency (PMM2-CDG), the most frequent congenital disorder of glycosylation, is an autosomal recessive disease caused by biallelic pathogenic variants in the PMM2 gene. There is no cure for this multisystemic syndrome. Some of the therapeutic approaches that are currently in development include mannose-1-phosphate replacement therapy, drug repurposing, and the use of small chemical molecules to correct folding defects. Preclinical models are needed to evaluate the efficacy of treatments to overcome the high lethality of the available animal model. In addition, the number of variants with unknown significance is increasing in clinical settings. This study presents the generation of a cellular disease model by knocking out the PMM2 gene in the hepatoma HepG2 cell line using CRISPR-Cas9 gene editing. The HepG2 knockout model accurately replicates the PMM2-CDG phenotype, exhibiting a complete absence of PMM2 protein and mRNA, a 90% decrease in PMM enzymatic activity, and altered ICAM-1, LAMP1 and A1AT glycoprotein patterns. The evaluation of PMM2 disease-causing variants validates the model's utility for studying new PMM2 clinical variants, providing insights for diagnosis and potentially for evaluating therapies. A CRISPR-Cas9-generated HepG2 knockout model accurately recapitulates the PMM2-CDG phenotype, providing a valuable tool for assessing disease-causing variants and advancing therapeutic strategies., Competing Interests: Declaration of competing interest The authors declare that they do not have any known personal or financial interests that could have affected the work that is described in this manuscript., (Copyright © 2024 Universidad Autónoma de Madrid. Published by Elsevier Inc. All rights reserved.)

A variety of CRISPR-Cas9-based gene editing technologies have been developed, including gene insertion and gene replacement, and applied to the study and treatment of diseases. While numerous studies have been conducted to improve the efficiency of gene insertion and to expand the system in various ways, there have been relatively few reports on gene replacement technology; therefore, further improvements are still needed in this context. Here, we developed the REMOVER-PITCh system to establish an efficient long-range gene replacement method and demonstrated its utility at two genomic loci in human cultured cells. REMOVER-PITCh depends on microhomology-assisted gene insertion technology called PITCh with highly multiplexed CRISPR-Cas9. First, we achieved gene replacement of about 20-kb GUSB locus using this system. Second, by applying the previously established knock-in-enhancing platform, the LoAD system, along with REMOVER-PITCh, we achieved the replacement of a longer gene region of about 200 kb at the ARSB locus. Our REMOVER-PITCh system will make it possible to remove and incorporate a variety of sequences from and into the genome, respectively, which will facilitate the generation of various disease and humanized models., (© 2024. The Author(s).)

Gene editing technologies have the potential to correct genetic disorders by modifying, inserting, or deleting specific DNA sequences or genes, paving the way for a new class of genetic therapies. While gene editing tools continue to be improved to increase their precision and efficiency, the limited efficacy of in vivo delivery remains a major hurdle for clinical use. An ideal delivery vehicle should be able to target a sufficient number of diseased cells in a transient time window to maximize on-target editing and mitigate off-target events and immunogenicity. Here, we review major advances in novel delivery platforms based on cell-derived vesicles - extracellular vesicles and virus-like particles - for transient delivery of gene editing payloads. We discuss major findings regarding packaging, in vivo biodistribution, therapeutic efficacy, and safety concerns of cell-derived vesicles delivery of gene editing cargos and their potential for clinical translation., Competing Interests: Declaration of Competing Interest B.P.K. is an inventor on patents or patent applications filed by MGB that describe genome engineering technologies. B.P.K. is a consultant for EcoR1 capital and Novartis Venture Fund, and is on the scientific advisory boards of Acrigen Biosciences, Life Edit Therapeutics, and Prime Medicine. B.P.K. has a financial interest in Prime Medicine, Inc., a company developing therapeutic CRISPR-Cas technologies for gene editing. B.P.K.'s interests were reviewed and are managed by MGH and MGB in accordance with their conflict-of-interest policies. L.P.A. laboratory has received funding from Bioblast, Wave Life Sciences, Roche, PTC Therapeutics. L.P.A. laboratory members are inventors on patents or patent applications filed by UC that describe gene therapy applications., (Copyright © 2024. Published by Elsevier B.V.)

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the joints. Although much remains unknown about the pathogenesis of RA, there is evidence that impaired immune tolerance and the development of RA are related. And it is precisely the restoration of immune tolerance at the site of the inflammation that is the ultimate goal of the treatment of RA. Over the past few decades, significant progress has been made in the treatment of RA, with higher rates of disease remission and improved long-term outcomes. Unfortunately, despite these successes, the proportion of patients with persistent, difficult-to-treat disease remains high, and the task of improving our understanding of the basic mechanisms of disease development and developing new ways to treat RA remains relevant. This review focuses on describing new treatments for RA, including cell therapies and gene editing technologies that have shown potential in preclinical and early clinical trials. In addition, we discuss the opportunities and limitations associated with the use of these new approaches in the treatment of RA.

Base editing represents a cutting-edge genome editing technique that utilizes the CRISPR system to guide base deaminases with high precision to specific genomic sites, facilitating the targeted alteration of individual nucleotides. Unlike traditional gene editing approaches, base editing does not require DNA double-strand breaks or donor templates. It functions independently of the cellular DNA repair machinery, offering significant advantages in terms of both efficiency and accuracy. In this review, we summarize the core design principles of various DNA base editors, their distinctive editing characteristics, and tactics to refine their efficacy. We also summarize their applications in crop genetic improvement and explore their potential contributions to forest genetic engineering.

Multiple abiotic stresses like extreme temperatures, water shortage, flooding, salinity, and exposure to heavy metals are confronted by crop plants with changing climatic patterns. Prolonged exposure to these adverse environmental conditions leads to stunted plant growth and development with significant yield loss in crops. CRISPR-Cas9 genome editing tool is being frequently employed to understand abiotic stress-responsive genes. Noteworthy improvements in CRISPR-Cas technology have been made over the years, including upgradation of Cas proteins fidelity and efficiency, optimization of transformation protocols for different crop species, base and prime editing, multiplex gene-targeting, transgene-free editing, and graft-based heritable CRISPR-Cas9 approaches. These developments helped to improve the knowledge of abiotic stress tolerance in crops that could potentially be utilized to develop knock-out varieties and over-expressed lines to tackle the adverse effects of altered climatic patterns. This review summarizes the mechanistic understanding of heat, drought, salinity, and metal stress-responsive genes characterized so far using CRISPR-Cas9 and provides data on potential candidate genes that can be exploited by modern-day biotechnological tools to develop transgene-free genome-edited crops with better climate adaptability. Furthermore, the importance of early-maturing crop varieties to withstand abiotic stresses is also discussed in this review., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Genetic studies in mushrooms, driven by innovations such as CRISPR-Cas9 genome editing and RNA interference, transform our understanding of these enigmatic fungi and their multifaceted roles in agriculture, medicine, and conservation. This comprehensive review explores the rationale and significance of genetic research in mushrooms, delving into the ethical, regulatory, and ecological dimensions of this field. CRISPR-Cas9 emerges as a game-changing technology, enabling precise genome editing, targeted gene knockouts, and pathway manipulation. RNA interference complements these efforts by downregulating genes for improved crop yield and enhanced pest and disease resistance. Genetic studies also contribute to the conservation of rare species and developing more robust mushroom strains, fostering sustainable cultivation practices. Moreover, they unlock the potential for discovering novel medicinal compounds, offering new horizons in pharmaceuticals and nutraceuticals. As emerging technologies and ethical considerations shape the future of mushroom research, these studies promise to revolutionize our relationship with these fungi, paving the way for a more sustainable and innovative world., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)