CRISPR-based genome editors are fundamentally limited by their requirement for double-strand DNA breaks (DSBs), restricted transgene cargo capacity, and reliance on error-prone endogenous DNA repair mechanisms. Non–long terminal repeat (non-LTR) retrotransposons—especially the site-specific R2 element—offer a mechanistically distinct and potentially safer choice for programmable genomic integration. These elements employ target-primed reverse transcription (TPRT)—an RNA-templated integration mechanism that circumvents DSB formation and supports amplification of self-copy. This review delineates the molecular mechanism of R2 retrotransposons, emphasizing their highly specific integration into the 28 S ribosomal DNA locus—a recognized genomic safe harbor. We describe the functional domains of the R2 protein, including the reverse transcriptase, restriction-like endonuclease, and nucleic acid-binding motifs, and explain how they coordinate to achieve precise DNA cleavage and cDNA synthesis. Recent cryo–electron microscopy (cryo-EM) structures have revealed discrete RNA-protein complex that orchestrate the stepwise progression of TPRT. Informed by these mechanistic insights, researchers have engineered programmable platforms—including PRINT and STITCHR—that enable RNA-directed transgene integration in mammalian systems. These platforms establish R2 as a viable all-RNA programmable system for targeted genomic integration. Future directions include reprogramming the DNA-binding specificity of R2 through protein engineering to target loci, optimizing integration fidelity and efficiency, and mining diverse R2-like elements from metagenomic data. With continued optimization and rigorous safety validation, R2-derived platforms could supplant current nuclease-dependent editors in applications requiring high-fidelity, large-cargo integration.
Chromatin presents a significant obstacle to CRISPR-Cas gene editing, as chromatin restricts nuclease access to DNA. Recent advances have produced a wide range of high-fidelity Cas9 and Cas12a variants with enhanced properties. However, their precision in targeting DNA within different contexts remains poorly understood. This gap limits our ability to predict and optimize Cas performance in the dynamic chromatin landscape. To elucidate how chromatin variability impacts Cas editing accuracy, we utilized GEMiNI-seq to systematically profile wild-type and engineered Cas9 and Cas12a nucleases across a range of nucleosome sequences. All nucleases showed reduced cleavage in nucleosomal DNA relative to naked DNA, with the strongest inhibition at dyad-proximal sites. Cleavage within nucleosomes was highly variable, with wtSpCas9 exhibiting up to 65-fold different activity depending on the nucleosome type. Editors with high catalytic activity (wtSpCas9, HIFIv2, LbCas12a ULTRA) consistently outperformed high-fidelity variants such as evoSpCas9, which displayed excellent specificity on naked DNA but poor performance in nucleosomal contexts. ROC and PRC analyses revealed that nucleosome sequence and orientation shape both sensitivity and specificity, with HIFIv1 emerging as the top-performing nuclease for nucleosomal targets, while evoSpCas9 excelled in exposed contexts. Our findings demonstrate that local nucleosome sequence and structure profoundly influence Cas nuclease accessibility and specificity. Variability in cleavage across nucleosome types underscores the need to consider chromatin context during target selection and nuclease design. These results provide a framework for selecting or engineering Cas editors optimized for therapeutic genome editing within chromatin.
BackgroundChimeric antigen receptor T-cell therapy has changed the treatment landscape of relapsed or refractory hematological malignancies, but primary non-response and post-infusion relapse remain frequent clinical problems. In aggressive B-cell lymphomas, acute leukemias, and multiple myeloma, treatment failure is often driven by overlapping mechanisms rather than a single resistance pathway. These include antigen loss or reduced antigen density, impaired immune recognition, defective inflammatory signaling, checkpoint-mediated suppression, metabolic stress, and limited effector-cell persistence within suppressive disease niches.Main BodyGenome engineering has become an important tool for both identifying and addressing these resistance mechanisms. CRISPR-based functional screening, single-cell perturbation approaches, and multi-omics profiling allow immune escape and tumor microenvironment-mediated resistance to be defined more functionally, rather than inferred only from correlative datasets. These insights can inform the design of CAR-T and CAR-NK therapies through multi-target or logic-gated receptors, checkpoint or exhaustion-pathway editing, cytokine-supported and armored constructs, metabolic fitness enhancement, and selected multiplex-editing strategies. In parallel, CAR-NK cells, universal allogeneic CAR-T products, and stem-cell-derived platforms may provide additional options in relapse-prone or heavily pretreated patients, particularly when autologous T-cell fitness, manufacturing feasibility, or repeat dosing is a concern.ConclusionA resistance-guided approach may help align engineered cellular therapy design with the dominant mechanisms of treatment failure in high-risk hematological malignancies. Rather than simply increasing engineering complexity, future CAR-T and CAR-NK development should link each modification to a measurable resistance mechanism, a feasible biomarker, and a clinically testable benefit. Prospective validation, genomic safety assessment, manufacturing consistency, and long-term monitoring will be essential before resistance-matched cellular immunotherapy can be broadly integrated into clinical practice.
Gene editing holds both hope and despair, necessitating vigilant, ethicolegal oversight to harness its advancement and avoid unintended effects like off-target effects and mosaicism. Despite efforts, governance fragmentation persists regionally and globally. Africa needs a phased, harmonized, continental governance framework for gene editing that combines regional oversight, national implementation, accountability, data governance, genomic sovereignty, and public participation. Policy harmonization is close to accomplishment, but obstacles persist. This paper proposes a practical continent-wide model anchored on African conformity, national legislative capacities, communal engagement, and data sharing. It presents an operational plan encompassing grassroots, nation-state, and regional regulatory bodies, centralized on a new pan-African agency for optimal oversight and governance. The model incorporates bioethical principlism and Ubuntu philosophy, forming the foundations of accountable and safe gene editing research and practice. Trespassing these foundations is a severe, inter-generational, and irresponsible act. This policy review critically analyzes the literature and evidence around gene editing, examining uncertainties, dilemmas, biases, and gaps in concepts. It presents an interpretation of the analysis in a narrative discourse with policy recommendations and governance reforms. The authors relied on empirical evidence and normative arguments. Gene editing is a multifaceted issue, yet a recourse for many patients with devastating diseases. Global progress has been hindered by procedural and operational defects and the controversy of enhancement versus therapy. Declarations, guidelines, and conventions should be prioritized to achieve universal benefits. A diligent consideration of collective risks is crucial to designing effective and inclusive policies. A multi-level approach is imperative for maintaining momentum and pace in technology development and localization.
Efficient delivery of CRISPR components remains a major determinant of genome editing outcomes. In this study, we compared conventional lipofection with magnetic nanoparticle-assisted gene delivery (magnetofection) for CRISPR-mediated genome editing efficiency using SpCas9 and AsCas12a systems. Based on the average values obtained from multiple independent targets, lipofection resulted in relatively low indel efficiencies, with mean values of average 8.1%–12.47%. In contrast, magnetofection markedly enhanced genome editing outcomes, yielding average indel efficiencies of average 42.29%–45.04%, representing a substantial increase (3.39- and 5.56-fold, respectively) compared with lipofection. This enhancement was consistently observed across both SpCas9-and AsCas12a-mediated editing, indicating that the improved efficiency conferred by magnetic nanoparticle delivery is independent of the nuclease platform. Furthermore, the increased performance of magnetofection was reproducible across multiple genomic loci and cell lines and was also effective under RNP-based delivery conditions, demonstrating its robustness and reliability. In addition to indel-based genome disruption, magnetofection also significantly improved prime editing efficiency (13.95% on average) compared to lipofection (3.81% on average). Overall, our results demonstrate that magnetic nanoparticle-mediated delivery enables highly efficient and reproducible CRISPR genome editing, substantially outperforming conventional lipofection for both indel formation and prime editing. Magnetofection therefore represents a powerful and broadly applicable delivery strategy for next-generation genome editing applications.
Sugarbeet (Beta vulgaris ssp. vulgaris, L.), is a vital temperate crop, supplying nearly 40% of the world's sugar. However, its high susceptibility to bacterial, fungal, and viral diseases creates an urgent need for improved, disease-resistant cultivars. The CRISPR/Cas9 system has rapidly advanced plant genetic engineering by enabling precise and targeted genome modifications. Our goal is to establish a gene-editing platform in sugarbeet to support future development of disease-resistant lines by targeting the candidate genes. In this study, we applied CRISPR/Cas9 to generate targeted mutations in two genes involved in chlorophyll biosynthesis and carotenoid-mediated leaf pigmentation: magnesium chelatase (Mg-chelatase) and phytoene desaturase (PDS). Two CRISPR/Cas9 constructs, each carrying an sgRNA targeting either Mg-chelatase or PDS, were developed and mobilized into Agrobacterium tumefaciens. A total of 233 and 200 hypocotyl explants were transformed with constructs targeting Mg-chelatase and PDS, resulting in regeneration efficiencies of 8% and 14% on kanamycin selection medium, respectively. Light green, yellow, variegated yellow-green, and albino phenotypes were observed among the putative transformants, whereas non-edited transformed lines resembled untransformed control plants. Targeted mutations, including insertions, deletions, and substitutions of nucleotides, were identified at both genomic loci, with editing efficiencies of 60.0% for Mg-chelatase and 68.75% for PDS underscoring the effectiveness of this approach in sugarbeet, a recalcitrant crop. Deletions ranged from 5 to 28 bp in Mg-chelatase and 2 to 21 bp in PDS, while insertion events consisted of single-base additions in Mg-chelatase edited lines and larger insertions of 7-16 bp in PDS mutants. The results demonstrate the successful deployment of CRISPR/Cas9 for targeted genome engineering in sugarbeet and establish a reliable platform for future gene-editing efforts aimed at enhancing resistance to a wide range of pathogens and diseases affecting the crop.
Introduction:The rapid development of genome editing technologies has enabled precise manipulation of livestock genomes for the improvement of production traits such as meat yield and milk quality. Myostatin (MSTN) and β-lactoglobulin (BLG) are key gene targets for enhancing muscle growth and reducing lactose intolerance, respectively. Methods:In this study, we employed an optimized CRISPR/EOCas12i system to simultaneously target MSTN and BLG in bovine fetal fibroblasts (BFFs) using a single plasmid. Results:T7E1 and Sanger sequencing confirmed efficient editing at multiple target sites, with EOCas12i producing deletions ranging from tens to over 100 bp. Furthermore, no off-target (OT) effects were detected at predicted loci, supporting the high specificity of this system in large animals. Gene-edited single-cell clones (SCCs) were expanded in conditioned medium, and selected double-knockout (DKO) clones served as nuclear donors for somatic cell nuclear transfer (SCNT) to produce MSTN/BLG double gene-edited cattle embryos. Discussion:Collectively, this study demonstrates the feasibility of generating MSTN/BLG double gene-edited cattle embryos using a single CRISPR/EOCas12i plasmid and SCNT, providing a robust platform for multiplex genome editing aimed at improving meat production and milk traits, with potential applications in both agricultural and biomedical research.
Domestic ferrets have served as experimental animals for over a century, offering a balance between practicality and biological relevance while bridging the gap between rodent and non-human primate models. Their anatomical and physiological similarities to humans, particularly in the respiratory and nervous systems, make ferrets an invaluable model for biomedical research across various disciplines. Recent advances in genome-editing technologies have enabled the efficient creation of genetically modified (GM) ferrets, significantly expanding their applications in basic and translational research. This review describes the unique biological characteristics of ferrets and their current applications in modeling human pathogenic infections, respiratory, gastrointestinal, and neurological disorders, as well as other complex diseases that rodent models fail to effectively recapitulate. We also discuss the challenges associated with ferret models and outline the future directions to enhance their utility, with the goal of advancing our understanding of human diseases and supporting the development of novel therapeutics.
CRISPR/Cas genome editing tools represent a promising technology for biomedicine with significant therapeutic potential for numerous human diseases. However, efficient delivery of these tools into primary cells, particularly in the form of ribonucleoprotein (RNP) complexes, remains a critical bottleneck that limits clinical translation. Virus-like particles (VLPs) derived from human immunodeficiency virus type 1 (HIV-1) or murine leukemia virus (MLV) have emerged as promising delivery vehicles for RNP complexes, yet their activity is limited by suboptimal nuclease and guide RNA packaging. Previously, we generated NanoMEDIC VLPs incorporating the AsCas12a nuclease with CMV-driven crRNA, which demonstrated substantially enhanced editing efficiency over SpCas9-VLPs with U6-driven gRNA. Here, we describe a detailed protocol for a small-scale production of AsCas12a-VLPs using three distinct transfection methods [cationic lipids, polyethyleneimine (PEI), and calcium-phosphate] and a large-scale production of VLPs using calcium-phosphate transfection. We show that both production scales yield comparable nuclease loading into VLPs and similar editing efficiencies, reaching up to 60% of CXCR4 knockout in Jurkat T cells.
Influenza A virus (IAV) and influenza B virus (IBV) remain major global public health threats because of their rapid antigenic evolution and efficient human-to-human transmission. In contrast, influenza C virus (ICV) and influenza D virus (IDV) generally exhibit narrower host ranges and milder pathogenicity, yet their potential for interspecies transmission and zoonotic spillover still warrants attention. Conventional prevention strategies, such as inactivated and live-attenuated vaccines, suffer from prolonged development timelines and diminished efficacy against rapidly evolving viral strains. However, antiviral drugs are increasingly limited by the rapid emergence of drug-resistant variants. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene-editing technology has emerged as a promising platform for influenza prevention and control owing to its programmability and precise targeting capability. In this paper, we summarize recent advances in CRISPR-based strategies for influenza prevention and control. The RNA-targeting CRISPR-associated protein 13 (Cas13) system can recognize conserved viral RNA sequences and suppress replication across influenza subtypes, whereas the DNA-targeting CRISPR-associated protein 9 (Cas9) system can edit host susceptibility genes and thereby reduce cellular permissiveness to infection. In addition, lipid nanoparticle (LNP)-based delivery systems have become important tools for improving the in vivo delivery and expression of CRISPR components by enhancing targeting efficiency and reducing immunogenicity. CRISPR-based diagnostics, such as Specific High-sensitivity Enzymatic Reporter unLOCKing (SHERLOCK), further expand the clinical utility of this technology by enabling rapid and sensitive detection of influenza viruses. Despite these advances, substantial challenges remain, including delivery inefficiency, off-target activity, long-term safety concerns, and the risk of viral escape. With continued technological refinement and careful translational development, CRISPR may become a versatile tool for influenza prevention, diagnosis, and therapy.
The processes of labor and birth have a complex evolutionary history, with substantial variation among species showing differences in gestational length, offspring number, anatomy, and rates of fetal development. Understanding the genomic basis of pregnancy is therefore a focus of evolutionary research, given the importance of reproductive success in processes such as natural selection, mutation, genetic drift, and migration. Disruptions to normal pregnancy processes include preterm birth, which can arise from multiple factors, including infection, anatomical variation, injury, age, parity, and multiple gestation and other obstetrical syndromes as well (e.g., preeclampsia, and stillbirth). These factors each influence unique and overlapping networks of candidate genes and biological pathways. Here we synthesize evidence from comparative genomics, population genetics, and vertebrate reproductive biology to show that many PTB-relevant genes, including those involved in progesterone signaling, innate immunity, placental regulation, and chromosome 19 gene clusters, have undergone lineage- or population-specific evolutionary change. Integrating evolutionary insights with functional genomics, machine learning, and modern genome-editing technologies, we provide a principled framework to distinguish conserved, high-risk targets from evolutionarily flexible loci, guiding safer mechanistic studies and future interventions to reduce PTB risk. From an initial list of approximately 1,500 genes involved in pregnancy, we identified those that show evidence of recent evolutionary change for which functional inference is possible. We review some specific nucleotide sites that, when disrupted via CRISPR gene editing, are likely to impact the processes of labor and birth. These loci fall within protein coding genes, transposable elements, transcription factor binding sites, and non-coding RNAs. They are found in nuclear hormone receptors (e.g., PGR), genes with placenta- and uterine-specific expression patterns (e.g., LGALS13), as well as signaling molecules and immunological loci. Finally, we provide evidence that gene activity and sequence variation differ across species and provide examples of pathway differences between chimpanzees (nociception) and humans (inflammation).
BackgroundSpalt-like transcription factor 2 (SALL2) has emerged as a potential tumor suppressor in various malignancies; however, its role in breast cancer remains underexplored. This study examines the effect of SALL2 overexpression on breast carcinogenesis, with a particular focus on the induction of apoptosis and inhibition of cell migration and invasion, using breast cancer receptor-positive and receptor-negative cell lines.MethodsBreast cancer cell lines (MCF-7 and MDA-MB-231) were transiently transfected with a SALL2 expression vector, and successful transfection was confirmed by real-time PCR and Western blot analysis. Functional assays performed included proliferation (MTT assay), wound healing, invasion (transwell Matrigel assay), apoptosis (flow cytometry), and assessment of mRNA expression levels of CDKN1A (p21), p16, PMAIP1 (NOXA), BAX, and MMP9 using quantitative real-time PCR.ResultsWe demonstrated that the transient upregulation of SALL2 expression markedly inhibited cell migration and invasion, processes central to tumor metastasis. This effect was accompanied by a reduction in MMP9, a key enzyme associated with extracellular matrix degradation and metastatic potential. Furthermore, upregulated SALL2 expression significantly promoted apoptosis, as evidenced by the upregulation of pro-apoptotic genes including PMAIP1 (NOXA), BAX, CDKN1A (p21), and p16. These changes suggest that SALL2 not only impedes metastatic capacity but also enhances apoptotic signaling pathways in breast cancer cells. Importantly, the tumor-suppressive nature of SALL2 was consistent in both types of breast cancer cell lines, underscoring its broad therapeutic relevance across different breast cancer subtypes.ConclusionOur findings indicate that SALL2 expression inhibits cell proliferation, migration, and invasion, while inducing apoptosis in breast cancer. These findings suggest that SALL2 may be a critical regulator of breast carcinogenesis and a potential target for therapeutics controlling tumor progression, invasion, and metastasis.
Pigeonpea (Cajanus cajan (L.) Millsp.; 2n = 2× = 22) is a drought-tolerant perennial grain legume commonly cultivated in India’s rain-fed and dry land zones, and it is a remarkable natural source of minerals and protein worldwide. Despite decades of research, the constraints associated with tissue culture continue to hinder the genetic improvement of the crop, including the explant’s inability to produce embryogenic calli, direct shoot formation, limited regeneration potential, and a lack of an effective transformation system. Moreover, traditional or molecular breeding approaches for crop improvement is time, resource, and labour-intensive. CRISPR/Cas9-mediated approach for targeted trait improvement has emerged as a robust technology for introducing desired genetic modifications in several crop plants. We sought to report an improved protocol for calli production, in vitro regeneration, and a CRISPR-mediated genome editing of the phytoene desaturase (PDS) gene via a biolistic-mediated transformation system in pigeonpea. The indigenously developed construct (CcPDS_NICTK-2_pCRISPR-Cas9) harboring pigeonpea codon-optimized Cas9 and target-specific sgRNA was used for transformation in pigeonpea explants (embryonic axis and cotyledonary nodes). The addition of tailored growth regulators and silver nitrate to shoot-induction media boosted plant regeneration to about 86% (±0.04) and transformation efficiency to 46% (±0.04). Sequencing analysis revealed the incurred mutations in the native CcPDS gene, with an editing efficiency of approximately 10%. Moreover, this optimized approach can be utilized in the future to generate marker-free genome-edited plants, addressing biosafety concerns and facilitating the acceptance and commercialization of genetically improved crops.
Myostatin (MSTN) gene knockout has attracted considerable interest for enhancing meat production in livestock species due to its well-established role as a negative regulator of skeletal muscle growth. In this study, we report the clinical outcome of a bi-allelic MSTN knockout buffalo produced via somatic cell nuclear transfer (SCNT) that failed to survive beyond 100 days of age. The MSTN knockout calf exhibited a double-muscling phenotype, which was validated by changes in mRNA expression of transcripts associated with increased muscle mass and reduced subcutaneous fat. In addition, the calf had a telomere length similar to that of age-matched calves produced through artificial insemination. However, serum biochemistry analysis revealed decreased levels of globulin, GGTP, ALP, and bilirubin, alongside increased concentrations of AST, urea, blood urea nitrogen, uric acid, phosphorus, and potassium. Cytokine profiling (including IFN-γ, IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17α, IL-36Rα, TNF-α, MIP-1α, MIP-1β, MCP-1, IP-10, and VEGF-α) demonstrated dysregulation of several inflammatory and chemotactic mediators. This case report highlights potential health and welfare challenges associated with producing MSTN knockout buffalo through cloning-based approaches.
Advancements in genome editing have established a new frontier for the treatment of various genetic diseases, including sickle cell disease (SCD). SCD, the most prevalent monogenic blood disorder, causes severe pain, organ damage, and reduced life expectancy. The recent clinical approval of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based gene therapies for severe sickle cell anemia marks a significant milestone in treating genetic diseases. Despite these breakthroughs, limitations in CRISPR technology persist, requiring further innovation. Alternative approaches, such as the Fanzor (Fz) system, are being developed to complement CRISPR’s capabilities. Unlike CRISPR, which is typically encoded within prokaryotic organisms, Fz is encoded in the eukaryotic genome, offering a universal RNA-guided mechanism applicable across all life kingdoms. Fz’s eukaryotic origin may facilitate more efficient delivery across diverse cell types and tissues, enhancing its therapeutic potential. Here, we will review the current successes and limitations of the CRISPR technology in editing mutation associated with SCD. Additionally, we will explore the potential role of Fz as a genome-editing tool for SCD, a field where its application has not yet been studied.
Caenorhabditis elegans is a powerful model for studying gene function and disease pathogenesis. While RNA interference effectively suppresses gene expression, CRISPR activation (CRISPRa) provides a robust tool for upregulating endogenous gene expression in C. elegans. Compared with traditional injection-based methods, feeding-based CRISPRa, similar to RNA interference, is easy, cost-effective and efficient. However, the traditional cloning workflows remain a bottleneck for high-throughput gene screening. Here, we present a pooled, one-step dual gRNA cloning protocol that enables rapid and efficient construction of gRNA expression vectors for CRISPRa. In a test case, by designing 55-mer and 54-mer primers, each containing one gRNA, we amplified and pooled 126 gRNA inserts in a single reaction pipeline. The 126 gRNA clones were completed in three pooled rounds, achieving 42%-56% coverage for each round, with remaining clones processed individually. This protocol dramatically reduces time, labor, and reagent consumption, while increasing scalability and maintaining reproducibility. It is particularly well suited for high-throughput screening of gene libraries or pathways and supports downstream applications such as phenotypic screening and lifespan analysis. This work advances CRISPRa-based functional genomics in C. elegans by providing a practical tool for large-scale gene activation studies.
Legumes are among the most important protein-rich crops in global agri-food systems. To meet the rising protein demand of a growing population, significant efforts are underway to enhance legume yield, nutritional quality, and resilience to environmental stresses through the manipulation of key genetic traits. Advanced technologies-including genetic engineering, gene editing, genomic selection, next-generation sequencing, single-cell genomics, and multi-omics-are accelerating legume improvement due to their high precision and efficiency. This review focuses on major gene-editing technologies, namely, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), TALENs (Transcription Activator-Like Effector Nucleases), ZFNs (Zinc Finger Nucleases), base editing (BE), and prime editing (PE), and their applications in key legume crops such as soybean (Glycine max), cowpea (Vigna unguiculata), chickpea (Cicer arietinum), groundnut (Arachis hypogaea), pea (Pisum sativum), barrel clover (Medicago truncatula), alfalfa (Medicago sativa), and Lotus japonicus. Among these platforms, CRISPR/Cas9 is the most widely adopted in legumes, largely due to its simplicity, versatility, and dependence on accurate genome sequence information and guide RNA (gRNA) design. Advances in next-generation sequencing and the growing availability of intuitive online gRNA design tools have streamlined CRISPR workflows, improving accessibility and precision. The present review indicates that CRISPR-P is the most used gRNA design tool in legume research, likely due to its early development for plant systems and integrated off-target prediction features. Therefore, alongside reviewing gene-editing applications, we emphasized the critical role of robust gRNA design tools as a foundation for successful genome editing. Future integration of artificial intelligence and large language models is expected to further enhance target prediction accuracy, minimize off-target effects, and enable more precise genome-editing strategies in legumes.
Two major processes are important for genome editing in plants: transformation by stable transfection, in which nucleic acids encoding genome-editing enzymes are introduced into plant cells and the regeneration of plant individuals from cells harboring mutations by genome-editing enzymes. The efficiency of transformation and regeneration by tissue culture varies across plant species, and is low in some practical crop species. In planta methods have been developed to exclude the need for tissue culture. However, few reports are available on methods that do not require stable transfection. Therefore, this study aimed to develop a new protocol for delivery genome editing tools that does not require transformation or tissue culture, by combining the in planta method with transient genome editing tools instead of stable transfection. Cas9, guide RNAs, and developmental regulators, which are factors involved in mitotic tissue induction, were transiently expressed by agroinfiltration of the stem tissue cut surfaces of tomatoes. New chimeric mutants, containing a mixture of cells with mutations introduced at or near the target sequence, were obtained. After examining conditions such as the concentration of Agrobacterium used for infection and post-infection treatment, we succeeded in obtaining chimeric mutants with an efficiency of 11.7%. In addition, most of the observed mutations were single base substitutions. These results indicate that the in planta method with transient expression of genome editing tools and induction of meristematic tissue can be used to introduce genome-edited mutations in tomatoes.
