Clustered Regularly Interspaced Short Palindromic repeats/CRISPR-associated Protein 9 Research Articles

Non-Invasive Delivery of CRISPR/Cas9 Ribonucleoproteins (Cas9 RNPs) into Cells via Nanoparticles for Membrane Transport

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is a promising biotechnology tool for genome editing. However, in living organisms, several pharmacokinetic challenges arise, including off-target side effects due to incorrect distribution, low bioavailability caused by membrane impermeability, and instability resulting from enzymatic degradation. Therefore, innovative delivery strategies must be developed to address these issues. Modified nanoparticles offer a potential solution for the non-invasive delivery of CRISPR/Cas9 ribonucleoproteins (Cas9 RNPs). Cas9 RNPs encapsulated in nanoparticles are protected from enzymatic degradation, similar to how microRNAs are shielded within exosomes. It is well-established that certain materials, including proteins, are expressed selectively in specific cell types. For example, the α-7 nicotinic receptor is expressed in endothelial and neuronal cells, while the αvβ3 integrin is expressed in cancer cells. These endogenous materials can facilitate receptor-mediated endocytosis or transcytosis. Nanoparticles encapsulating Cas9 RNPs and coated with ligands targeting such receptors may be internalized through receptor-mediated mechanisms. Once internalized, Cas9 RNPs could perform the desired gene editing in the nucleus after escaping the endosome through mechanisms such as the proton sponge effect or membrane fusion. In this review, I discuss the potential and advantages of delivering Cas9 RNP-encapsulated nanoparticles coated with ligands through receptor-mediated endocytosis or transcytosis.

Advancing vegetable genetics with gene editing: a pathway to food security and nutritional resilience in climate-shifted environments.

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 stresstolerance, 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.

CRISPR/Cas9-mediated homology-directed repair for precise insertion of constitutive promoter: Overexpression of OsNRAMP7 in TBR225 rice

Precise gene insertion using homology-directed repair (HDR) in Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene editing has emerged as a powerful tool for plant genome modification, offering unprecedented control over genetic alterations. This study successfully applied this technology to TBR225, a major rice variety in Vietnam, aiming to overexpress the OsNRAMP7 gene by inserting a 35S constitutive promoter. Our research addressed the need for efficient methods to enhance specific gene expression in crop plants, with a focus on improving this economically important rice variety. We designed and implemented a single vector system comprising gRNA and Cas9 expression constructs, along with a donor DNA repair template containing the 35S promoter. The system was delivered via Agrobacterium-mediated transformation to TBR225 rice. Our results demonstrated the successful insertion of the 35S promoter upstream of the OsNRAMP7 open reading frame in TBR225, leading to significantly increased OsNRAMP7 expression in edited lines. Notably, we obtained homozygous transgene-free OsNRAMP7-overexpressing TBR225 lines in the T1 generation, confirming stable transmission of the desired modification in this Vietnamese rice variety. These results validate the effectiveness of CRISPR/Cas9-mediated HDR for precise genetic modifications in TBR225 rice. This successful research on Vietnam's major rice variety offers a valuable approach for crop improvement and functional genomics studies, potentially accelerating the development of rice varieties with enhanced traits such as stress tolerance or nutrient use efficiency.

Statistical Analysis of Microarray Data to Identify Key Gene Expression Patterns in Primary Hyperoxaluria

This study aims to utilize microarray data deposited by Romero et al. and conduct bioinformatic analysis for identifying differentially expressed genes (DEGs) associated with a novel method involving gene correction at the Alanine–Glyoxylate Aminotransferase (AGXT) locus and direct conversion of fibroblasts from primary hyperoxaluria type 1 (PH1) patients into healthy induced hepatocytes (iHeps) using Clustered Regularly Interspaced Short Palindromic Repeats - CRISPR-associated protein 9 (CRISPR-Cas9) technology. Additionally, the study aims to elucidate the molecular mechanisms underlying hyperoxaluria compared to oxalate crystal formation. Romero et al.'s GSE226019 microarray data was retrieved from Gene Expression Omnibus. Statistical analysis was done in R and Bioconductor, utilizing rigorous methods to ensure robust and reproducible results. The limma program compared gene expression levels across groups. Pathway analysis, protein-protein interaction (PPI) network creation, and miRNA-target interaction network analysis were constructed. The top ten DEGs included ANGPTL3, SLC38A3, KNG1, BDH1, GC, ADH1C, ARG1, CYP3A4, AMBP, and CYP2C9. Enrichment analysis revealed significant associations with various biological pathways, including Linoleic acid metabolism and Retinol metabolism. Volcano plots and mean difference plots highlighted significant gene expression changes between different sample groups. Protein-protein interaction networks and miRNA-target interaction networks provided insights into molecular interactions and regulatory mechanisms. The top ten differentially expressed genes include ANGPTL3, SLC38A3, KNG1, BDH1, GC, ADH1C, ARG1, CYP3A4, AMBP, and CYP2C9—emerge as key players with strong associations to critical biological pathways like Linoleic acid metabolism and drug metabolism-cytochrome P450. Understanding the regulatory role of specific miRNAs (hsa-miR-4501, hsa-miR-5692c, hsa-miR-6731-3p, hsa-miR-6867-5p, hsa-miR-616-3p, hsa-miR-4468, hsa-miR-3692-3p, hsa-miR-4277, hsa-miR-4763-5p, hsa-miR-4797-5p) in gene expression could provide further insights into disease mechanisms and potential therapeutic avenues. The statistical findings provide a foundation for predictive modeling, hypothesis testing, and exploring personalized therapeutic strategies.

Deciphering the Role of the SREBF1 Gene in the Transcriptional Regulation of Porcine Adipogenesis Using CRISPR/Cas9 Editing.

Sterol regulatory element-binding protein 1 (SREBP1) is an important transcription factor that controls lipid metabolism and adipogenesis. Two isoforms, SREBP1a and SREBP1c, are generated by alternative splicing of the first exon of the SREBF1 gene. The porcine SREBF1 gene has mainly been studied for its role in lipid metabolism in adipose tissues, but little is known about its involvement, and the role of its two isoforms, in adipogenesis. The aim of the present study was to introduce a deletion in the 5'-regulatory region of the SREBF1c gene, considered crucial for adipogenesis, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. This approach allows for the evaluation of how inhibiting SREBF1c transcription affects the expression of other genes essential for adipocyte differentiation, particularly PPARG, CEBPA, CEBPB, CEBPD, GATA2, and FABP4. It was observed that disrupting the SREBF1c isoform had no effect on the GATA2 gene but did result in a decrease in the expression of the CEBPA and CEBPD genes, an increase in the expression of CEBPB, and an inhibition in the expression of the PPARG and FABP4 genes. These changes in gene expression blocked adipogenesis, as could be seen by the failure of lipid droplets to accumulate. Our results provide evidence highlighting the pivotal role of the SREBP1c isoform in the regulation of porcine adipogenesis.

Endocrine pathology in young rabbits with cystic fibrosis

BackgroundCystic fibrosis (CF) is an autosomal recessive genetic disorder caused by loss-of-function mutations in the CF transmembrane conductance regulator gene. CF-related pancreatic lesions are known to cause exocrine dysfunctions such...

Exploring the role of FAT genes in Solanaceae species through genome-wide analysis and genome editing.

Plants produce numerous fatty acid derivatives, and some of these compounds have significant regulatory functions, such as governing effector-induced resistance, systemic resistance, and other defense pathways. This study systematically identified and characterized eight FAT genes (Acyl-acyl carrier protein thioesterases), four in the Solanum lycopersicum and four in the Solanum tuberosum genome. Phylogenetic analysis classified these genes into four distinct groups, exhibiting conserved domain structures across different plant species. Promoter analysis revealed various cis-acting elements, most of which are associated with stress responsiveness and growth and development. Micro-RNA (miRNA) analysis identified specific miRNAs, notably miRNA166, targeting different FAT genes in both species. Utilizing clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated knockout, mutant lines for SlFATB1 and SlFATB3 were successfully generated and exhibited diverse mutation types. Biochemical evaluation of selected mutant lines revealed significant changes in fatty acid composition, with linoleic and linolenic acid content variations. The study also explored the impact of FAT gene knockout on tomato leaf architecture through scanning electron microscopy, providing insights into potential morphological alterations. Knocking out of FAT genes resulted in a significant reduction in both trichome and stoma density. These findings contribute to a comprehensive understanding of FAT genes in Solanaceous species, encompassing genetic, functional, and phenotypic aspects.

Applications of CRISPR/Cas9 technology in mice and livestock genome editing: Current research

Abstract Clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a novel and one of the most promising gene editing methods. Here we review the current research on the use of CRISPR/Cas9 technology to edit the genomes of mice, rabbits, pigs, sheep, goats, and cattle, as well as current obstacles and perspectives that may arise thanks to this technology. CRISPR/Cas9 has an advantage over other gene editing methods because of its simple design and the ease of modifying multiple target sites (multiplexing). This technology allows for the creation of in vitro and in vivo models of many diseases, which may contribute to the development of new drugs and effective therapies. CRISPR/Cas9 can also revolutionize the meat, dairy, leather, and fur industries by genetically modifying animals to obtain different products.

An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. Conventional gene editing platforms that rely on nuclease activity, such as Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point™ base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared to the conventional CRISPR-Cas9 system. Site-specific knock-in of a chimeric antigen receptor (CAR) and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

Generation of New β-Conglycinin-Deficient Soybean Lines by Editing the lincRNA lincCG1 Using the CRISPR/Cas9 System.

Soybean β-conglycinin is a major allergen that adversely affects the nutritional properties of soybean. Soybean deficient in β-conglycinin is associated with low allergenicity and high nutritional value. Long intergenic noncoding RNAs (lincRNAs) regulate gene expression and are considered important regulators of essential biological processes. Despite increasing knowledge of the functions of lincRNAs, relatively little is known about the effects of lincRNAs on the accumulation of soybean β-conglycinin. The current study presents the identification of a lincRNA lincCG1 that was mapped to the intergenic noncoding region of the β-conglycinin α-subunit locus. The full-length lincCG1 sequence was cloned and found to regulate the expression of soybean seed storage protein (SSP) genes via both cis- and trans-acting regulatory mechanisms. Loss-of-function lincCG1 mutations generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system led to the deficiency of the allergenic α'-, α-, and β-subunits of soybean β-conglycinin as well as higher content of proteins, sulfur-containing amino acids, and free arginine. The dominant null allele LincCG1, and consequently, the β-conglycinin-deficient phenotype associated with the lincCG1-gene-edited line was stably inherited by the progenies in a Mendelian fashion. The dominant null allele LincCG1 may therefore be exploited for engineering/developing novel hypoallergenic soybean varieties. Furthermore, Cas9-free and β-conglycinin-deficient homozygous mutant lines were obtained in the T1 generation. This study is the first to employ the CRISPR/Cas9 technology for editing a lincRNA gene associated with the soybean allergenic protein β-conglycinin. Moreover, this study reveals that lincCG1 plays a crucial role in regulating the expression of the β-conglycinin subunit gene cluster, besides highlighting the efficiency of employing the CRISPR/Cas9 system for modulating lincRNAs, and thereby regulating soybean seed components.

A Coiled-Coil Nucleotide-Binding Domain Leucine-Rich Repeat Receptor Gene MeRPPL1 Plays a Role in the Replication of a Geminivirus in Cassava.

Disease resistance gene (R gene)-encoded nucleotide-binding leucine-rich repeat proteins (NLRs) are critical players in plant host defence mechanisms because of their role as receptors that recognise pathogen effectors and trigger plant effector-triggered immunity (ETI). This study aimed to determine the putative role of a cassava coiled-coil (CC)-NLR (CNL) gene MeRPPL1 (Manes.12G091600) (single allele) located on chromosome 12 in the tolerance or susceptibility to South African cassava mosaic virus (SACMV), one of the causal agents of cassava mosaic disease (CMD). A transient protoplast system was used to knock down the expression of MeRPPL1 by clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9). The MeRPPL1-targeting CRISPR vectors and/or SACMV DNA A and DNA B infectious clones were used to transfect protoplasts isolated from leaf mesophyll cells from the SACMV-tolerant cassava (Manihot esculenta) cultivar TME3. The CRISPR/Cas9 silencing vector significantly reduced MeRPPL1 expression in protoplasts whether with or without SACMV co-infection. Notably, SACMV DNA A replication was higher in protoplasts with lower MeRPPL1 expression levels than in non-silenced protoplasts. Mutagenesis studies revealed that protoplast co-transfection with CRISPR-MeRPPL1 silencing vector + SACMV and transfection with only SACMV induced nucleotide substitution mutations that led to altered amino acids in the highly conserved MHD motif of the MeRPPL1-translated polypeptide. This may abolish or alter the regulatory role of the MHD motif in controlling R protein activity and could contribute to the increase in SACMV-DNA A accumulation observed in MeRPPL1-silenced protoplasts. The results herein demonstrate for the first time a role for a CNL gene in tolerance to a geminivirus in TME3.

Delivering CRISPR to the HIV-1 reservoirs.

Human immunodeficiency virus type 1 (HIV-1) infection is well known as one of the most complex and difficult viral infections to cure. The difficulty in developing curative strategies arises in large part from the development of latent viral reservoirs (LVRs) within anatomical and cellular compartments of a host. The clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9 (CRISPR/Cas9) system shows remarkable potential for the inactivation and/or elimination of integrated proviral DNA within host cells, however, delivery of the CRISPR/Cas9 system to infected cells is still a challenge. In this review, the main factors impacting delivery, the challenges for delivery to each of the LVRs, and the current successes for delivery to each reservoir will be discussed.

Conditional knockout mouse model reveals a critical role of peroxiredoxin 1 in oral leukoplakia carcinogenesis

Peroxiredoxin 1 (Prx1) is an antioxidant protein that may promote the carcinogenesis in oral leukoplakia (OLK). To investigate the effect of Prx1 on the oral mucosal epithelium of OLK, we generated a Prx1 conditional knockout (cKO) mouse model. The mRNA and gRNA were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technique. An infusion cloning method was used to construct a homologous recombination vector. To obtain the F0 generation mice, fertilized eggs of C57BL/6J mice were microinjected with Cas9 mRNA, gRNA, and a donor vector. Polymerase chain reaction (PCR) amplification and sequencing were used to identify F1 generation mice. Using the cyclization recombination-enzyme-locus of the X-overP1 (Cre-loxP) system, we created a Prx1 cKO mouse model, and the effectiveness of the knockout was confirmed through immunohistochemistry. We examined the influence of Prx1 knockout on the occurrence of OLK in mice by constructing a model of tongue mucosa carcinogenesis induced by 4-nitroquinoline-1-oxide (4NQO). Prx1 modification was present in the F1 generation, as evidenced by PCR amplification and sequencing. Prx1flox/flox: Cre + mice exhibited normal growth and fertility. Immunohistochemical analysis revealed that tongue epithelial cells in Prx1flox/flox: Cre + mice displayed a distinct deletion of Prx1. An examination of the heart, liver, spleen, lung, and kidney tissues revealed no visible histological changes. Histological analysis showed a reduction in the occurrence of the malignant transformation of OLK in the tongue tissues of Prx1flox/flox: Cre + mice. Ki67 immunostaining showed that Prx1 knockout significantly inhibited cell proliferation in the tongue epithelial. Our research developed a conditional knockout mouse model for Prx1. The obtained results provide insights into the function of Prx1 in the development of oral cancer and emphasize its potential as a therapeutic target for precancerous oral lesions.

Reproductive Characteristics and Suitability of Sterile dead end Knockout Nibe Croaker as a Recipient for Intraperitoneal Germ Cell Transplantation.

The use of sterile recipients is crucial for efficiently producing donor-derived offspring through surrogate broodstock technology for practical aquaculture applications. Although knockout (KO) of the dead end (dnd) gene has been used in previous studies as a sterilization method, it has not been reported in marine fish. In this study, nibe croaker was utilized as a model for marine teleosts that produce small pelagic eggs, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized to produce dnd KO fish. The F1 generation, which carried a nonsense mutation in the dnd gene, was produced by mating founder individuals with wild-type counterparts. Subsequently, the F2 generation was produced by mating the resulting males and females. Among the F2 generations, 24.0% consisted of homozygous KO individuals. Histological analysis revealed that primordial germ cells (PGCs) were present in homozygous KO individuals at 10days post-hatching (dph), similar to wild-type individuals. However, by 20 dph, PGCs were absent in KO individuals. Furthermore, no germ cells were observed in the gonads of both sexes of homozygous KO individuals at 6months old, which is the typical maturity age for wild-type individuals of both sexes. In addition, when cryopreserved donor nibe croaker testicular cells were transplanted, only donor-derived offspring were successfully obtained through the spontaneous mating of homozygous KO recipient parents. Results indicate that dnd KO nibe croaker lacks germ cells and can serve as promising recipients, producing only donor-derived gametes as surrogate broodstock.

An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.

Prediction of CRISPR/Cas9 off-target activity using multi-scale convolutional neural network

Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) is a new generation of gene editing technology, which relies on single guide RNA to identify specific gene sites and guide Cas9 nuclease to edit specific location in the genome. However, the off-target effect of this technology hampers its development. In recent years, several deep learning models have been developed for predicting the CRISPR/Cas9 off-target activity, which contributes to more efficient and safe gene editing and gene therapy. However, the prediction accuracy remains to be improved. In this paper, we proposed a multi-scale convolutional neural network-based method, designated as CnnCRISPR, for CRISPR/Cas9 off-target prediction. First, we used one-hot encoding method to encode the sgRNA-DNA sequence pair, followed by a bitwise or operation on the two binary matrices. Second, the encoded sequence was fed into the Inception-based network for training and evaluating. Third, the well-trained model was applied to evaluate the off-target situation of the sgRNA-DNA sequence pair. Experiments on public datasets showed CnnCRISPR outperforms existing deep learning-based methods, which provides an effective and feasible method for addressing the off-target problems.

Mitigating the Off-target Effects in CRISPR/Cas9-mediated Genetic Editing with Bioinformatic Technologies

The biological and clinical fields have recognized the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9) system as a precise and efficient tool for editing the genome. Despite its merits, the system poses crucial challenges, notably the off-target effects which could lead to unintended mutations - a substantial impediment for clinical applications that may potentially compromise the validity of research and the safety of therapeutic applications. Bioinformatics plays a pivotal role in mitigating this risk. Utilizing more refined bioinformatic tools and algorithms, researchers can reduce off-target mutations remarkably. These instruments, powered by machine learning and computational modelling, are able to predict off-target effects and provide aid for the design of more efficient sgRNA. Despite these advancements, it remains crucial to continue to focus on the improvement and assessment of such bioinformatics strategies. This review aims to holistically explore the mechanism and applications of CRISPR/Cas9 genome editing, its off-target effects, and the consequent impacts, along with the potential of bioinformatics techniques to identify off-target risks and facilitate sgRNA design. This review will also incorporate a clinical trial on HIV-1 treatment as a case study to highlight the potential of bioinformatics in devising solutions to mitigate the potential off-target effects of CRISPR/Cas9-mediated genetic editing.

Challenges and Opportunities of Gene Therapy in Cancer

Challenges and Opportunities of Gene Therapy in Cancer

Fine mapping and identification of two NtTOM2A homeologs responsible for tobacco mosaic virus replication in tobacco (Nicotiana tabacum L.)

BackgroundTobacco mosaic virus (TMV) is a widely distributed viral disease that threatens many vegetables and horticultural species. Using the resistance gene N which induces a hypersensitivity reaction, is a common strategy for controlling this disease in tobacco (Nicotiana tabacum L.). However, N gene-mediated resistance has its limitations, consequently, identifying resistance genes from resistant germplasms and developing resistant cultivars is an ideal strategy for controlling the damage caused by TMV.ResultsHere, we identified highly TMV-resistant tobacco germplasm, JT88, with markedly reduced viral accumulation following TMV infection. We mapped and cloned two tobamovirus multiplication protein 2A (TOM2A) homeologs responsible for TMV replication using an F2 population derived from a cross between the TMV-susceptible cultivar K326 and the TMV-resistant cultivar JT88. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated loss-of-function mutations of two NtTOM2A homeologs almost completely suppressed TMV replication; however, the single gene mutants showed symptoms similar to those of the wild type. Moreover, NtTOM2A natural mutations were rarely detected in 577 tobacco germplasms, and CRISPR/Cas9-mediated variation of NtTOM2A led to shortened plant height, these results indicating that the natural variations in NtTOM2A were rarely applied in tobacco breeding and the NtTOM2A maybe has an impact on growth and development.ConclusionsThe two NtTOM2A homeologs are functionally redundant and negatively regulate TMV resistance. These results deepen our understanding of the molecular mechanisms underlying TMV resistance in tobacco and provide important information for the potential application of NtTOM2A in TMV resistance breeding.

Nanotechnology‐based CRISPR/Cas9 delivery system for genome editing in cancer treatment

AbstractThe clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (CRISPR/Cas9) systems initiate a revolution in genome editing, which have a significant potential for treating cancer. A significant amount of research has been conducted regarding genetic modification using CRISPR/Cas9 systems, and 33 clinical trials using ex vivo or in vivo CRISPR/Cas9 gene editing techniques have been carried out to treat cancer. Despite its potential advantages, the main obstacle to convert CRISPR/Cas9 technology into clinical genome editing applications is the safe and efficient transport of genetic material owing to various extra‐ and intracellular biological hurdles. We outline the characteristics of three forms of CRISPR/Cas9 cargos, plasmids, mRNA/sgRNA, and ribonucleoprotein (RNP) complexes in this review. The recent in vivo nanotechnology‐based delivery techniques for these three categories to treat cancer are then reviewed. In the end, we outline the prerequisites for effective and secure in vivo CRISPR/Cas9 delivery in clinical contexts and discuss challenges with current nanocarriers. This review offers a thorough overview of the CRISPR/Cas9 nano‐delivery system for the treatment of cancer, serving as a resource for the design and building of CRISPR/Cas9 delivery systems and offering fresh perspectives on the treatment of tumors.