Multi-omics analysis reveal the fall armyworm Spodoptera frugiperda tolerate high temperature by mediating chitin-related genes.

Climate change facilitates the rapid invasion of agricultural pests, threatening global food security. The fall armyworm Spodoptera frugiperda is a highly polyphagous migratory pest tolerant to high temperatures, allowing its proliferation in harsh thermal environments. We aimed to demonstrate mechanisms of its high-temperature tolerance, particularly transcriptional and metabolic regulation, which are poorly understood. To achieve the aim, we examined the impact and mechanism of heat events on S. frugiperda by using multiple approaches: ecological measurements, transcriptomics, metabolomics, RNAi, and CRISPR/Cas9 technology. We observed that several physiological indices (larval survival rate, larval period, pupation rate, pupal weight, eclosion rate, and average fecundity) decreased as the temperature increased, with the 32 °C treatment displaying a significant difference from the control group at 26 °C. Significantly upregulated expression of genes encoding endochitinase and chitin deacetylase was observed in the chitin-binding, extracellular region, and carbohydrate metabolic process GO terms of hemolymph, fat body, and brain, exhibiting a tissue-specific pattern. Significantly enriched pathways (e.g., cutin, suberin, and wax biosynthesis; oxidative phosphorylation and cofactor biosynthesis; diverse amino acid biosynthesis and degradation; carbon metabolism; and energy metabolism), all of which are essential for S. frugiperda larvae to tolerate temperature, were found in metabolites that were expressed differently. Successful RNA interference targeting of the three chitin-related genes reduced gene expression levels and larval survival rate. Knockout of the endochitinase gene by using the CRISPR/Cas9 system significantly reduced the relative gene expression and increased sensitivity to high-temperature exposure. On the basis of our findings, theoretical foundations for understanding the high-temperature tolerance of S. frugiperda populations and latent genetic control strategies were established.

Receptors and Host Factors for Enterovirus Infection: Implications for Cancer Therapy.

Enteroviruses, with their diverse clinical manifestations ranging from mild or asymptomatic infections to severe diseases such as poliomyelitis and viral myocarditis, present a public health threat. However, they can also be used as oncolytic agents. This review shows the intricate relationship between enteroviruses and host cell factors. Enteroviruses utilize specific receptors and coreceptors for cell entry that are critical for infection and subsequent viral replication. These receptors, many of which are glycoproteins, facilitate virus binding, capsid destabilization, and internalization into cells, and their expression defines virus tropism towards various types of cells. Since enteroviruses can exploit different receptors, they have high oncolytic potential for personalized cancer therapy, as exemplified by the antitumor activity of certain enterovirus strains including the bioselected non-pathogenic Echovirus type 7/Rigvir, approved for melanoma treatment. Dissecting the roles of individual receptors in the entry of enteroviruses can provide valuable insights into their potential in cancer therapy. This review discusses the application of gene-targeting techniques such as CRISPR/Cas9 technology to investigate the impact of the loss of a particular receptor on the attachment of the virus and its subsequent internalization. It also summarizes the data on their expression in various types of cancer. By understanding how enteroviruses interact with specific cellular receptors, researchers can develop more effective regimens of treatment, offering hope for more targeted and efficient therapeutic strategies.

Oral Microto-Nano Genome-Editing System Enabling Targeted Delivery and Conditional Activation of CRISPR-Cas9 for Gene Therapy of Inflammatory Bowel Disease.

The potent CRISPR-Cas9 technology can correct genes in human mutated cells to achieve the treatment of multiple diseases, but it lacks safe and effective delivery systems. Herein, we proposed an oral microto-nano genome-editing system aiming at the enteric excessive level of TNF-α for specific gene therapy of inflammatory bowel disease (IBD). This editing system facilitated the assembly of Cas9/sgRNA ribonucleoprotein (RNP) into nanoclusters (NCs) through the bridging of disulfide bonds. RNP-NCs were subsequently encapsulated within inflammatory cell-targeted lipopolysaccharide-deleted outer membrane vesicles (dOMVs) sourced from Escherichia coli Nissle 1917, which were further shielded by an outer layer of calcium alginate microspheres (CAMs). By leveraging the protection effect of CAMs, the oral administration system withstood gastric acid degradation upon entry into the stomach, achieving targeted delivery to the intestines with high efficiency. As the pH gradually rose, the microscale CAMs swelled and disintegrated, releasing nanoscale RNP-NCs encapsulated in dOMVs into the intestines. These RNP-NCs@dOMVs could traverse the mucosal barrier and target inflammatory macrophages where conditionally activated Cas9/sgRNA RNPs effectively perform genomic editing of TNF-α within the nucleus. Such oral microto-nano genome-editing systems represent a promising translational platform for the treatment of IBD.

CRISPR/Cas9-mediated genome editing technique to control fall armyworm (Spodoptera frugiperda) in crop plants with special reference to maize.

Fall Armyworm imposes a major risk to agricultural losses. Insecticides have historically been used to manage its infestations, but it eventually becomes resistant to them. To combat the pest, a more recent strategy based on the use of transgenic maize that expresses Bt proteins such as Cry1F from the bacteria has been used. Nonetheless, there have been numerous reports of Cry1F maize resistance in FAW populations. Nowadays, the more effective and less time-consuming genome editing method known as CRISPR/Cas9 technology has gradually supplanted these various breeding techniques. This method successfully edits the genomes of various insects, including Spodoptera frugiperda. On the other hand, this new technique can change an insect's DNA to overcome its tolerance to specific insecticides or to generate a gene drive. The production of plant cultivars resistant to fall armyworms holds great potential for the sustainable management of this pest, given the swift advancement of CRISPR/Cas9 technology and its varied uses. Thus, this review article discussed and critically assessed the use of CRISPR/Cas9 genome-editing technology in long-term fall armyworm pest management. However, this review study focuses primarily on the mechanism of the CRISPR-Cas9 system in both crop plants and insects for FAW management.

Regulatory Assessment of Casgevy for the Treatment of Transfusion-Dependent ß-Thalassemia and Sickle Cell Disease with Recurrent Vaso-Occlusive Crises.

Sickle cell disease (SCD) and transfusion-dependent ß-thalassemia (TDT) are hereditary haemoglobinopathies characterized by a reduction in functional ß-globin chains. Both conditions cause tiredness and increase susceptibility to infection, which can lead organ failure, significantly reducing life expectancy and typically requiring those affected to undergo regular erythrocyte transfusion. Recently, a novel therapeutic treatment for SCD and TDT was approved by the UK regulatory body (Medicines and Healthcare products Regulatory Agency; MHRA). Exagamglogene autotemcel (Casgevy) is the first licensed therapy globally to utilize CRIPSR/Cas9 technology and induces an increase in expression of γ-globin chains to compensate for the reduction in functional ß-globin. Casgevy represents a first-in-class therapeutic, and numerous considerations were made by the MHRA throughout its assessment of the medicine. These include, but are not limited to, the risk of tumorigenicity and off-target editing, a limited cohort size, the validity of proposed dosing and the conduction of only single-arm studies. The MHRA's analyses of the data to support the proposed indications are presented and discussed throughout this manuscript. Overall, the sponsors claims were considered well supported by their data, and Casgevy was licensed for the treatment of TDT or SCD in patients 12 years of age and older for whom hematopoietic stem cell (HSC) transplantation is appropriate, but a human leukocyte antigen-matched related HSC donor is not available.

AdNAC20 Regulates Lignin and Coumarin Biosynthesis in the Roots of Angelica dahurica var. Formosana.

Angelica dahurica var. formosana (ADF), which belongs to the Umbelliferae family, is one of the original plants of herbal raw material Angelicae Dahuricae Radix. ADF roots represent an enormous biomass resource convertible for disease treatment and bioproducts. But, early bolting of ADF resulted in lignification and a decrease in the coumarin content in the root, and roots lignification restricts its coumarin for commercial utility. Although there have been attempts to regulate the synthesis ratio of lignin and coumarin through biotechnology to increase the coumarin content in ADF and further enhance its commercial value, optimizing the biosynthesis of lignin and coumarin remains challenging. Based on gene expression analysis and phylogenetic tree profiling, AdNAC20 as the target for genetic engineering of lignin and coumarin biosynthesis in ADF was selected in this study. Early-bolting ADF had significantly greater degrees of root lignification and lower coumarin contents than that of the normal plants. In this study, overexpression of AdNAC20 gene plants were created using transgenic technology, while independent homozygous transgenic lines with precise site mutation of AdNAC20 were created using CRISPR/Cas9 technology. The overexpressing transgenic ADF plants showed a 9.28% decrease in total coumarin content and a significant 12.28% increase in lignin content, while knockout mutant plants showed a 16.3% increase in total coumarin content and a 33.48% decrease in lignin content. Furthermore, 29,671 differentially expressed genes (DEGs) were obtained by comparative transcriptomics of OE-NAC20, KO-NAC20, and WT of ADF. A schematic diagram of the gene network interacting with AdNAC20 during the early-bolting process of ADF was constructed by DEG analysis. AdNAC20 was predicted to directly regulate the transcription of several genes with SNBE-like motifs in their promoter, such as MYB46, C3H, and CCoAOMT. In this study, AdNAC20 was shown to play a dual pathway function that positively enhanced lignin formation but negatively controlled coumarin formation. And the heterologous expression of the AdNAC20 gene at Arabidopsis thaliana proved that the AdNAC20 gene also plays an important role in the process of bolting and flowering.

CRISPR/Cas9 Gene Editing: A Novel Approach Towards Alzheimer's Disease Treatment.

In defiance of the vast amount of information regarding Alzheimer's disease (AD) that has been learned over the past thirty years, progress toward developing an effective therapy has been difficult. A neurological ailment that progresses and cannot be reversed is Alzheimer's disease, which shows neurofibrillary tangles, beta-amyloid plaque, and a lack of cognitive processes that is created by tau protein clumps with hyperphosphorylation that finally advances to neuronal damage without a recognized treatment, which has stimulated research into new therapeutic strategies. The protein CAS9 is linked to CRISPR, which is a clustered Regularly Interspaced Short Palindromic Repeat that inactivates or corrects a gene by recognizing a gene sequence that produces a doublestranded break has enchanted a whole amount of interest towards its potency to cure gene sequences in AD. The novel CRISPR-Cas9 applications for developing in vitro and in vivo models to the benefit of AD investigation and therapies are thoroughly analyzed in this work. The discussion will also touch on the creation of delivery methods, which is a significant obstacle to the therapeutic use of CRISPR/Cas9 technology. By concentrating on specific genes, such as those that are significant early- onset AD risk factors and late-onset AD risk factors, like the apolipoprotein E4 (APOE4) gene, this study aims to evaluate the potential application of CRISPR/Cas9 as a possible treatment for AD.

From Gene Editing to Biofilm Busting: CRISPR-CAS9 Against Antibiotic Resistance-A Review.

In recent decades, the development of novel antimicrobials has significantly slowed due to the emergence of antimicrobial resistance (AMR), intensifying the global struggle against infectious diseases. Microbial populations worldwide rapidly develop resistance due to the widespread use of antibiotics, primarily targeting drug-resistant germs. A prominent manifestation of this resistance is the formation of biofilms, where bacteria create protective layers using signaling pathways such as quorum sensing. In response to this challenge, the CRISPR-Cas9 method has emerged as a ground-breaking strategy to counter biofilms. Initially identified as the "adaptive immune system" of bacteria, CRISPR-Cas9 has evolved into a state-of-the-art genetic engineering tool. Its exceptional precision in altering specific genes across diverse microorganisms positions it as a promising alternative for addressing antibiotic resistance by selectively modifying genes in diverse microorganisms. This comprehensive review concentrates on the historical background, discovery, developmental stages, and distinct components of CRISPR Cas9 technology. Emphasizing its role as a widely used genome engineering tool, the review explores how CRISPR Cas9 can significantly contribute to the targeted disruption of genes responsible for biofilm formation, highlighting its pivotal role in reshaping strategies to combat antibiotic resistance and mitigate the challenges posed by biofilm-associated infectious diseases.

The GPI-anchor biosynthesis pathway is critical for syncytiotrophoblast differentiation and placental development.

The glycosylphosphatidylinositol (GPI) biosynthetic pathway in the endoplasmic reticulum (ER) is crucial for generating GPI-anchored proteins (GPI-APs), which are translocated to the cell surface and play a vital role in cell signaling and adhesion. This study focuses on two integral components of the GPI pathway, the PIGL and PIGF proteins, and their significance in trophoblast biology. We show that GPI pathway mutations impact on placental development impairing the differentiation of the syncytiotrophoblast (SynT), and especially the SynT-II layer, which is essential for the establishment of the definitive nutrient exchange area within the placental labyrinth. CRISPR/Cas9 knockout of Pigl and Pigf in mouse trophoblast stem cells (mTSCs) confirms the role of these GPI enzymes in syncytiotrophoblast differentiation. Mechanistically, impaired GPI-AP generation induces an excessive unfolded protein response (UPR) in the ER in mTSCs growing in stem cell conditions, akin to what is observed in human preeclampsia. Upon differentiation, the impairment of the GPI pathway hinders the induction of WNT signaling for early SynT-II development. Remarkably, the transcriptomic profile of Pigl- and Pigf-deficient cells separates human patient placental samples into preeclampsia and control groups, suggesting an involvement of Pigl and Pigf in establishing a preeclamptic gene signature. Our study unveils the pivotal role of GPI biosynthesis in early placentation and uncovers a new preeclampsia gene expression profile associated with mutations in the GPI biosynthesis pathway, providing novel molecular insights into placental development with implications for enhanced patient stratification and timely interventions.

Generation of human induced pluripotent stem cell line derived from Becker muscular dystrophy patient with CRISPR/Cas9-mediated correction of DMD gene mutation.

Becker muscular dystrophy (BMD) is an X-linked recessive disorder caused by in-frame deletions in the dystrophin gene (DMD), leading to progressive muscle degeneration and weakness. We generated a human induced pluripotent stem cell (hiPSC) line from a BMD patient. BMD hiPSCs were then engineered by CRISPR/Cas9-mediated knock-in of missing exons 3-9 of DMD gene. Obtained hiPSC line may be a valuable tool for investigating the mechanisms underlying BMD pathogenesis.

Downregulation of MGMT expression by targeted editing of DNA methylation enhances temozolomide sensitivity in glioblastoma.

Glioblastoma is the most common and aggressive primary tumor of the central nervous system with poor outcome. Current gold standard treatment is surgical resection followed by a combination of radio- and chemotherapy. Efficacy of temozolomide (TMZ), the primary chemotherapeutic agent, depends on the DNA methylation status of the O6-methylguanine DNA methyltransferase (MGMT), which has been identified as a prognostic biomarker in glioblastoma patients. Clinical studies revealed that glioblastoma patients with hypermethylated MGMT promoter have a better response to TMZ treatment and a significantly improved overall survival. In this study, we thus used the CRISPRoff genome editing tool to mediate targeted DNA methylation within the MGMT promoter region. The system carrying a CRISPR-deactivated Cas9 (dCas9) fused with a methyltransferase (Dnmt3A/3L) domain downregulated MGMT expression in TMZ-resistant human glioblastoma cell lines through targeted DNA methylation. The reduction of MGMT expression levels reversed TMZ resistance in TMZ-resistant glioblastoma cell lines resulting in TMZ induced dose-dependent cell death rates. In conclusion, we demonstrate targeted RNA-guided methylation of the MGMT promoter as a promising tool to overcome chemoresistance and improve the cytotoxic effect of TMZ in glioblastoma.

Integrating Omics and CRISPR Technology for Identification and Verification of Genomic Safe Harbor Loci in the Chicken Genome.

BACKGROUND: One of the most prominent questions in the field of transgenesis is 'Where in the genome to integrate a transgene?'. Escape from epigenetic silencing and promoter shutdown of the transgene needs reliable genomic safe harbor (GSH) loci. Advances in genome engineering technologies combined with multi-omics bioinformatics data have enabled rational evaluation of GSH loci in the host genome. Currently, no validated GSH loci have been evaluated in the chicken genome. RESULTS: Here, we analyzed and experimentally examined two GSH loci in the genome of chicken cells. To this end, putative GSH loci including chicken HIPP-like (cHIPP; between DRG1 and EIF4ENIF1 genes) and chicken ROSA-like (cROSA; upstream of the THUMPD3 gene) were predicted using multi-omics bioinformatics data. Then, the durable expression of the transgene was validated by experimental characterization of continuously-cultured isogenous cell clones harboring DsRed2-ΔCMV-EGFP cassette in the predicted loci. The weakened form of the CMV promoter (ΔCMV) allowed the precise evaluation of GSH loci in a locus-dependent manner compared to the full-length CMV promoter. CONCLUSIONS: cHIPP and cROSA loci introduced in this study can be reliably exploited for consistent bio-manufacturing of recombinant proteins in the genetically-engineered chickens. Also, results showed that the genomic context dictates the expression of transgene controlled by ΔCMV in GSH loci.

Identification of two novel rice S genes through combination of association and transcription analyses with gene-editing technology.

Traditional rice blast resistance breeding largely depends on utilizing typical resistance (R) genes. However, the lack of durable R genes has prompted rice breeders to find new resistance resources. Susceptibility (S) genes are potential new targets for resistance genetic engineering using genome-editing technologies, but identifying them is still challenging. Here, through the integration of genome-wide association study (GWAS) and transcriptional analysis, we identified two genes, RNG1 and RNG3, whose polymorphisms in 3'-untranslated regions (3'-UTR) affected their expression variations. These polymorphisms could serve as molecular markers to identify rice blast-resistant accessions. Editing the 3'-UTRs using CRISPR/Cas9 technology affected the expression levels of two genes, which were positively associated with rice blast susceptibility. Knocking out either RNG1 or RNG3 in rice enhanced the rice blast and bacterial blight resistance, without impacting critical agronomic traits. RNG1 and RNG3 have two major genotypes in diverse rice germplasms. The frequency of the resistance genotype of these two genes significantly increased from landrace rice to modern cultivars. The obvious selective sweep flanking RNG3 suggested it has been artificially selected in modern rice breeding. These results provide new targets for S gene identification and open avenues for developing novel rice blast-resistant materials.

AFP deletion leads to anti-tumorigenic but pro-metastatic roles in liver cancers with concomitant CTNNB1 mutations.

HCC remains one of the most prevalent and deadliest cancers. Serum AFP level is a biomarker for clinical diagnosis of HCC, instead the contribution of AFP to HCC development is clearly highly complex. Here, we discussed the effect of AFP deletion in the tumorigenesis and progression of HCC. AFP deletion in HepG2 cells inhibited the cell proliferation by inactivating PI3K/AKT signaling. Surprisingly, AFP KO HepG2 cells appeared the increasing metastatic capacity and EMT phenotype, which was attributed to the activation of WNT5A/ß-catenin signal. Further studies revealed that the activating mutations of CTNNB1 was closely related with the unconventional pro-metastatic roles of AFP deletion. Consistently, the results of DEN/CCl4-induced HCC mouse model also suggested that AFP knockout suppressed the growth of HCC primary tumors, but promoted lung metastasis. Despite the discordant effect of AFP deletion in HCC progression, a drug candidate named OA showed the potent suppression of HCC tumor growth by interrupting AFP-PTEN interaction and, importantly, reduced the lung metastasis of HCC via angiogenesis suppression. Thus, this study demonstrates an unconventional effect of AFP in HCC progression, and suggests a potent candidate strategy for HCC therapy.

CRISPR/Cas9 and piggyBac Transposon-Based Conversion of a Pathogenic Biallelic TBCD Variant in a Patient-Derived iPSC Line Allows Correction of PEBAT-Related Endophenotypes.

Induced pluripotent stem cells (iPSCs) have been established as a reliable in vitro disease model system and represent a particularly informative tool when animal models are not available or do not recapitulate the human pathophenotype. The recognized limit in using this technology is linked to some degree of variability in the behavior of the individual patient-derived clones. The development of CRISPR/Cas9-based gene editing solves this drawback by obtaining isogenic iPSCs in which the genetic lesion is corrected, allowing a straightforward comparison with the parental patient-derived iPSC lines. Here, we report the generation of a footprint-free isogenic cell line of patient-derived TBCD-mutated iPSCs edited using the CRISPR/Cas9 and piggyBac technologies. The corrected iPSC line had no genetic footprint after the removal of the selection cassette and maintained its "stemness". The correction of the disease-causing TBCD missense substitution restored proper protein levels of the chaperone and mitotic spindle organization, as well as reduced cellular death, which were used as read-outs of the TBCD KO-related endophenotype. The generated line represents an informative in vitro model to understand the impact of pathogenic TBCD mutations on nervous system development and physiology.

Bioengineering of fungal endophytes through the CRISPR/Cas9 system.

The CRISPR/Cas9 system is a genome-editing tool that allows for precise and efficient modifications to the DNA of a cell. This technology can be used in endophytic fungi, which live within plants and can have beneficial effects on their host, making them important for agriculture. Using CRISPR/Cas9, researchers can introduce specific genetic changes into endophytic fungal genomes, allowing them to study the function of genes, improve their plant-growth-promoting properties, and create new, more beneficial endophytes. This system works by using the Cas9 protein, which acts as a pair of molecular scissors, to cut DNA at specific locations determined by a guide RNA. Once the DNA is cut, the cell's natural repair mechanisms can be used to insert or delete specific genes, allowing for precise editing of the fungal genome. This article discusses the mechanism and applications of CRISPR/Cas9 to fungal endophytes.

[The Effect of the Knockout of Major Transsulfuration Genes on the Pattern of Protein Synthesis in D. melanogaster].

The enzymes involved in the transsulfuration pathway and hydrogen sulfide production-cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) - play an important cytoprotective role in the functioning of the organism. Using CRISPER/Cas9 technology, we obtained Drosophila strains with deleted cbs, cse, and mst genes as well as with double deletion of cbs and cse genes. We analyzed the effect of these mutations on the pattern of protein synthesis in the salivary glands of third instar larvae and in the ovaries of mature flies. In the salivary glands of strains with cbs and cse deletions, a decrease was found in the accumulation of the FBP2 storage protein containing 20% methionine amino acid residues. In the ovaries, changes were detected in the level of expression and isofocusing points of proteins involved in cell protection against oxidative stress, hypoxia, and protein degradation. It was shown that in the strains with deletions of transsulfuration enzymes the proteins have a similar degree of oxidation to that of the control strain. A decrease in the total number of proteasomes and their activity was found in the strains with deletions of the cbs and cse genes.