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Mutations in NF-κB-related molecules result in combined immunodeficiency characterized by recurrent infection. In this study, we aimed to investigate the association between mutations in NF-κB family members, RELA, RELB, and RELC, and regulatory T cell (Treg) function in humans. RELA, RELB, and RELC were knocked out using CRISPR/Cas9-mediated homologous recombination in CD4+/CD8+ T cells and Tregs isolated from healthy donors. The RELA, RELB, and RELC knockouts did not alter the phenotype or cytokine production profile of CD4+/CD8+ T cells or Jurkat cells. Similar to that observed in knockout mice, RELA or RELC knockout in MT-2 cells and freshly isolated Tregs reduced FOXP3 expression and the immune suppressive function of Tregs. Additionally, PD-L1 expression in effector T cells and Tregs decreased considerably following RELC knockdown. These findings demonstrated that the deletion of RELA or RELC resulted in the loss of Treg-like phenotype and function owing to the downregulation of FOXP3 expression.

Genetic disorders, particularly autosomal recessive diseases such as sickle cell disease and thalassemia, pose significant public health challenges in Saudi Arabia. High consanguinity rates contribute to their prevalence, necessitating advanced diagnostic approaches. Computational genomics offers innovative solutions for early diagnosis and personalized healthcare. Recent advancements in genomics, such as whole-genome sequencing and CRISPR-Cas9 gene editing, have transformed our understanding of molecular interventions for hereditary diseases. This research focuses on developing computational gene models tailored to the genetic profile of Saudi Arabia, with a particular emphasis on autosomal recessive (AR) diseases. This study aims to develop a hybrid Convolutional Neural Network-Gated Recurrent Unit (CNN-GRU) model specifically designed for diagnosing genetic illnesses prevalent in Saudi Arabia. The model seeks to leverage the strengths of CNNs for spatial feature extraction and GRUs for temporal dependency analysis, providing a robust tool for genomic data interpretation. The proposed CNN-GRU model was applied to a dataset representative of genetic disorders, demonstrating superior performance metrics with an accuracy of 98.8 %, precision of 98 %, recall of 99 %, and an F1-score of 98.5 %. These results surpassed traditional methods such as SVM, RNN, and LSTM-RNN, underscoring the model's efficacy in diagnosing genetic illnesses. Correlation analysis further validated the model's robustness by highlighting the independence of most medical and test parameters across diverse datasets. However, the analysis of the ROC curve identified areas for optimization, suggesting potential improvements to enhance the model's discriminatory power for even more precise diagnostics.

High-throughput genetic screening serves as an indispensable approach for deciphering gene functions and the intricate relationships between phenotypes and genotypes. The CRISPR/Cas9 system, with its ability to precisely edit genomes on a large scale, has revolutionized the field by enabling the construction of comprehensive genomic libraries. This technology has become a cornerstone for genome-wide screenings in disease research. This review offers a comprehensive examination of how CRISPR/Cas9-based genetic screening has been leveraged to uncover genes that play a role in disease mechanisms, focusing on areas such as cancer development and viral replication processes. The insights presented in this review hold promise for the development of novel therapeutic strategies and precision medicine approaches.

As a member of the single-fluorophore genetically encoded calcium indicators (GECIs), jGCaMP7f is widely applied to investigate intracellular Ca2+ concentrations. Here, we established an INS-jGCaMP7f knock-in H1 human embryonic stem cell (hESC) line by integrating jGCaMP7f gene into insulin locus via CRISPR/Cas9 system. The reporter cell line not only effectively labelled the insulin-producing cells induced from hESC, but also reflected the cytosolic change of Ca2+ level in response to different stimuli. This reporter cell line is a valuable research tool for studying functional maturation of hESC-derived insulin-producing cells, conducting drug screenings, and exploring the mechanisms of diabetes.

Agricultural production is affected by the worsening effects of climate change with severe yield losses caused by rising temperatures, water scarcity and consequent modifications in the interactions between crops, pests, and pathogens. The availability of stress-tolerant plants will thus be a key point to guarantee the world food security in the next future. To this purpose, a significant contribution might be provided by the New Genomic Techniques (NGTs), such as CRISPR/Cas9, which allow to insert targeted modifications in the plants’ genomes offering new opportunities for crop improvement. Susceptibility genes encode proteins that pathogens can take advantage of during the colonization process, and their disabling confers a broad-spectrum and long-lasting pathogen tolerance to the plant. Among the S-genes, Downy Mildew Resistance 6 (DMR6) encodes an enzyme involved in Salicylic Acid (SA) degradation, and its inactivation in other Solanaceae species has proven to increase SA levels and confer tolerance to a broad spectrum of pathogens. We identified two orthologs of this gene in eggplant's genome, namely SmDMR6–1 and SmDMR6–2. In the ‘Black Beauty’ cultivar, only SmDMR6–1 expression significantly increased upon infection by the two oomycetes Phytophthora infestans and Phytophthora capsici, suggesting its involvement in the regulation of plant responses to biotic stresses. Here we report, for the first time in eggplant, the knockout of SmDMR6–1 gene through CRISPR/Cas9 technology. The regenerated T0 plants were screened by Sanger sequencing and one was selected and self-pollinated to generate T1 and then T2 plants. The mutant lines were subjected to pathogen assays which highlighted an increased tolerance to infection by P. infestans and P. capsici, if compared to non-edited plants.

Megalobrama amblycephala is susceptible to hypoxia, which results high mortality rates in aquaculture. A preliminary study on a new variety of M. amblycephala called ''Pujiang No.2'' (GS-01–002–2020) discovered that its tolerance to hypoxia was significantly improved compared with that of wild breed. The objective of this study was to identify the key genes responsible for the enhanced hypoxia tolerance of this new variety. Based on previous RNA sequencing results, the function of btr02 (bloodthirsty-like tripartite motif [TRIM] 02 gene), a key gene for hypoxia tolerance, was explored using CRISPR/Cas9. The results demonstrated that the mutation rate of M. amblycephala btr02+/- was 39.38 %. The 5-month-old mutant was of the same size as normal individuals; however, its tolerance to hypoxia decreased substantially. Specifically, the LOEcrit of knockout mutants increased significantly (P

Yogurt is a coagulated dairy product that results from the fermentation of milk and milk products by lactic acid bacteria, specifically Lactobacillus bulgaricus and Streptococcus thermophilus. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is an emerging tool in molecular biology research which can be used extensively in prokaryotes and eukaryotes. Lactate dehydrogenase (LDH) is an important enzyme of glycolytic pathway. In this research, we have edited L. bulgaricus d-lactate dehydrogenase (ldh) gene using CRISPR/Cas9 system. Genome editing was accomplished through synthesizing gDNA on the basis of PAM region of ldh gene and ligation of synthesized gDNA with pCRISPR-SacB plasmid which was later co-transformed with mutagenic donor DNA into competent cells containing pCasRed. This method combined CRISPR/Cas9 with γ Red machinery to achieve editing of ldh gene. The PCR screening and sequencing results confirmed that, base deletion has been occurred in the target region of ldh gene. Differences in several characteristics including texture, pH were observed on the yogurt samples from newly developed L. bulgaricus strains compared to normally produced yogurt. Our results suggest that, other genes of lactobacillus can be edited through CRISPR/Cas9 technology which can be extensively used for metabolic engineering.

Induced pluripotent stem cells (iPSCs) harboring patient derived SAMD9 mutation offer a unique platform to study the multi-organ involvement observed in this rare disease, referred to as myelodysplasia, infections, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy (MIRAGE) syndrome. The pluripotent nature of iPSCs allows in vitro differentiation into various somatic cell types representing multiple organ systems affected in SAMD9-mutated patients. Hence, in this paper, we present a CRISPR/Cas9-engineered iPSC model carrying SAMD9 c.2948T>G, p.I983S mutation previously reported in two patients with severe MIRAGE syndrome.

Genome editing by CRISPR-Cas holds promise for the treatment of retinal dystrophies. For therapeutic gene editing, transient delivery of CRISPR-Cas9 is preferable to viral delivery which leads to long-term expression with potential adverse consequences. Cas9 protein and its guide RNA, delivered as ribonucleoprotein (RNP) complexes, have been successfully delivered into the retinal pigment epithelium in vivo. However, the delivery into photoreceptors, the primary focus in retinal dystrophies, has not been achieved. Here, we investigate the feasibility of direct RNP delivery into photoreceptors and retinal pigment epithelium cells. We demonstrate that Cas9 or adenine-base editors complexed with guide RNA, can enter retinal cells without the addition of any carrier compounds. Once in the retinal cells, editing rates vary based on the efficacy of the guide RNA and the specific location edited within the genes. Cas9 RNP delivery at high concentrations, however, leads to outer retinal toxicity. This underscores the importance of improving delivery efficiency for potential therapeutic applications in the future.

Combination immunotherapy has shown promising potential for enhancing the objective response rate compared to immune checkpoint blockade (ICB) monotherapy. However, combination therapy with multi-drugs is limited by the different properties of the agents and inconsistent synergistic targeted delivery. Herein, based on a universal triterpene template and the anticancer active agent ursolic acid (UA), a cytomembrane-coated biomimetic delivery nanoplatform (UR@M) prepared by the self-assembly of a PD-L1 targeted CRISPR/Cas9 system and UA was designed for hepatocellular carcinoma (HCC) treatment. UR@M showed enhanced tumor accumulation in vivo with homologous tumor targeting, and CRISPR in the nanosystem exhibited potent gene-editing efficiency of 76.53% in vitro and 62.42% in vivo with no off-target effects. UA activated the natural immune system through the TLR-2-MyD88-TRAF6 pathway, which synergistically enhanced the proliferation of natural killer cells and dendritic cells and realized excellent immune cytotoxic T cell infiltration by combining with the ICB of PD-L1. The strategy of work along both lines based on innate immune and adaptive immunity displayed a significant effect in tumor regression. Overall, the UA-templated strategy “killed three birds with one stone” by establishing a self-assembly nanosystem, inducing tumor cell death, and promoting synergistic immunostimulation for HCC treatment.

Gene editing facilitated by homology-directed repair represents a promising strategy for precisely correcting pathogenic variants underlying monogenic disorders, including the life-threatening skin blistering condition junctional epidermolysis bullosa (JEB). Frequent reports of unintended off-target genotoxicity associated with conventional Cas9 nuclease editing have increasingly led to the adoption of dual-Cas9 nickases (dual-Cas9n) owing to their improved safety profile. However, rates of precise repair obtained with such strategies remain low. In this study, we establish a dual-Cas9n approach targeting LAMB3, using electroporation to deliver Cas9-nickase ribonucleoproteins and modified single-stranded oligodeoxynucleotide repair templates into primary JEB keratinocytes. Targeting a hotspot pathogenic variant (c.1903C>T, p.R635∗), we report perfect correction efficiencies of up to 54% based on standard next-generation sequencing. Using a high-fidelity Cas9 nuclease, we also report perfect repair of up to 74% when using a small-molecule modulator of DNA repair. Dual-Cas9n–corrected JEB keratinocytes demonstrated restored laminin-332 expression and secretion in vitro, leading to improved cellular adhesion and accurate laminin-332 localization in engineered skin equivalents. This protocol represents a significant improvement in precision gene repair using Cas9 nickases for epidermolysis bullosa, with the potential to be applied to a large cohort of patients harboring this prevalent pathogenic variant.

Largemouth bass (LMB, Microterus salmoides) is an important commercial freshwater fish, and body color is a key economic trait for quality improvement. Gene editing has not been established in LMB due to the unavailability of one-cell fertilized eggs. In this study, three types of pigment cells, melanophores, iridophores and xanthophores were identified in LMB and melanophores were found first appeared in the iris of eyes in larvae 1.5 days after hatching. The fertilized eggs were obtained through hormonal induction (5 μg/kg LHRH-A3, 600 IU/kg hCG and 2 mg/kg DOM) and artificial insemination, and the tyrb (tyrosinase b) or csf1ra (colony-stimulating factor 1 receptor a) gRNA and Cas9 mRNA were microinjected into the fertilized eggs at the one-cell stage for targeted gene editing. The target site was designed on the first exon of tyrb and the second exon of csf1ra, respectively. The efficiency of mutation was gRNA and Cas9 mRNA concentration dependent, and 400 ng/μl gRNA and 800 ng/μl Cas9 mRNA were recommended as the appropriate injection dose for genome editing in LMB. Enzyme-digestion and sanger-sequencing demonstrated that the CRISPR/Cas9 system is available for the gene editing of LMB. The tyrb mutation resulted in reduced melanin and even albinism in eye and body of larvae, while the csf1ra mutation led to reddish body color, reduced xanthophores and melanophores in juvenile and adult fish with high mutation rate. Our results indicated that the CRISPR/Cas9 gene-editing system was successfully established in LMB. The mutation of body color related genes provided great convenience for the observation of mutant phonotypes and produced improved strains of LMB. It is the first successful application of gene-editing technology in LMB, which has taken a key step in studying the gene function and molecular mechanism and provided a novel method for improving the economic traits of LMB aquaculture.

The soybean crop, known as a ''miracle crop'' for its versatility as an oilseed, legume, and protein-rich source, is facing yield curtailments due to fluctuating global temperatures, emphasizing a deeper understanding of its heat stress tolerance mechanisms ply both conventional and non-conventional methods. Cutting edges techniques such as CRISPR/Cas9, genetic engineering, QTL mapping, and transcriptome studies are in limelight. The core motif is to supercharge heat tolerance by manipulating heat shock proteins (HSPs), transcription factors, and epigenetic mechanisms. Soybean response to heat stress entails complex molecular and cellular processes including hormone signaling, ROS detoxification, antioxidant synthesis, and gene expression regulation. Additionally, transcriptome and proteome perusal has shown the significant role of transcriptional changes in heat stress response. Abiotic stresses, including drought and nutrient deficiencies, also pose risks to global food security by reducing crop yields. Advanced approaches that enhance stress resilience in soybean are critical for buoy future production amid unpredictable climate challenges.

Freshwater shortages, exacerbated by climate change and unpredictable rainfall, significantly hinder global food security. AP2/ERF transcription factors regulate genes that help plants survive and adapt to harsh environmental stresses. Although ERF109 is linked to drought tolerance, its exact role remains unknown. To investigate this, we generated mutant alleles of the OsERF109 gene in an indica rice cultivar and studied their response to simulated drought conditions. Our findings show that the Oserf109 mutation exhibits no visible effect on plant growth and development under optimum environmental conditions. However, the Oserf109 null mutant demonstrated improved drought tolerance and survival rates. This improvement is due to reduced transpiration, lower canopy temperature, and better plant water status. The Oserf109 mutant maintains cellular hydration and membrane stability by increasing proline content under drought stress. The Oserf109 mutant also exhibits enhanced water use efficiency and improved gaseous exchange, which boosts photosynthetic efficiency, strengthens antioxidant defenses, and preserves chloroplast integrity, protecting the plant from oxidative damage during drought. Additionally, Oserf109 knockout plants showed increased tolerance to osmotic stress and reduced sensitivity to ABA, resulting in better seedling growth and higher germination rates. Our research reveals that the Oserf109 mutation enhances rice crop resilience and yield under adverse conditions. These findings illustrated the inhibitory regulatory role of OsERF109 in the drought tolerance mechanism, with the mutant form of this allele demonstrating potential implications in developing resilient rice cultivars for drought stress.

Remarkable progress has been made in the field of genome engineering after the discovery of CRISPR/Cas9 in 2012 by Jennifer Doudna and Emmanuelle Charpentier. Compared to any other gene-editing tools, CRISPR/Cas9 attracted the attention of the scientific community because of its simplicity, specificity, and multiplex editing possibilities for which the inventors were awarded the Nobel prize for chemistry in 2020. CRISPR/Cas9 allows targeted alteration of the genomic sequence, gene regulation, and epigenetic modifications using an RNA-guided site-specific endonuclease. Though the impact of CRISPR/Cas9 was undisputed, some of its limitations led to key modifications including the use of miniature-Cas proteins, Cas9 Retron precise Parallel Editing via homologY (CRISPEY), Cas-Clover, or development of alternative methods including retron-recombineering, Obligate Mobile Element Guided Activity(OMEGA), Fanzor, and Argonaute proteins. As cancer is caused by genetic and epigenetic alterations, gene-editing was found to be highly useful for knocking out oncogenes, editing mutations to regain the normal functioning of tumor suppressor genes, knock-out immune checkpoint blockade in CAR-T cells, producing ‘off-the-shelf’ CAR-T cells, identify novel tumorigenic genes and functional analysis of multiple pathways in cancer, etc. Advancements in nanoparticle-based delivery of guide-RNA and Cas9 complex to the human body further enhanced the potential of CRISPR/Cas9 for clinical translation. Several studies are reported for developing novel delivery methods to enhance the tumor-specific application of CRISPR/Cas9 for anticancer therapy. In this review, we discuss new developments in novel gene editing techniques and recent progress in nanoparticle-based CRISPR/Cas9 delivery specific to cancer applications.

The fluorescence ubiquitination cell cycle inhibitor (FUCCI) has been introduced to monitor cell cycle activity in living cells, including human induced pluripotent stem cells (hiPSC) and derived cell types. We have recently developed hiPSC with stable expression of dCas9VPR for endogenous gene activation and a Citrine-tagged ACTN2 cell line to monitor sarcomere development and function in muscle cells. Here, we present dual and triple transgenic hiPSC lines developed by genomic integration of FUCCI with and without dCas9VPR into the ROSA26 and AAVS1 loci, respectively, in the previously introduced ACTN2-Citrine line. Functionality of the transgenes was demonstrated in the novel hiPSC line, which we introduce as Myo-CCER and CraCCER.

The Growth Hormone Receptor (GHR) gene encodes a protein that is essential for mediating the biological effects of growth hormone (GH). A series of molecular events are set off when GH binds to its receptor, resulting in a variety of physiological reactions linked to development, growth, and metabolism. Recently a particular genetic variation, within the GHR gene that is labeled as the “d3GHR,” which lacks exon 3 was associated with longevity. This specific deletion isoform was connected to changes in the structure of the GHR protein, which may have an impact on the GHR's function. To test in vitro the advantage of the d3 carrier that may link to longevity, we employed the CRISPR/Cas9 technique to produce two isoforms: the homozygotes isoform (d3/d3) and the heterozygotes isoform (d3/fl) using HEK293 cell line. The CRISPR editing effectiveness was >85 %, indicating that we had successfully built the Cas9-gRNA complex that is appropriate for the GHR gene. The viability of the resulted isoform cells was examined under three environmental stressors that mimic some aging processes. In addition, we examined the GHR signaling pathway by selecting potential downstream genes in the GHR signaling cascade. The results show that heterozygotes cells demonstrated higher survival rates under UV radiation compared with the WT cells (87 % compared with 67 % for the WT cells when exposed to 2 min of UV radiation), and in fasting conditions, the d3GHR cells showed a 15 % greater viability than the WT cells. Moreover, the baseline expression levels (without intervention) of the IGF1 and JAK/STAT genes signaling pathways significantly declined in the homozygotes cells compared with the WT (p

Mucopolysaccharidosis (MPS) syndromes are a group of heterogeneous genetic disorders in terms of genetic basis and clinical manifestations, ranging from mild to fatal forms. There are a number of applied or prospective treatment modalities for MPS, including bone marrow transplantation, enzyme replacement therapy, targeted gene therapy and substrate reduction therapy. Recently, CRISPR/Cas9 technology has emerged as a novel tool for several metabolic disorders, such as MPS. This review concentrates on the application of this technique in the treatment of MPS, particularly MPS I, and modeling of disease-causing mutations.

Vitamin D deficiency poses a widespread health challenge, shaped by environmental and genetic determinants. A recent discovery identified a genetic regulator, rs11542462, in the SDR42E1 gene, though its biological implications remain largely unexplored. Our bioinformatic assessments revealed pronounced SDR42E1 expression in skin keratinocytes and the analogous HaCaT human keratinocyte cell lines, prompting us to select the latter as an experimental model. Employing CRISPR/Cas9 gene-editing technology and multi-omics approach, we discovered that depleting SDR42E1 showed a 1.6-fold disruption in steroid biosynthesis pathway (P-value = 0.03), considerably affecting crucial vitamin D biosynthesis regulators. Notably, SERPINB2 (P-value = 2.17 × 10−103), EBP (P-value = 2.46 × 10−13), and DHCR7 (P-value = 8.03 × 10−09) elevated by ∼2–3 fold, while ALPP (P-value

Virus-induced gene silencing (VIGS) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology. However, the investigation of gene silencing and editing in radish remains limited. In this study, a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus (TRV)- and turnip yellow mosaic virus (TYMV)-mediated gene silencing vectors. The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish. The expression level of RsPDS was significantly inhibited using VIGS in ‘NAU-067’ radish leaves. The rootless seedlings of ‘NAU-067’ were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences. Nine adventitious roots were blue with GUS staining, and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing. Furthermore, albino lines were generated with A. tumefaciens-mediated transformation of radish cotyledons. Five base substitutions and three base deletions occurred at target sequence 2 in Line 1, and three base insertions and three base substitutions occurred at target sequence 1 in Line 2. This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish, which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.

Organic anion-transporting polypeptides 1B1 (OATP1B1) plays a crucial role in the transport of statins. However, there are too few animal models related to OATP1B1, especially humanized animal models. In this study, the human SLCO1B1 cDNA was inserted into the second exon of the rat Slco1b2 gene using CRISPR/Cas9 technology. Pharmacokinetic characteristics of statins were conducted in wild-type (WT), humanized OATP1B1 (hOATP1B1), and OATP1B2 knockout (OATP1B2 KO) rats, respectively. The results showed that human OATP1B1 was successfully expressed in rat liver and exhibited transport function. Furthermore, the pharmacokinetic results revealed that OATP1B1 exhibited varying uptake levels of pivastatin, rosuvastatin, and fluvastatin, leading to different levels of exposure within the body. These results were consistent with those obtained from in vitro experiments using overexpressed cell lines. In conclusion, we established a novel humanized SLCO1B1 transgenic rat model to assess the role of human OATP1B1 in the uptake of different statins. The different uptake mediated by OATP1B1 may be an important reason for the different efficacy of statins. The hOATP1B1 rat is a promising model for improving the prediction of human drug transport.

Introduction: As one of the common psychiatric diseases, depression poses serious threats to human health. Although many genes have been nominated for depression, few of them were investigated in details at the molecular level. Objectives: To demonstrate Frizzled class receptor 6 (FZD6) functions in depression through disrupting Wnt/β-catenin signal pathway. Methods: The FZD6 edited cell line and mouse model were generated by using CRISPR/Cas9 technique. The expression of key genes and proteins in Wnt/β-catenin pathway was determined by qRT-PCR and Western blotting, respectively. Animal behavioral tests, including open field test (OFT), elevated plus maze test (EPM), forced swimming test (FST), tail suspension test (TST), and sucrose preference test (SPT), were employed to determine anxiety- and depressive-like behaviors. Immunofluorescent staining was used to assess cell proliferation in the hippocampus of mouse brain. Results: Among patients with depression, FZD6, one of the receptors of Wnt ligand, was significantly decreased. In CRISPR/Cas9-based FZD6 knockdown cells, we showed that FZD6 plays a significant role in regulating expression of genes involved in Wnt/β-catenin pathway. Subsequently behavioral studies on Fzd6 knockdown mice (with a 5-nucleotide deletion; Fzd6-Δ5) revealed significant changes in depressive symptoms, including increased immobility duration in FST, less preference of sucrose in SPT, reduction of distance traveled in OFT, and decreased time spent in open arms in EPM. Immunofluorescent staining showed decreased cell proliferation in the hippocampus of Fzd6-Δ5 mice with reduced number of Ki67+ and PCNA+ cells.Moreover, decreased Gsk3β mRNA expression, phosphorylated GSK3β, and cytoplasmic β-catenin in the hippocampus of Fzd6-Δ5 mice provided further evidence supporting the role of Fzd6 in depression. Conclusion: Together, above findings proved the significant role of FZD6 in depression through its effect on hippocampal cell proliferation and its ability to regulate canonical Wnt/β-catenin pathway.

Extracellular vesicles (EVs) have recently emerged as a promising delivery platform for CRISPR/Cas9 ribonucleoproteins (RNPs), owing to their ability to minimize off-target effects and immune responses. However, enhancements are required to boost the efficiency and safety of Cas9 RNP enrichment within EVs. In response, we employed the Fc/Spa interaction system, in which the human Fc domain was fused to the intracellular domain of PTGFRN-Δ687 and anchored to the EV membrane. Simultaneously, the B domain of the Spa protein was fused to the C domain of cargos such as Cre or spCas9. Due to the robust interaction between Fc and Spa, this method enriched nearly twice the amount of cargo within the EVs. EVs loaded with spCas9 RNP targeting the HSV1 genome exhibited significant inhibition of viral replication in vitro and in vivo. Moreover, following neuron-targeting peptide RVG modification, the in vivo dosage in neural tissues substantially increased, contributing to the clearance of the HSV1 virus in neural tissues and exhibiting a lower off-target efficiency. These findings establish a robust platform for efficient EV-based SpCas9 delivery, offering potential therapeutic advantages for HSV1 infections and other neurological disorders.

The recent years have witnessed the rapid advancement of clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9)-mediated gene editing in cancer therapy. The success of cancer gene therapy relies heavily on the development of safe and effective delivery vectors. We focus on organic nanocarriers because of their flexible customizability, which permits them to accomplish excellent delivery efficiency and performance. In this review, the pharmacokinetic behavior of CRISPR/Cas9 nanocarriers serves as a guide to outline the strategies for improving the bioavailability of nanocarriers through physical, chemical, and biological means. Furthermore, the potential of organic nanocarriers for simultaneous delivery of therapeutic agents and gene editing tools is highlighted. Finally, we discuss future directions for the development of cancer therapeutic nanosystems, paving the way for the construction of effective CRISPR/Cas9 delivery systems.

An effective CRISPR/Cas9 reagent delivery system has been developed in a commercially significant crop, the chilli pepper using a construct harboring two distinct gRNAs targeting exons 14 and 15 of the Phytoene desaturase (CaPDS) gene, whose loss-of-function mutation causes a photo-bleaching phenotype and impairs the biosynthesis of carotenoids. The construct carrying two sgRNAs was observed to create visible albino phenotypes in cotyledons regenerating on a medium containing 80 mg/L kanamycin, and plants regenerated therefrom after biolistic-mediated transfer of CRISPR/Cas9 reagents into chilli pepper cells. Analysis of CRISPR/Cas9 genome-editing events, including kanamycin screening of mutants and assessing homozygosity using the T7 endonuclease assay (T7E1), revealed 62.5 % of transformed plants exhibited successful editing at the target region and displayed both albino and mosaic phenotypes. Interestingly, the sequence analysis showed that insertions and substitutions were present in all the plant lines in the targeted CaPDS region. The detected mutations were mostly 12- to 24-bp deletions that disrupted the exon–intron junction, along with base substitutions and the insertion of 1-bp at the protospacer adjacent motif (PAM) region of the target site. The reduction in essential photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoid) in knockout chilli pepper lines provided further evidence that the CaPDS gene had been functionally disrupted. In this present study, we report that the biolistic delivery of CRISPR/Cas9 reagents into chilli peppers is very effective and produces multiple mutation events in a short span of time.

Iron (Fe) deficiency is a pressing global health concern, particularly affecting vulnerable groups like women and children in resource-limited areas. Addressing this challenge requires innovative solutions, and biofortified crops, like Fe-enriched wheat, can offer a sustainable solution to improve nutrition in cereal-based diets. While conventional breeding methods have yielded competitive Fe-biofortified wheat varieties across various nations, the imminent challenges in securing food and nutritional security for the future necessitate a delicate balance: maintaining genetic progress in grain yield while concurrently elevating grain Fe content. Despite substantial strides in elucidating the intricacies of Fe homeostasis, there remains a substantial knowledge gap, especially in the context of wheat and similar crop species. It is paramount to gain a comprehensive understanding of the hurdles impeding Fe enrichment in plant tissues and delve into the diverse mechanisms governing Fe uptake, translocation, transport, and storage within wheat. To surmount these challenges, researchers have explored a multitude of strategies, including mutagenesis, QTL mapping, meta-QTL analysis, GWAS, transgenesis, and genome editing. Furthermore, harnessing the potential of microorganisms, particularly engineered endophytes coupled with plant genes associated with Fe accumulation, emerges as a promising and pragmatic tool for augmenting Fe biofortification in wheat. This comprehensive review underscores the significant advancements made in unravelling the genetic and genomic aspects of Fe accumulation in wheat, while also delineating the future research directions in this field. By synergistically deploying these multifaceted approaches, scientists hold the potential to develop wheat varieties characterized by enhanced grain Fe content, improved bioavailability, and reduced anti-nutritional factors. Such innovations can play a pivotal role in advancing nutrition and health outcomes for populations reliant on wheat-based diets, particularly in resource-scarce regions.

Iron is an essential plant nutrient, and a continuous supply of it is required as it is a key factor in various metabolic processes, including photosynthesis, chlorophyll synthesis, and respiration. Various transcription factors are known to regulate iron homeostasis in plants, and the bHLH transcription factor family is one of them. The StbHLH47 is a homologue of the Arabidopsis POPEYE (PYE), which is known to repress iron homeostasis-related genes in Arabidopsis. Potato is the most consumed vegetable in the world and is low in iron content. We have generated CRISPR/Cas9-edited StbHLH47 lines and performed a detailed analysis of these lines. The analysis revealed that the roots of StbHLH47 edited lines have decreased ferric chelate reductase (FCR) activity compared to the roots of the wild-type (WT) plant. We also observed that CRISPR/Cas9 edited lines have fewer trichomes when compared to the WT plant. The expression of genes associated with iron homeostasis was also measured. Compared to the control, the expression of StbHLH47 was downregulated in the edited lines, while the expression of StNAS4, StOPT3, and StFRO3 was upregulated. This suggests the negative regulation of StbHLH47 in modulating iron. The iron content was also quantified using inductively coupled plasma mass spectrometry (ICP-MS) and found to be increased in the generated transgenic lines when compared to WT plants. Overall, this study reveals that StbHLH47 negatively regulates the expression of iron homeostasis-related genes. StbHLH47 edited lines exhibited decreased FCR activity, changes in phenotype, and increased iron content in the potato plants. Key message: This study provides novel insight into the role of StbHLH47 in modulating iron content in Solanum tuberosum and controlling the expression of various iron homeostasis-related genes.

Autologous chimeric antigen receptor (CAR) T cell therapy has revolutionised the management of certain B-cell malignancies. However, as bespoke therapies, challenges include complex manufacturing logistics and risks ranging from suboptimal harvests to inadvertent transduction and masking of blast populations. Premanufactured, ready -to -use allogeneic CAR T cells could mitigate some of these hurdles if barriers created by HLA (Human leukocyte antigen) mismatching can be addressed. Genome editing to disrupt TCRαβ (T-cell receptor αβ) expression has been shown to be effective in addressing alloreactivity and avoiding graft versus host disease (GVHD). Platforms including transcription activator-like effector nucleases (TALENs), homing endonucleases and clustered regularly interspersed short palindromic repeats (CRISPR) / Cas9 have allowed multiplex editing of TCR genes in combination with CD52, the target antigen of alemtuzumab, as a strategy to evade lymphodepletion used to prevent host v graft rejection effects. Alternative approaches have targeted pathways to prevent HLA expression on donor T cells, and have also allowed targeted insertion of CAR genes, including placing transgene expression under the control of endogenous transcriptional machinery. These tools have rapidly progressed to clinical trials, and applications have extended beyond B-cell malignancies, showing promising early results in other settings, including relapsed/refractory(r/r) T-cell leukaemia. Short term immunological effects and toxicities have been generally manageable, and long-term monitoring is ongoing to help build confidence in safety over time.

Antimicrobial peptides (AMPs) show promise in enhancing resistance against pathogens. Previously, we integrated two AMP genes, cathelicidin (Cath) from alligator (Alligator mississippiensis or Alligator sinensis) and cecropin (Cec) from moth (Hyalophora cecropia), into the channel catfish (Ictalurus punctatus) genome. This study examines the efficacy of exogenous AMP gene integration in improving bacterial resistance in transgenic channel catfish and assesses the direct and pleiotropic effects of gene replacement/knockout on survival and growth based on insertion site. Transgenic Cath- and Cec-expressing fish exhibited similar or higher survival rates (P ​> ​0.05) compared to controls during the initial culture. Integration of the Cec transgene doubled the survival rate when challenged with Edwardsiella ictaluri, with knock-in (KI) of Cath further increasing bacterial resistance. Coupling Cec KI with mstn knockout (KO) increased survival 3-fold after E. ictaluri infection and growth by 50% at 4 months post-fertilization (mpf). However, random integration of Cec had a minimal effect on disease resistance and did not enhance growth. Random integration of Cath increased survival 2.5-fold and 4-fold against E. ictaluri and Flavobacterium covae, respectively, without affecting growth. Cath KI at the lh locus increased survival 4-fold when challenged with F. covae and reduced growth by 10% (P ​> ​0.05) at 24 mpf, whereas Cath KI coupled with mc4r KO resulted in a 2.5-fold increase in survival following F. covae infection compared with controls, and increased growth by 80% at 3 mpf. Simultaneous KI of Cath and Cec, along with KO of mc4r and mstn, increased survival 4-fold against E. ictaluri, while increasing growth by 50% at 3 mpf. Dual insertion of AMP genes yielded the greatest resistance to disease. These direct and pleiotropic effects may increase comprehension and societal acceptance of genetic engineering in aquaculture.

Clinical application of CRISPR-Cas9 technology for large deletions of somatic mutations is inefficient, and methods to improve utility suffer from our inability to rapidly assess mono- vs. biallelic deletions. Here we establish a model system for investigating allelic heterogeneity at the single-cell level and identify indel scarring from non-simultaneous nuclease activity at gRNA cut sites as a major barrier to CRISPR-del efficacy both in vitro and in vivo. We show that non-simultaneous nuclease activity is partially prevented via restriction of CRISPR-Cas9 expression via inducible adeno-associated viruses (AAVs) or lipid nanoparticles (LNPs). Inducible AAV-based expression of CRISPR-del machinery significantly improved mono- and biallelic deletion frequency in vivo, supporting the use of the Xon cassette over traditional constitutively expressing AAV approaches. These data depicting improvements to deletions and insight into allelic heterogeneity after CRISPR-del will inform therapeutic approaches for phenotypes that require either large mono- or biallelic deletions, such as autosomal recessive diseases or where mutant allele-specific gRNAs are not readily available, or in situations where the targeted sequence for excision is located multiple times in a genome.

β-Thalassemia is brought about by defective β-globin (HBB [hemoglobin subunit β]) formation and, in severe cases, requires regular blood transfusion and iron chelation for survival. Genome editing of hematopoietic stem cells allows correction of underlying mutations as curative therapy. As potentially safer alternatives to double-strand-break–based editors, base editors (BEs) catalyze base transitions for precision editing of DNA target sites, prompting us to reclone and evaluate two recently published adenine BEs (ABEs; SpRY and SpG) with relaxed protospacer adjacent motif requirements for their ability to correct the common HBBIVSI-110(G>A) splice mutation. Nucleofection of ABE components as RNA into patient-derived CD34+ cells achieved up to 90% editing of upstream sequence elements critical for aberrant splicing, allowing full characterization of the on-target base-editing profile of each ABE and the detection of differences in on-target insertions and deletions. In addition, this study identifies opposing effects on splice correction for two neighboring context bases, establishes the frequency distribution of multiple BE editing events in the editing window, and shows high-efficiency functional correction of HBBIVSI-110(G>A) for our ABEs, including at the levels of RNA, protein, and erythroid differentiation.

The yellow seed trait is preferred by breeders for its potential to improve the seed quality and commercial value of Brassica napus. In the present study, we produced yellow seed mutants using a CRISPR/Cas9 system when the two BnPAP2 homologs were knocked out. Histochemical staining of the seed coat demonstrated that proanthocyanidin accumulation was significantly reduced in the pap2 double mutants and decreased specifically in the endothelial and palisade layer cells of the seed coat. Transcriptomic and metabolite profiling analysis suggested that disruption of the BnPAP2 genes could reduce the expression of structural and regulated genes in the phenylpropanoid and flavonoid biosynthetic pathways. The broad suppression of these genes might hinder proanthocyanidin accumulation during seed development, and thereby causing the yellow seed trait in B. napus. These results indicate that BnPAP2 might play a vital role in the regulatory network controlling proanthocyanidin accumulation.

Introduction: Chimeric Antigen Receptor (CAR) T cells have tremendous potentials for cancer treatment; however, various challenges impede their universal use. These restrictions include the poor function of T cells in tumor microenvironments, the shortage of tumor-specific antigens and, finally, the high cost and time-consuming process, as well as the poor scalability of the method. Creative gene-editing tools have addressed each of these limitations and introduced next generation products for cell therapy. The clustered regularly interspaced short palindromic repeats-associated endonuclease 9 (CRISPR/Cas9) system has triggered a revolution in biology fields, as it has a great capacity for genetic manipulation. Method: In this review, we considered the latest development of CRISPR/Cas9 methods for the chimeric antigen receptor T cell (CAR T)-based immunotherapy. Results: The ability of the CRISPR/Cas9 system to generate the universal CAR T cells and also potent T cells that are persistent against exhaustion and inhibition was explored. Conclusion: We explained CRISPR delivery methods, as well as addressing safety concerns related to the use of the CRISPR/Cas9 system and their potential solutions.

The identification of functional midgut receptors for pesticidal proteins produced by Bacillus thuringiensis (Bt) is critical for deciphering the molecular mechanism of Bt resistance in insects. Reduced expression of the PxABCB1 gene was previously found to be associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.). To directly validate the potential receptor role of PxABCB1 and its contribution to Bt Cry1Ac toxicity in P. xylostella, we used CRISPR/Cas9 to generate a homozygous knockout ABCB1KO strain with a 5-bp deletion in exon 3 of its gene. The ABCB1KO strain exhibited a 63-fold resistance to Cry1Ac toxin compared to the parental DBM1Ac-S strain. Intriguingly, the ABCB1KO strain also exhibited significant increases in susceptibility to abamectin and emamectin benzoate. No changes in susceptibility to various other Bt Cry proteins or synthetic insecticides were observed. The knockout strain exhibited no significant fitness costs. Overall, our study indicates that PxABCB1 can protect the insect against avermectin insecticides on one hand, while on the other hand it facilitates the toxic effect of the Bt Cry1Ac toxin. The results of this study will help to inform integrated pest management approaches against this destructive pest.

The objective of this study was to determine the role of SLC15A4 in the muramyl dipeptide (MDP)-mediated inflammatory response of bovine rumen epithelial cells (BRECs). First, changes in the mRNA expression of pro-inflammatory factor genes in BRECs following 10 μg mL–1 MDP treatments were examined. RT-qPCR results showed that the expression of pro-inflammatory factor (IL-1β, IL-6, and TNF-α) mRNAs were significantly increased under MDP stimulation (P

Objective: To construct models of high-risk human papillomavirus (HPV) infection with precancerous lesions or cervical cancer and explore the immune function. Methods: Using CRISPR/Cas9, the expression vector HPV16-E6-E7-Rosa26 was microinjected into fertilized eggs of C57BL/6 N mice using homologous recombination, and the F0 generation was obtained for reproduction. Then, the formation of precancerous lesions was promoted via intramuscular injection of estradiol. Presence of precancerous cervical-vaginal intraepithelial lesions, Ki67 and p16 expression levels, and CD8+ T cell proportions in the spleen were evaluated. Results: Two F0 generation mice exhibited correct the homologous recombination. Seven positive mice were identified in the F1 generation. After breeding and mating, 25 homozygous and 11 heterozygous HPV16-E6-E7-engineered mice were obtained from the F2 generation. After estradiol benzoate treatment, the cervical-vaginal epithelium appeared as precancerous lesions with positive Ki67 and p16 expression. The percentage of CD8+ T cells decreased. Conclusion: HPV16-E6-E7-Rosa26 induced low immune function in mice, and provides a good model for the basic research of the mechanisms of action of HPV infection-associated precancerous lesions or cervical cancer.

Despite the success of combination antiretroviral therapy (cART) in HIV treatment, a cure for HIV remains elusive. Scientists postulate that HIV latent reservoirs may be a vital target in curative strategies. Vorinostat is a latency-reversing agent that has demonstrated some effectiveness in reactivating latent HIV, but complementary therapies may be essential to enhance its efficacy. One such approach may utilize the CRISPR-Cas9 system, which has evolved to include transcriptional activators such as dCas9-VPR. In this study, we explored the effects of combining vorinostat coupled with gesicle-mediated delivery of dCas9-VPR in promoting the transcription of integrated HIV proviruses in HIV-NanoLuc CHME-5 microglia and J-Lat 10.6 lymphocytes. We confirmed that dCas9-VPR ribonucleoprotein complexes can be packaged into gesicles and application to cells successfully induced HIV transcription through interactions with the HIV LTR. Vorinostat also induced significant increases in proviral transcription but generated inhibition of cellular proliferation (microglia) or cell viability (lymphocytes) starting at 1,000 nM and higher concentrations. Experiments combining dCas9-VPR gesicles and vorinostat confirmed the enhanced transcriptional activation of the HIV provirus in microglia but not lymphocytes. Thus, a combination of dCas9-VPR gesicles with other latency-reversing agents may provide a complementary method to activate latent HIV in future studies utilizing patient-derived cells or small animal models.

Abstract Background About one-third of patients with estrogen receptor alpha (ERα)-positive breast cancer have tumors which are progesterone receptor (PR) negative. PR is an important prognostic factor in breast cancer. Patients with ERα-positive/PR-negative tumors have shorter disease-free and overall survival than patients with ERα-positive/PR-positive tumors. New evidence has shown that progesterone (P4) has an anti-proliferative effect in ERα-positive breast cancer cells. However, the role of PR in breast cancer is only poorly understood. Methods We disrupted the PR gene (PGR) in ERα-positive/PR-positive T-47D cells using the CRISPR/Cas9 system. This resulted in cell pools we termed PR-low as P4 mediated effects were inhibited or blocked compared to control T-47D cells. We analyzed the gene expression profiles of PR-low and control T-47D cells in the absence of hormone and upon treatment with P4 alone or P4 together with estradiol (E2). Differentially expressed (DE) genes between experimental groups were characterized based on RNA-seq and Gene Ontology (GO) enrichment analyses. Results The overall gene expression pattern was very similar between untreated PR-low and untreated control T-47D cells. More than 6000 genes were DE in control T-47D cells upon stimulation with P4 or P4 plus E2. When PR-low pools were subjected to the same hormonal treatment, up- or downregulation was either blocked/absent or consistently lower. We identified more than 3000 genes that were DE between hormone-treated PR-low and control T-47D cells. GO analysis revealed seven significantly enriched biological processes affected by PR and associated with G protein-coupled receptor (GPCR) pathways which have been described to support growth, invasiveness, and metastasis in breast cancer cells. Conclusions The present study provides new insights into the complex role of PR in ERα-positive/PR-positive breast cancer cells. Many of the genes affected by PR are part of central biological processes of tumorigenesis.

Odorant receptor (OR) is crucial for insects to detect and recognize external chemical cues closely related to their survival. The insect OR forms a heteromeric complex composed of a ligand-specific receptor and a ubiquitously odorant receptor coreceptor (Orco). This study used the CRISPR/Cas9 technique to knock out (KO) Orco and reveal its essential role in acting on OR-meditated olfactory behavior in a critical invasive agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda. Electroantennogram (EAG) results suggested that the Orco mutants of both male and female moths severely reduced their electrophysiological responses to the eight tested plant volatiles and two sex pheromones. However, the Orco gene played distinct roles in mating behavior between sexes: the mating behavior was fully disrupted in mutated males but not in mutated females. The oviposition result indicated that the Orco KO females displayed reduced egg laying by 24.1% compared with the mated wild type (WT) females. Overall, these results strongly suggest that Orco is an excellent target for disrupting FAW’s normal behavior and provides a feasible pest control approach.

Aedes aegypti (Ae. aegypti) is a major vector of dengue virus (DENV) and Zika virus (ZIKV). Understanding the complex interaction mechanisms between mosquito vectors and arboviruses is essential to interrupt virus transmission. This study constructed CYP4C21 knockout (KO) Aag2 cells (Ae. aegypti cells) and confirmed that CYP4C21 KO reduced DENV2 and ZIKV copies in Aag2 cells, which suggests that CYP4C21 may play an important role in mosquito infection with arboviruses. Furthermore, it is the first report of the CYP4 family related to viral infection, which lays the foundation for exploring the role of the CYP4C21 in the interaction of Ae. aegypti and arbovirus and provides novel insights into the function of cytochrome family proteins.

TGF-β-activated kinase 1 binding protein 2 (TAB2) is an intermediate protein that connects TNFR1 and other receptor signals to the TGF-β-activated kinase 1 (TAK1) signaling complex. TAB2 has been proved clinically relevant to congenital heart defects (CHD) and cardiomyopathy. In this study, we created a TAB2 knockout human embryonic stem cell line by CRISPR/Cas9 technology. The WAe009-A-Z cell line displayed stem cell morphology, pluripotency and normal karyotype, which could develop into three germ layers in vitro.

Background & Aims: Human sporadic colorectal cancer (CRC) results from a multistep pathway with sequential acquisition of specific genetic mutations in the colorectal epithelium. Modeling CRC in vivo is critical for understanding the tumor microenvironment. To accurately recapitulate human CRC pathogenesis, mouse models must include these multi-step genetic abnormalities. The aim of this study was to generate a sporadic CRC model that more closely mimics this multi-step process and to use this model to study the role of a novel Let7 target PLAGL2 in CRC pathogenesis. Methods: We generated a CRISPR/Cas9 somatic mutagenesis mouse model that is inducible and multiplexed for simultaneous inactivation of multiple genes involved in CRC pathogenesis. We used both a doxycycline-inducible transcriptional activator and a doxycycline-inactivated transcriptional repressor to achieve tight, non-leaky expression of the Cas9 nickase. This mouse has transgenic expression of multiple guide RNAs to induce sporadic inactivation in the gut epithelium of 4 tumor suppressor genes commonly mutated in CRC, Apc, Pten, Smad4, and Trp53. These were crossed to Vil-LCL-PLAGL2 mice, which have Cre-inducible overexpression of PLAGL2 in the gut epithelium. Results: These mice exhibited random somatic mutations in all 4 targeted tumor suppressor genes, resulting in multiple adenomas and adenocarcinomas in the small bowel and colon. Crosses with Vil-LCL-PLAGL2 mice demonstrated that gut-specific PLAGL2 overexpression increased colon tumor growth. Conclusions: This conditional model represents a new CRISPR/Cas9-mediated mouse model of colorectal carcinogenesis. These mice can be used to investigate the role of novel, previously uncharacterized genes in CRC, in the context of multiple commonly mutated tumor suppressor genes and thus more closely mimic human CRC pathogenesis.

Salmonella enterica serovar Typhimurium manipulates cellular Rho GTPases for host cell invasion by effector protein translocation via the Type III Secretion System (T3SS). The two Guanine nucleotide exchange (GEF) mimicking factors SopE and –E2 and the inositol phosphate phosphatase (PiPase) SopB activate the Rho GTPases Rac1, Cdc42 and RhoA, thereby mediating bacterial invasion. S. Typhimurium lacking these three effector proteins are largely invasion-defective. Type III secretion is crucial for both early and later phases of the intracellular life of S. Typhimurium. Here we investigated whether and how the small GTPase RhoB, known to localize on endomembrane vesicles and at the invasion site of S. Typhimurium, contributes to bacterial invasion and to subsequent steps relevant for S. Typhimurium lifestyle. We show that RhoB is significantly upregulated within hours of Salmonella infection. This effect depends on the presence of the bacterial effector SopB, but does not require its phosphatase activity. Our data reveal that SopB and RhoB bind to each other, and that RhoB localizes on early phagosomes of intracellular S. Typhimurium. Whereas both SopB and RhoB promote intracellular survival of Salmonella, RhoB is specifically required for Salmonella-induced upregulation of autophagy. Finally, in the absence of RhoB, vacuolar escape and cytosolic hyper-replication of S. Typhimurium is diminished. Our findings thus uncover a role for RhoB in Salmonella-induced autophagy, which supports intracellular survival of the bacterium and is promoted through a positive feedback loop by the Salmonella effector SopB.

Hepatitis C virus (HCV) infections frequently recur after liver transplantation in patients with HCV-related liver diseases. Approximately 30% of these patients progress to cirrhosis within 5 years after surgery. In this study, we proposed an effective therapeutic strategy to overcome the recurrence of HCV. CRISPR-Cas9 was used to insert an expression cassette encoding an RNA aptamer targeting HCV NS5B replicase as an anti-HCV agent into adeno-associated virus integration site 1 (AAVS1), known as a “safe harbor,” in a hepatocellular carcinoma cell line to confer resistance to HCV. The RNA aptamer expression system based on a dihydrofolate reductase minigene was precisely knocked in into AAVS1, leading to the stable expression of aptamer RNA in the developed cell line. HCV replication was effectively inhibited at both the RNA and protein levels in cells transfected with HCV RNA or infected with HCV. RNA immunoprecipitation and competition experiments strongly suggested that this HCV inhibition was due to the RNA aptamer–mediated sequestration of HCV NS5B. No off-target insertion of the RNA aptamer expression construct was observed. The findings suggest that HCV-resistant liver cells produced by genome editing technology could be used as a new alternative in the development of a treatment for HCV-induced liver diseases.

The methylotrophic yeast Pichia pastoris (also known as Komagataella phaffii) is widely used as a yeast cell factory for producing heterologous proteins. Recently, it has gained attention for its potential in producing chemicals from inexpensive feedstocks, which requires efficient genetic engineering platforms. This review provides an overview of the current advances in developing genetic tools for metabolic engineering of P. pastoris. The topics cover promoters, terminators, plasmids, genome integration sites, and genetic editing systems, with a special focus on the development of CRISPR/Cas systems and their comparison to other genome editing tools. Additionally, this review highlights the prospects of multiplex genome integration, fine-tuning gene expression, and single-base editing systems. Overall, the aim of this review is to provide valuable insights into current genetic engineering and discuss potential directions for future efforts in developing efficient genetic tools in P. pastoris.

Regulatory T cells (Tregs) play a central role in modulating adaptive immune responses in humans and mice. The precise biological role of non-canonical nuclear factor ‘κ-light-chain-enhancer’ of activated B cells (NFKB) signaling in human Tregs has yet to be fully elucidated. To gain insight into this process, a Treg-like cell line (MT-2) was genetically modified using CRISPR/Cas9. Interestingly, NFKB2 knockout MT-2 cells exhibited downregulation of FOXP3, while NFKB1 knockout did not. Additionally, mRNA expression of FOXP3-dependent molecules was significantly reduced in NFKB2 knockout MT-2 cells. To better understand the functional role of the NFKB signaling, the NFKB1/NFKB2 loci of human primary Tregs were genetically edited using CRISPR/Cas9. Similar to MT-2 cells, NFKB2 knockout human Tregs displayed significantly reduced FOXP3 expression. Furthermore, NFKB2 knockout human Tregs showed downregulation of FOXP3-dependent molecules and a diminished suppressive function compared to wild-type and NFKB1 knockout Tregs. These findings indicate that non-canonical NFKB signaling maintains a Treg-like phenotype and suppressive function in human Tregs through the FOXP3-dependent regulatory T cell program.

Abstract Background Sudangrass (Sorghum sudanense) is a major biomass producer for livestock feed and biofuel in many countries. It has a wide range of adaptations for growing on marginal lands under biotic and abiotic stresses. The immature inflorescence is an explant with high embryogenic competence and is frequently used to regenerate different sorghum cultivars. Caffeic acid O-methyl transferase (COMT) is a key enzyme in the lignin biosynthesis pathway, which limits ruminant digestion of forage cell walls and is a crucial barrier in the conversion of plant biomass to bioethanol. Genome editing by CRISPR/Cas9-mediated mutagenesis without a transgenic footprint will accelerate the improvement and facilitate regulatory approval and commercialization of biotech crops. Methods and results We report the overcome of the recalcitrance in sudangrass transformation and regeneration in order to use genome editing technique. Hence, an efficient regeneration system has been established to induce somatic embryogenesis from the immature inflorescence of two sudangrass cultivars on four MS-based media supplemented with different components. Our results indicate an interaction between genotype and medium composition. The combination of Giza-1 cultivar and M4 medium produces the maximum frequency of embryogenic calli of 80% and subsequent regeneration efficiency of 22.6%. Precise mutagenesis of the COMT gene is executed using the CRISPR/Cas9 system with the potential to reduce lignin content and enhance forage and biomass quality in sudangrass. Conclusion A reliable regeneration and transformation system has been established for sudangrass using immature inflorescence, and the CRISPR/Cas9 system has demonstrated a promising technology for genome editing. The outcomes of this research will pave the road for further improvement of various sorghum genotypes to meet the global demand for food, feed, and biofuels, achieving sustainable development goals (SDGs).

Small airway epithelial cells (SAECs) play a central role in the pathogenesis of lung diseases and are now becoming a crucial cellular model for target identification and validation in drug discovery. However, primary cell lines such as SAECs are often difficult to transfect using traditional lipofection methods; therefore, gene editing using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is often carried out through ribonucleoprotein (RNP) electroporation. Here we have established a robust, scalable, and automated arrayed CRISPR nuclease (CRISPRn) screening workflow for SAECs which can be combined with a myriad of disease-specific endpoint assays.

Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors. Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects. However, relatively few safe harbor loci are available in pigs, a fact which has impeded the development of multi-transgenic pig research. We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain (COL1A1) gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. After the knock-in of a 2A peptide-green fluorescence protein (2A-GFP) transgene in the last codon of COL1A1 in multiple porcine cells, including porcine kidney epithelial (PK15), porcine embryonic fibroblast (PEF) and porcine intestinal epithelial (IPI-2I) cells, quantitative PCR (qPCR), Western blotting, RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus. The qPCR results showed that the GFP knock-in had no effect (P=0.29, P=0.66 and P=0.20 for PK15, PEF and IPI-2I cells, respectively) on the mRNA expression of COL1A1 gene. Similarly, no significant differences (P=0.64, P=0.48 and P=0.80 for PK15, PEF and IPI-2I cells, respectively) were found between the GFP knock-in and wild type cells by Western blotting. RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation (P