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

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Phosphorus concentration on the surface of seawater varies greatly with different environments, especially in coastal. The molecular mechanism by which cyanobacteria adapt to fluctuating phosphorus bioavailability is still unclear. In this study, transcriptomes and gene knockouts were used to investigate the adaptive molecular mechanism of a model coastal cyanobacterium Synechococcus sp. PCC 7002 during periods of phosphorus starvation and phosphorus recovery (adding sufficient phosphorus after phosphorus starvation). The findings indicated that phosphorus deficiency affected the photosynthesis, ribosome synthesis, and bacterial motility pathways, which recommenced after phosphorus was resupplied. Even more, most of the metabolic pathways of cyanobacteria were enhanced after phosphorus recovery compared to the control which was kept in continuous phosphorus replete conditions. Based on transcriptome, 54 genes potentially related to phosphorus-deficiency adaptation were selected and knocked out individually or in combination. It was found that five mutants showed weak growth phenotype under phosphorus deficiency, indicating the importance of the genes (A0076, A0549-50, A1094, A1320, A1895) in the adaptation of phosphorus deficiency. Three mutants were found to grow better than the wild type under phosphorus deficiency, suggesting that the products of these genes (A0079, A0340, A2284-86) might influence the adaptation to phosphorus deficiency. Bioinformatics analysis revealed that cyanobacteria exposed to highly fluctuating phosphorus concentrations have more sophisticated phosphorus acquisition strategies. These results elucidated that Synechococcus sp. PCC 7002 have variable phosphorus response mechanisms to adapt to fluctuating phosphorus concentration, providing a novel perspective of how cyanobacteria may respond to the complex and dynamic environments. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00244-y.

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Escherichia coli depletion of chaperone trigger factor and DnaK/J were not viable at 37°C, but viable below 30°C. Among the engineered E. coli depleted of trigger factor and DnaK/J, one strain Z625, exhibited survival at 37°C, while another strain Z629 only survived below 30°C. Comparative analysis of fatty acid profiles of Z625 and Z629 revealed absence of numerous saturated fatty acids in Z625 as compared to the wild-type E. coli BW25113. In addition, increased unsaturated fatty acids were present in Z625, whereas the fatty acids profile of Z629 closely resembled that of BW25113. Whole genome sequencing revealed a 9-bp insertion in rpoB of Z625. Combined structural analysis of simulated RpoB protein bearing the amino acid sequence L451G452N453 insertion and susceptibility analysis to rifampicin suggested that the insertion did not disturb the individual RpoB structure as beta subunit of RNA polymerase. Comparative transcriptomic analysis of Z625 and Z629 suggested that this insertion impacted transcription of the overall RNA polymerase in Z625, leading to potential repression of some genes whose overexpression was toxic to E. coli. Additionally, Z625 exhibited distinctive metabolic adaptations, likely contributing to its survival at 37°C. In summary, our study elucidated one LGN insertion in rpoB that impacts transcriptional regulation in E. coli, thereby explaining the survival of E. coli depletion of trigger factor and DnaK/J at 37°C, and these founding suggested that some simple mutations in critical genes like rpoB might play an important role in driving adaptive evolution.

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Metarhizium rileyi is a filamentous entomopathogenic fungus that is highly pathogenic to lepidopteran insects. In our study, we constructed an Agrobacterium tumefaciens-mediated transgene system using the hygromycin resistance gene (Hyg R) as a selection marker in M. rileyi through homologous recombination. Binary knockout vectors for two genes (NOR_03501, longevity assurance gene, and NOR_03153, ATP-binding domain protein domain gene) in the M. rileyi strain SZCY201010 were successfully developed. We compared the genetic transformation efficiency using five kinds of asexual spores. The initial genetic transformation rates using a competent blastospore for NOR_03501 and NOR_03153 were 54.35 and 47.19%, respectively. Subsequently, both genes were successfully knocked out, and the transformed fungi were verified by PCR, RT-qPCR, and green fluorescent protein labeling. The biological phenotypes of the two genes were analyzed. The NOR_03501 gene plays a crucial role in carbon source utilization, stress resistance, and cuticle infection of fungal mycelium growth, while the NOR_03153 gene is significant for conidial production, stress resistance, and body wall infection. This study provides a promising tool for gene manipulation in M. rileyi, enhancing research in functional genomics and the exploration of fungal gene resources.

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In vitro and ex-vivo target identification strategies often fail to predict in vivo efficacy, particularly for glioblastoma (GBM), a highly heterogenous tumor rich in resistant cancer stem cells (GSCs). An in vivo screening tool can improve prediction of therapeutic efficacy by considering the complex tumor microenvironment and the dynamic plasticity of GSCs driving therapy resistance and recurrence. This study proposes lipid nanoparticles (LNPs) as an efficient in vivo CRISPR-Cas9 gene editing tool for target validation in mesenchymal GSCs. LNPs co-delivering mRNA (mCas9) and single-guide RNA (sgRNA) were successfully formulated and optimized facilitating both in vitro and in vivo gene editing. In vitro, LNPs achieved up to 67 % reduction in green fluorescent protein (GFP) expression, used as a model target, outperforming a commercial transfection reagent. Intratumoral administration of LNPs in GSCs resulted in ~80 % GFP gene knock-out and a 2-fold reduction in GFP signal by day 14. This study showcases the applicability of CRISPR-Cas9 LNPs as a potential in vivo screening tool in GSCs, currently lacking effective treatment. By replacing GFP with a pool of potential targets, the proposed platform presents an exciting prospect for therapeutic target validation in orthotopic GSCs, bridging the gap between preclinical and clinical research.

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Objectives: Hepatic ischemia-reperfusion (HIR) is a severe process in pathophysiology that occurs clinically in hepatectomy, and hepatic transplantations. The present study aimed to investigate the effect of PKC θ deletion against HIR injury and elucidate its mechanism in pathophysiology. Materials and Methods: HIR injury was induced in wild-type and PKC θ deletion mice treated with or without heme. The ALT and AST levels were determined to evaluate liver function. HIR injury was observed via histological examination. Oxidative stress and inflammatory response markers, and their signaling pathways were detected. Results: The study found that PKC θ knockout decreased serum AST and ALT levels when compared to the WT mice. Furthermore, heme treatment significantly reduced the ALT and AST levels of the PKC θ deletion mice compared with the untreated PKC θ deletion mice. PKC θ deletion markedly elevated superoxide dismutase activity in the liver tissue, reduced malondialdehyde content in the tissue, and the serum TNF-α and IL-6 levels compared with the WT mice. Heme treatment was observed to elevate the activity of SOD and reduced MDA content and serum of TNF-α and IL 6 in the PKC θ deletion animals. Meanwhile, heme treatment increased HO-1 and Nrf 2 protein expression, and reduced the levels of TLR4, phosphorylated NF-κB, and IKB-α. Conclusion: These findings suggested that PKC θ deletion ameliorates HIR, and heme treatment further improves HIR, which is related to regulation of PKC θ deletion on Nrf 2/HO-1 and TLR4/NF-κB/IKB α pathway.

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Loss of ovarian homeostasis is associated with ovary dysfunction and female diseases; however, the underlying mechanisms responsible for the establishment of homeostasis and its function in the ovary have not been fully elucidated. Here, we showed that conditional knockout of Rab37 in oocytes impaired macroautophagy/autophagy proficiency in the ovary and interfered with follicular homeostasis and ovary development in mice. Flunarizine treatment upregulated autophagy, thus rescuing the impairment of follicular homeostasis and ovarian dysfunction in rab37 knockout mice by reprogramming of homeostasis. Notably, both the E2F1 and EGR2 transcription factors synergistically activated Rab37 transcription and promoted autophagy. Thus, RAB37-mediated autophagy ensures ovary function by maintaining ovarian homeostasis.Abbreviations: AMH: anti-Mullerian hormone; ATG: autophagy related; BECN1: beclin 1; cKO: conditional knockout; Cre: cyclization recombination enzyme; dpp: days postpartum; E2: estradiol; E2F1: E2F transcription factor 1; EBF1: EBF transcription factor 1; EGR2: early growth response 2; FSH: follicle stimulating hormone; LH: luteinizing hormone; mpp: months postpartum; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; RAB37: RAB37, member RAS oncogene family; SQSTM1: sequestosome 1; TFEB: transcription factor EB; Zp3: zona pellucida glycoprotein 3.

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BACKGROUND: The dimerizable Cre recombinase system (DiCre) exhibits increased leaky activity in Cryptosporidium, leading to unintended gene editing in the absence of induction. Therefore, optimization of the current DiCre technique is necessary for functional studies of essential Cryptosporidium genes. METHODS: Based on the results of transcriptomic analysis of Cryptosporidium parvum stages, seven promoters with different transcriptional capabilities were screened to drive the expression of Cre fragments (FKBP-Cre59 and FRB-Cre60). Transient transfection was performed to assess the effect of promoter strength on leakage activity. In vitro and in vivo experiments were performed to evaluate the leaky activity and cleavage efficiency of the optimized DiCre system by polymerase chain reaction (PCR), nanoluciferase, and fluorescence analyses. RESULTS: The use of promoters with lower transcriptional activity, such as pcgd6_4110 and pcgd3_260, as opposed to strong promoters such as pActin, pα-Tubulin, and pEnolase, reduced the leakage rate of the system from 35-75% to nearly undetectable levels, as verified by transient transfection. Subsequent in vitro and in vivo experiments using stable lines further demonstrated that the optimized DiCre system had no detectable leaky activity. The system achieved 71% cleavage efficiency in vitro. In mice, a single dose of the inducer resulted in a 10% conditional gene knockout and fluorescent protein expression in oocysts. These fluorescently tagged transgenic oocysts could be enriched by flow sorting for further infection studies. CONCLUSIONS: A DiCre conditional gene knockout system for Cryptosporidium with good cleavage efficiency and reduced leaky activity has been successfully established.

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Recent studies reveal that biosynthesis of iron-sulfur clusters (Fe-Ss) is essential for cell proliferation, including that of cancer cells. Nonetheless, it remains unclear how Fe-S biosynthesis functions in cell proliferation/survival. Here, we report that proper Fe-S biosynthesis is essential to prevent cellular senescence, apoptosis, or ferroptosis, depending on cell context. To assess these outcomes in cancer, we developed an ovarian cancer line with conditional KO of FDX2, a component of the core Fe-S assembly complex. FDX2 loss induced global downregulation of Fe-S-containing proteins and Fe2+ overload, resulting in DNA damage and p53 pathway activation, and driving the senescence program. p53 deficiency augmented DNA damage responses upon FDX2 loss, resulting in apoptosis rather than senescence. FDX2 loss also sensitized cells to ferroptosis, as evidenced by compromised redox homeostasis of membrane phospholipids. Our results suggest that p53 status and phospholipid homeostatic activity are critical determinants of diverse biological outcomes of Fe-S deficiency in cancer cells.

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There are approximately 20,000 protein-coding genes in humans and mice. More than 1000 of these genes are predominantly expressed in the testis or are testis-specific and thought to play an important role in male reproduction. Through the production of gene knockout mouse models and phenotypic evaluations, many genes essential for spermatogenesis, sperm maturation, and fertilization have been discovered, greatly contributing to the elucidation of their molecular mechanisms. On the other hand, there are many cases in which single-gene knockout models do not affect fertility, indicating that tissue-specific genes are not always critical. Here, we selected 18 genes whose mRNA expression is restricted to the testis or higher than in other tissues, but whose function in male reproduction is unknown. We then created single-gene KO mouse models using the CRISPR/Cas9 system. The established KO males were subjected to mating tests and screened for effects on fecundity, revealing that these genes were not essential for spermatogenesis and male fertility. This knowledge will contribute to understanding the functions of genes characteristic of the testis and identify the cause of male infertility.

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Ras-related Rap1A GTPase is implicated in pancreas ß-cell insulin secretion and is stimulated by the cAMP sensor Epac2, a guanine exchange factor and activator of Rap1 GTPase. In this study, we examined the differential proteomic profiles of pancreata from C57BL/6 Rap1A-deficient (Null) and control wild-type (WT) mice with nanoLC-ESI-MS/MS to assess targets of Rap1A potentially involved in insulin regulation. We identified 77 overlapping identifier proteins in both groups, with 8 distinct identifier proteins in Null versus 56 distinct identifier proteins in WT mice pancreata. Functional enrichment analysis showed four of the eight Null unique proteins, ERO1-like protein ß (Ero1lß), triosephosphate isomerase (TP1), 14-3-3 protein γ, and kallikrein-1, were exclusively involved in insulin biogenesis, with roles in insulin metabolism. Specifically, the mRNA expression of Ero1lß and TP1 was significantly (p < 0.05) increased in Null versus WT pancreata. Rap1A deficiency significantly affected glucose tolerance during the first 15-30 min of glucose challenge but showed no impact on insulin sensitivity. Ex vivo glucose-stimulated insulin secretion (GSIS) studies on isolated Null islets showed significantly impaired GSIS. Furthermore, in GSIS-impaired islets, the cAMP-Epac2-Rap1A pathway was significantly compromised compared to the WT. Altogether, these studies underscore an essential role of Rap1A GTPase in pancreas physiological function.

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Constructing accurate gene regulatory network s (GRNs), which reflect the dynamic governing process between genes, is critical to understanding the diverse cellular process and unveiling the complexities in biological systems. With the development of computer sciences, computational-based approaches have been applied to the GRNs inference task. However, current methodologies face challenges in effectively utilizing existing topological information and prior knowledge of gene regulatory relationships, hindering the comprehensive understanding and accurate reconstruction of GRNs. In response, we propose a novel graph neural network (GNN)-based Multi-Task Learning framework for GRN reconstruction, namely MTLGRN. Specifically, we first encode the gene promoter sequences and the gene biological features and concatenate the corresponding feature representations. Then, we construct a multi-task learning framework including GRN reconstruction, Gene knockout predict, and Gene expression matrix reconstruction. With joint training, MTLGRN can optimize the gene latent representations by integrating gene knockout information, promoter characteristics, and other biological attributes. Extensive experimental results demonstrate superior performance compared with state-of-the-art baselines on the GRN reconstruction task, efficiently leveraging biological knowledge and comprehensively understanding the gene regulatory relationships. MTLGRN also pioneered attempts to simulate gene knockouts on bulk data by incorporating gene knockout information.

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Heme is a crucial component in endowing plant-based meat analogs with flavor and color. This study aimed to develop a green strategy for heme production by reducing fermentation off-odor and accelerating heme synthesis. First, an efficient CRISPR/Cas9n system was constructed in Bacillus amyloliquefaciens to construct the odor-reducing chassis cell HZC9nΔGPSU, and the odor substances including the branched-chain short fatty acids, putrescine, and ammonia were reduced by 62, 70, and 88%, respectively. Meanwhile, the hemA gene was confirmed to be the key gene for enhanced heme synthesis. Various hemA genes were compared to obtain the best gene dhemA, and the catalysis mechanism was explained by molecular docking simulation. After further expression of dhemA in HZC9nΔGPSU, the heme titer of HZC9nΔGPSU/pHY-dhemA reached 11.31 ± 0.51 mg/L, 1.70-fold higher than that of HZC9n/pHY-dhemA. The knockout of off-odor-related genes reduced the odor substances and enhanced the heme synthesis, which is promising for the green production of high-quality heme.

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Macroautophagy, simply referred to below as autophagy, is an intracellular degradation system that is highly conserved in eukaryotes. Since the processes involved in autophagy are accompanied by membrane dynamics, RAB small GTPases, key regulators of membrane trafficking, are generally thought to regulate the membrane dynamics of autophagy. Although more than half of the mammalian RABs have been reported to be involved in canonical and selective autophagy, no consensus has been reached in regard to the role of RABs in mammalian autophagy. Here, we comprehensively analyzed a rab-knockout (KO) library of MDCK cells to reevaluate the requirement for each RAB isoform in basal and starvation-induced autophagy. The results revealed clear alteration of the MAP1LC3/LC3-II level in only four rab-KO cells (rab1-KO, rab2-KO, rab7a-KO, and rab14-KO cells) and identified RAB14 as a new regulator of autophagy, specifically at the autophagosome maturation step. The autophagy-defective phenotype of two of these rab-KO cells, rab2-KO and rab14-KO cells, was very mild, but double KO of rab2 and rab14 caused a severer autophagy-defective phenotype (greater LC3 accumulation than in single-KO cells, indicating an overlapping role of RAB2 and RAB14 during autophagosome maturation. We also found that RAB14 is phylogenetically similar to RAB2 and that it possesses the same properties as RAB2, i.e. autophagosome localization and interaction with the HOPS subunits VPS39 and VPS41. Our findings suggest that RAB2 and RAB14 overlappingly regulate the autophagosome maturation step through recruitment of the HOPS complex to the autophagosome.Abbreviation: AID2: auxin-inducible degron 2; ATG: autophagy related; BafA1: bafilomycin A1; CKO: conditional knockout; EBSS: Earle's balanced salt solution; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; HRP: horseradish peroxidase; IP: immunoprecipitation; KD: knockdown; KO: knockout; LAMP2: lysosomal-associated membrane protein 2; MDCK: Madin-Darby canine kidney; mAb: monoclonal antibody; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; 5-Ph-IAA: 5-phenyl-indole-3-acetic acid; pAb: polyclonal antibody; siRNA: small interfering RNA; SNARE: soluble NSF-attachment protein receptor; TF: transferrin; WT: wild-type.

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Effectively managing foodborne pathogens is imperative in food processing, where probiotics play a crucial role in pathogen control. This study focuses on the Lactiplantibacillus plantarum AR113 and its gene knockout strains, exploring their antimicrobial properties against Escherichia coli O157:H7 and Staphylococcus aureus. Antimicrobial assays revealed that the inhibitory effect of AR113 increases with its growth and the potential bacteriostatic substance is acidic. AR113Δldh, surpassed AR113Δ0273&2024, exhibited a complete absence of bacteriostatic properties, which indicates that lactic acid is more essential than acetic acid in the bacteriostatic effect of AR113. However, the exogenous acid validation test affirmed the equivalent superior bacteriostatic effect of lactic acid and acetic acid. Notably, AR113 has high lactate production and deletion of the ldh gene not only lacks lactate production but also affects acetic production. This underscores the ldh gene's pivotal role in the antimicrobial activity of AR113. In addition, among all the selected knockout strains, AR113ΔtagO and ΔccpA also had lower antimicrobial effects, suggesting the importance of tagO and ccpA genes of AR113 in pathogen control. This study contributes insights into the antimicrobial potential of AR113 and stands as the pioneering effort to use knockout strains for comprehensive bacteriostatic investigations.

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[This corrects the article DOI: 10.3389/fcell.2023.1290876.].

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Aging is an inevitable physiological process, often accompanied by age-related bone loss and subsequent bone-related diseases that pose serious health risks. Research on skeletal diseases caused by aging in humans is challenging due to lengthy study durations, difficulties in sampling, regional variability, and substantial investment. Consequently, mice are preferred for such studies due to their similar motor system structure and function to humans, ease of handling and care, low cost, and short generation time. In this review, we present a comprehensive overview of the characteristics, limitations, applicability, bone phenotypes, and treatment methods in naturally aging mice and prematurely aging mouse models (including SAMP6, POLG mutant, LMNA, SIRT6, ZMPSTE24, TFAM, ERCC1, WERNER, and KL/KL-deficient mice). We also summarize the molecular mechanisms of these aging mouse models, including cellular DNA damage response, senescence-related secretory phenotype, telomere shortening, oxidative stress, bone marrow mesenchymal stem cell (BMSC) abnormalities, and mitochondrial dysfunction. Overall, this review aims to enhance our understanding of the pathogenesis of aging-related bone diseases.

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Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. In 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 with the conventional CRISPR-Cas9 system. Site-specific knockin of a chimeric antigen receptor 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.

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OBJECTIVES: To investigate the effects of myelin- and lymphocyte-associated protein (MAL) gene knockout on the morphological structure of lung tissue and the expression of E-cadherin (E-cad) and alpha-smooth muscle actin (α-SMA) in an asthmatic mouse model. METHODS: Twenty-four specific pathogen-free (SPF) C57BL/6J mice were divided into four groups: the wild-type normal (WT/SAL), wild-type asthmatic (WT/OVA), gene knockout normal (MAL-/-/SAL), and gene knockout asthmatic (MAL-/-/OVA) groups. The establishment of the asthma mouse models was confirmed by evaluating behavioral symptoms and histopathological H&E and Masson staining. Western blotting and RT-qPCR were used to measure E-cad and α-SMA expression levels in lung tissues. RESULTS: H&E staining of mouse lung tissues from WT/OVA, MAL-/-/SAL, and MAL-/-/OVA groups revealed a thickened bronchial wall, irregular lumen edge, locally fallen mucosal epithelium, and inflammatory cell infiltration compared with those of the WT/SAL group. In the WT and MAL-/- groups, the proportion of Masson-stained tissues in the OVA group was greater than that in the SAL group (p < 0.05). Compared with those in the WT/SAL group, the expression levels of α-SMA mRNA and protein were increased, while those of E-cad were decreased in the WT/OVA group (p < 0.01). Similarly, compared with those in the MAL-/-/SAL group, the expression levels of E-cad mRNA and protein were increased, while those of α-SMA were decreased in the MAL-/-/OVA group (p < 0.01). All these differences were statistically significant (p < 0.01). CONCLUSIONS: The MAL gene contributes to EMT inhibition and the stability of the airway barrier under normal physiological conditions by regulating E-cad and α-SMA expression.