CRISPR-Cas9 Technology Research Articles

Endogenous interactomes of MFN1 and MFN2 provide novel insights into interorganelle communication and autophagy

ABSTRACT MFN1 (mitofusin 1) and MFN2 are key players in mitochondrial fusion, endoplasmic reticulum (ER)-mitochondria juxtaposition, and macroautophagy/autophagy. However, the mechanisms by which these proteins participate in these processes are poorly understood. Here, we studied the interactomes of these two proteins by using CRISPR-Cas9 technology to insert an HA-tag at the C terminus of MFN1 and MFN2, and thus generating HeLa cell lines that endogenously expressed MFN1-HA or MFN2-HA. HA-affinity isolation followed by mass spectrometry identified potential interactors of MFN1 and MFN2. A substantial proportion of interactors were common for MFN1 and MFN2 and were regulated by nutrient deprivation. We validated novel ER and endosomal partners of MFN1 and/or MFN2 with a potential role in interorganelle communication. We characterized RAB5C (RAB5C, member RAS oncogene family) as an endosomal modulator of mitochondrial homeostasis, and SLC27A2 (solute carrier family 27 (fatty acid transporter), member 2) as a novel partner of MFN2 relevant in autophagy. We conclude that MFN proteins participate in nutrient-modulated pathways involved in organelle communication and autophagy.

Principles and research progress of gene editing technology

Gene editing is a new genetic engineering technology. Given the quick development of molecular biology technology, genome editing tools have made great progress from the initial ZFNs and TALEN technology to CRISPR-Cas9 technology in recent years. Gene editing is the modification of specific targets in the genome of an organism to change its genetic information and phenotypic characteristics. This study analyzed the principle and research progress of gene editing technology through the data. For example, CRISPR-Cas9 can protect against secondary viral attacks, and ZFNs can be cleaved on any genomic sequence. Studies have found that there are still great challenges in the clinical field of genetic technology. Including the need for standardized safety protocols, regulatory transparency, and pricing frameworks, which increase the risk of further mutations. This article elaborates on the role and future development of gene editing in medicine, and provides a reference for the future field of gene editing technology.

Using Biotechnology to Create Transgenic Crops that Resist Climate Change

Climate change is a major threat to food security, especially for farmers in vulnerable areas facing the challenges of climate change. This study aimed to modify the plant genome to increase tolerance to extreme climatic conditions such as drought and high temperatures using genetic engineering methods based on CRISPR-Cas9 technology. Transgenic and control plants were grown in a laboratory experimental design with a quantitative approach. The results showed that the transgenic plants consistently outperformed the control plants under drought, high temperature, and low nutrient conditions. The transgenic plant showed a growth rate of 15.2 and average productivity of 25.7, higher than the control (12.3 and 19.1). Under high temperature conditions, the increase in productivity was even greater, reaching 33.2%, indicating that the inserted HSP gene successfully protects important proteins from heat stress damage. Genetic modification through the expression of genes such as DREB1A, HSP70, and PHR1 was shown to increase plant resistance to environmental stress. With these findings, biotechnology provides a real opportunity to build more adaptive and sustainable agricultural systems, supporting global food needs amid the intensifying challenges of global warming. This research is very relevant in answering the big challenge in the future, which is how to ensure biotechnology can be utilised sustainably to meet the world's growing food needs, without compromising ecosystems and biodiversity.

Calcium sensing receptor regulate claudin-14 via PKA-STAT3 pathway in rat model of nephrolithiasis.

The calcium-sensitive receptor (CaSR) has been identified as a key factor in the formation of kidney stones. A substantial body of research has illuminated the function of CaSR in stone formation with respect to oxidative stress, epithelial injury, crystal adhesion, and stone-associated proteins. Nevertheless, as a pivotal molecule in renal calcium excretion, its pathway that contributes to stone formation by regulating calcium supersaturation remains underexplored. An in vitro rat calcium oxalate kidney stone model was established through the co-cultivation of calcium oxalate monohydrate (COM) with NRK-52E cells, while an in vivo model was constructed using the ethylene glycol method. Subsequently, the level of the CaSR-claudin-14 pathway was determined. To further elucidate the molecular pathway of CaSR-mediated regulation of claudin-14, drugs were selectively added to the in vitro and ex vivo kidney stone models, and the expression of claudin-14 and the levels of stone formation were detected. Moreover, the direct regulation of claudin-14 by CaSR with STAT3 serving as a transcription factor was examined via the dual luciferase assay. Eventually, a Cldn-14 knockout rat model and a model of kidney stone induction by ethylene glycol were generated using CRISPR-Cas9 technology to further clarify the role of claudin-14 in the CaSR-regulated formation of kidney stones. In vitro and in vivo observations revealed that calcium oxalate induces high expression of CaSR-claudin-14. Specifically, CaSR regulates claudin-14 expression through phosphorylation modification of STAT3 via protein kinase A (PKA). In vitro, the intervention of PKA and STAT3 reversed the elevated claudin-14 levels and stone formation induced by CaSR. Finally, we generated cldn-14 knockout rats using CRISPR-Cas9 technology and observed that ethylene glycol still induced stone formation in these animals. Nevertheless, the specific activation or inhibition of CaSR demonstrated no notable impact on stone formation. The results of our study indicate that calcium oxalate crystals induce the activation of the pro-stone pathway of CaSR. That is, activated CaSR regulates claudin-14 levels via the PKA-STAT3 pathway, which further promotes calcium salt stone formation. The role of CaSR in the regulation of stone homeostasis is further enriched.

Nitrate assimilation pathway is impacted in young tobacco plants overexpressing a constitutively active nitrate reductase or displaying a defective CLCNt2

BackgroundWe have previously shown that the expression of a constitutively active nitrate reductase variant and the suppression of CLCNt2 gene function (belonging to the chloride channel (CLC) gene family) in field-grown tobacco reduces tobacco-specific nitrosamines (TSNA) accumulation in cured leaves and cigarette smoke. In both cases, TSNA reductions resulted from a strong diminution of free nitrate in the leaf, as nitrate is a precursor of the TSNA-producing nitrosating agents formed during tobacco curing and smoking. These nitrosating agents modify tobacco alkaloids to produce TSNAs, the most problematic of which are NNN (N-nitrosonornicotine) and NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone). The expression of a deregulated nitrate reductase enzyme (DNR) that is no longer responsive to light regulation is believed to diminish free nitrate pools by immediately channeling incoming nitrate into the nitrate assimilation pathway. The reduction in nitrate observed when the two tobacco gene copies encoding the vacuolar nitrate transporter CLCNt2 were down-regulated by RNAi-mediated suppression or knocked out using the CRISPR-Cas technology was mechanistically distinct; likely attributable to the inability of the tobacco cell to efficiently sequester nitrate into the vacuole where this metabolite is protected from further assimilation. In this study, we used transcriptomic and metabolomic analyses to compare the nitrate assimilation response in tobacco plants either expressing DNR or lacking CLCNt2 function.ResultsWhen grown in a controlled environment, both DNR and CLCNt2-KO (CLCKO) plants exhibited (1) reduced nitrate content in the leaf; (2) increased N-assimilation into the amino acids Gln and Asn; and (3) a similar pattern of differential regulation of several genes controlling stress responses, including water stress, and cell wall metabolism in comparison to wild-type plants. Differences in gene regulation were also observed between DNR and CLCKO plants, including genes encoding nitrite reductase and asparagine synthetase.ConclusionsOur data suggest that even though both DNR and CLCKO plants display common characteristics with respect to nitrate assimilation, cellular responses, water stress, and cell wall remodeling, notable differences in gene regulatory patterns between the two low nitrate plants are also observed. These findings open new avenues in using plants fixing more nitrogen into amino acids for plant improvement or nutrition perspectives.

Assessing the Feasibility and Challenges of CRISPR-Cas9 for Treating Genetic Disorders in Colombia

This study explores the potential applications of CRISPR-Cas9 technology in treating genetic disorders in Colombia, with a specific focus on thalassemia and sickle cell anemia, which disproportionately affect Afro-Colombians. Given the country’s rich genetic diversity, which includes Afro-Colombians, mestizos, and indigenous populations, the study examines how CRISPR could be tailored to address region-specific genetic mutations and improve health outcomes. The objective of this research is to assess the feasibility, challenges, and strategic implications of CRISPR for genetic disorders in Colombia, considering factors such as healthcare infrastructure, social resistance, and genetic variation. The methodology employed includes a comprehensive review of existing literature, comparative analysis of global CRISPR applications, and a synthesis of data from genetic studies specific to Colombia’s population. The findings indicate that while CRISPR offers significant promise for addressing genetic diseases, challenges such as healthcare accessibility, high costs, and the country’s genetic diversity must be overcome. Furthermore, ethical concerns regarding genetic modification must be addressed through public education and engagement. Future research should focus on expanding genetic databases, particularly for underrepresented populations, and exploring the economic and social implications of CRISPR therapies in Colombia's rural and marginalized communities. These efforts will help ensure that CRISPR technology is effectively and equitably applied to improve public health outcomes.

Potential regulatory mechanism and clinical significance of synaptotagmin binding cytoplasmic RNA interacting protein in colorectal cancer.

Colorectal cancer (CRC) causes many deaths worldwide. Synaptotagmin binding cytoplasmic RNA interacting protein (SYNCRIP) is an RNA-binding protein that plays an important role in multiple cancers by epigenetically targeting some genes. Our study will examine the expression, potential effect, biological function and clinical value of SYNCRIP in CRC. To examine the expression, potential effect, biological function and clinical value of SYNCRIP in CRC. The expression of SYNCRIP was examined by immunohistochemistry arrays and high-throughput data. The effect of SYNCRIP gene in CRC cell growth was evaluated by CRISPR-Cas9 technology. The target genes of SYNCRIP were calculated using various algorithms, and the molecular mechanism of SYNCRIP in CRC was explored by mutation analysis and pathway analysis. The clinical value of SYNCRIP in prognosis and radiotherapy was revealed via evidence-based medicine methods. The protein and mRNA levels of SYNCRIP were both highly expressed in CRC samples compared to nontumorous tissue based on 330 immunohistochemistry arrays and 3640 CRC samples. Cells grew more slowly in eleven CRC cell lines after knocking out the SYNCRIP gene. SYNCRIP could epigenetically target genes to promote the occurrence and development of CRC by boosting the cell cycle and affecting the tumor microenvironment. In addition, CRC patients with high SYNCRIP expression are more sensitive to radiotherapy. SYNCRIP is upregulated in CRC, and highly expressed SYNCRIP can accelerate CRC cell division by exerting its epigenetic regulatory effects. In addition, SYNCRIP is expected to become a potential biomarker to predict the effect of radiotherapy.

TBCC Domain-Containing Protein Regulates Sporulation and Virulence of Phytophthora capsici via Nutrient-Responsive Signaling.

Phytopathogenic oomycetes, particularly Phytophthora capsici, the causal agent of Phytophthora blight disease in essential vegetables and fruit crops, remains a persistent challenge in the vegetable production industry. However, the core molecular regulators of the pathophysiology and broad-range host characteristics of P. capsici remain unknown. Here, we used transcriptomics and CRISPR-Cas9 technology to functionally characterize the contributions of a novel gene (PcTBCC1) coding for a hypothetical protein with a tubulin-binding cofactor C domain with a putative chloroplast-targeting peptide (cTP) to the pathophysiological development of P. capsici. We observed significant upregulation in the expression of PcTBCC1 during pathogen-host interactions. However, the vegetative growth of the ∆Pctbcc1 strains was not significantly different from the wild-type strains. PcTBCC1 gene replacement significantly compromised the sporulation, pathogenic differentiation, and virulence of P. capsici. At the same time, ∆Pctbcc1 strains were sensitive to cell wall stress-inducing osmolytes. These observations, coupled with the close evolutionary ties between PcTBCC1 and pathogenic oomycetes and algae, partly support the notion that PcTBCC1 is a conserved determinant of pathogenesis. This study provides insights into the significance of tubulin-binding cofactors in P. capsici and underscores the potential of PcTbcc1 as a durable target for developing anti-oomycides to control phytopathogenic oomycetes.

Abstract B006: Rescuing expression of a tumor suppressor gene – restoring FH in Hereditary leiomyomatosis and renal cell carcinoma

Abstract Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a disorder characterized by the loss of fumarate hydratase (FH) activity, leading to the development of cutaneous leiomyomas, uterine leiomyomas, and aggressive early-onset type 2 papillary renal cell carcinoma. In HLRCC, the disease is driven by the somatic loss of the remaining functional FH allele in individuals carrying a germline pathogenic variant in one copy of the FH gene. The complete loss of FH activity disrupts the tricarboxylic acid (TCA) cycle, resulting in the accumulation of fumarate, which disrupts signaling pathways and contributes to the oncogenic phenotype. We recently identified a single nucleotide polymorphism (SNP) in an intronic region of the fumarate hydratase (FH) gene that leads to the loss of FH activity (Crooks et al., 2023). This SNP introduces a novel splice-acceptor site, resulting in the inclusion of an aberrant exon between wild-type exons 9 and 10. This ‘poison-like’ exon encodes a premature STOP codon, producing a nonfunctional truncated FH protein. Previous studies have shown that restoring FH activity can abrogate pathogenic behavior in patient-derived cells, highlighting the potential for therapeutic interventions targeting FH activity restoration. Our goal is to restore FH enzymatic activity by preventing the inclusion of this cryptic exon in carriers of this variant. To achieve this, we are exploring strategies to restore functional FH production by interfering with the inclusion of the pathogenic intron during splicing by removing the splice acceptor using CRISPR-Cas9 or base editing technologies. We tested these approaches by generating a reporter cell line that recapitulates the splicing pattern observed in patients, where expression of GFP depends on correct splicing between exon 9 and 10. We successfully identified antisense oligonucleotide (ASO) and single-guide RNA (sgRNA) sequences that restore GFP expression in our reporter cell lines. We are now evaluating the efficacy of these reagents in patient-derived cells. An alternative approach involves using synthetic RNA to restore FH expression. The expression of proteins from synthetic RNAs is a promising strategy for treating cancer and infectious diseases. We are currently investigating the efficiency of restoring FH activity through in-vitro transcribed (IVT) mRNA. This approach has the advantage of enabling FH function restoration in cells with mutations that are not amenable to antisense oligonucleotide (ASO) or gene editing techniques. This work serves as an avenue to investigate the application of nucleic acid therapies to restoring activity tumor suppressor genes in kidney cancer. Citation Format: Siddhardha S. Maligireddy, Mariana D. Mandler, Jodie C. Lunger, Christina M. Fitzsimmons, Daniel R. Crooks, Geetha M. Cawthon, Christopher J. Ricketts, Cathy D. Vocke, W. Marson Linehan, Pedro J. Batista. Rescuing expression of a tumor suppressor gene – restoring FH in Hereditary leiomyomatosis and renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B006.

TMEM232 is required for the formation of sperm flagellum and male fertility in mice

Asthenoteratozoospermia is a major cause of male infertility. Thus far, the identified related genes can explain only a small share of asthenoteratozoospermia cases, suggesting the involvement of other genes. The transmembrane protein TMEM232 is highly expressed in mouse testes. In the present study, to determine its function of TMEM232 in testes, we constructed a Tmem232-null mouse model using CRISPR–Cas9 technology. Tmem232 knockout (KO) male mice was completely infertile, and their sperm were immotile, with morphological defects of the flagellum. Electron microscopy revealed an aberrant midpiece-principal junction and the loss of the fourth outer microtubule doublet in the sperm of Tmem232−/− mice. Sperm cells presented an 8 + 2 conformation and an irregular arrangement of the mitochondrial sheath. Proteomic analysis revealed altered expression of proteins related to flagellar motility, sperm capacitation, the integrity and stability of sperm structure, especially an upregulated expression of multiple ribosome components in TMEM232-deficient spermatids. Additionally, TMEM232 was observed to be involved in autophagy by interacting with autophagy-related proteins, such as ATG14, to regulate ribosome homeostasis during spermiogenesis. These results suggest that TMEM232, as a potential scaffold protein involving in the correct assembly, distribution, and stability maintenance of certain functional complexes by recruiting key intracellular proteins, is essential for the formation of a highly structured flagellum and plays an important role in the autophagic elimination of cytosolic ribosomes to provide energy for sperm motility.

CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant Staphylococcus aureus.

Antibiotic resistance poses a global health crisis limiting the efficacy of available therapeutic agents. We explored CRISPR-Cas-based antimicrobials to combat multidrug resistance in methicillin-resistant Staphylococcus aureus (MRSA), targeting methicillin (mecA), gentamicin (aacA), and ciprofloxacin (grlA, grlB) resistance genes. Engineered CRISPR plasmids with specific single-guide RNAs were electroporated into MRSA strains. Real-time polymerase chain reaction assessed gene expression changes, while antibiotic susceptibility tests (ASTs) evaluated resistance status. Results showed a 1.5-fold decrease in mecA, a 5.5-fold decrease in grlA, a 6-fold decrease in grlB, and a 4-fold decrease in aacA expression. ASTs demonstrated the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis revealed a 70% decrease in penicillin-binding protein 2a expression. Sanger sequencing confirmed point mutations in the grlB and aacA genes. Our findings highlight the potential of CRISPR-Cas9 technology to restore antibiotic efficacy against multidrug-resistant pathogens.

Engineered production of 5-aminolevulinic acid in recombinant Escherichia coli BL21.

5-aminolevulinic acid (ALA) is a non-protein amino acid that has been widely used in the fields of medicine and agriculture. This study aims to engineer the C5 pathway of the ALA biosynthesis in Escherichia coli BL21 to enhance ALA production. The ALA synthase genes gltX, hemA, and hemL were overexpressed in E. coli BL21 to lead to the increase in the production of ALA. The sRNA RyhB was also overexpressed to downregulate the expression of ALA dehydratase to reduce the downstream bioconversion of ALA to porphobilinogen. Next, the gene arcA was knocked out by CRISPR-Cas9 technology to open the TCA cycle to promote the respiratory metabolism of the strain to reduce the feedback inhibition of heme to ALA. The fermentation conditions of the engineered strain were optimized by response surface experiments. The time-course analysis of the ALA production was carried out in a 1 L shake flask. Through these efforts, the production of ALA in engineered strain reached 2953 mg/L in a 1 L shake flask. This study contributes to the industrial production of ALA by the engineered E. coli in the future.

LncRNA Taurine Up-Regulated 1 Knockout Provides Neuroprotection in Ischemic Stroke Rats by Inhibiting Nuclear-Cytoplasmic Shuttling of HuR.

Background: Long non-coding RNA taurine-upregulated gene 1 (TUG1) is involved in various cellular processes, but its role in cerebral ischemia-reperfusion injury remains unclear. This study investigated TUG1's role in regulating the nucleocytoplasmic shuttling of human antigen R (HuR), a key apoptosis regulator under ischemic conditions. Methods: CRISPR-Cas9 technology was used to generate TUG1 knockout Sprague Dawley rats to assess TUG1's impact on ischemic injury. The infarct area and neuronal apoptosis were evaluated using TUNEL, hematoxylin and eosin (HE), and TTC staining, while behavioral functions were assessed. Immunofluorescence staining with confocal microscopy was employed to examine TUG1-mediated HuR translocation and expression changes in the apoptosis-related proteins COX-2 and Bax. Results: TUG1 knockout rats showed significantly reduced cerebral infarct areas, decreased neuronal apoptosis, and improved neurological functions compared to controls. Immunofluorescence staining revealed that HuR translocation from the nucleus to the cytoplasm was inhibited, leading to decreased COX-2 and Bax expression levels. Conclusions: TUG1 knockout reduces ischemic damage and neuronal apoptosis by inhibiting HuR nucleocytoplasmic shuttling, making TUG1 a potential therapeutic target for ischemic stroke.

CRISPR-Mediated SRY Gene Mutation Increases the Expression of Female Lineage-Specific Gene in Pre-Implantation Buffalo Embryo.

In mammals, sex determination is governed by the SRY gene on the Y chromosome, redirecting gonadal development from forming ovaries to testes. Mutations or alterations in the SRY gene can significantly affect phenotypic changes and lineage-specific markers. This study aims to elucidate the role of the SRY gene in buffalo embryos using CRISPR-Cas9 technology. We designed a crRNA targeting the HMG domain of the SRY gene using the CRISPOR algorithm. Nucleofection of sgRNA-Cas9 RNPs into buffalo fibroblasts confirmed efficient cleavage at the targeted site. Using this validated guide, we investigated the role of the SRY gene in sexual determination by electroporating CRISPR-Cas9-RNPs into single-stage zygotes of buffalo. Genetic changes in the SRY gene were confirmed through sequencing, revealing mosaic blastocysts with multiple alleles and non-mosaic mutants. Mutations in SRY gene increased the expression of female lineage-specific gene Wnt4 whereas decreased the expression of male specific gene SOX9 in blastocysts, suggesting reprogramming towards female sex determination pathways. Our findings provide insights into buffalo sex differentiation mechanisms and potential applications in reproductive strategies for breeding programmes.

Cutting-Edge Strategies for Overcoming Therapeutic Barriers in Alzheimer's Disease.

Alzheimer's disease (AD) remains one of the hardest neurodegenerative diseases to treat due to its enduring cognitive deterioration and memory loss. Despite extensive research, few viable treatment approaches have been found; these are mostly due to several barriers, such as the disease's complex biology, limited pharmaceutical efficacy, and the BBB. This presentation discusses current strategies for addressing these therapeutic barriers to enhance AD treatment. Innovative drug delivery methods including liposomes, exosomes, and nanoparticles may be able to pass the blood-brain barrier and allow medicine to enter specific brain regions. These innovative strategies of medicine distribution reduce systemic side effects by improving absorption. Moreover, the development of disease-modifying treatments that target tau protein tangles, amyloid-beta plaques, and neuroinflammation offers the chance to influence the course of the illness rather than only treat its symptoms. Furthermore, gene therapy and CRISPR-Cas9 technologies have surfaced as potentially groundbreaking methods for addressing the underlying genetic defects associated with AD. Furthermore, novel approaches to patient care may involve the utilization of existing medications having neuroprotective properties, such as those for diabetes and cardiovascular conditions. Furthermore, biomarker research and personalized medicine have made individualized therapy approaches possible, ensuring that patients receive the best care possible based on their unique genetic and molecular profiles.

PAMPHLET: PAM prediction HomoLogous-Enhancement toolkit for precise PAM prediction in CRISPR-Cas systems

The CRISPR-Cas technology has revolutionized our ability to understand and engineer organisms, evolving from a singular Cas9 model to a diverse CRISPR toolbox. A critical bottleneck in developing new Cas proteins is identifying protospacer adjacent motif (PAM) sequences. Due to the limitations of experimental methods, bioinformatics approaches have become essential. However, existing PAM prediction programs are limited by the small number of spacers in CRISPR-Cas systems, resulting in low accuracy. To address this, we develop PAMPHLET, a novel pipeline that uses homology searches to identify additional spacers, significantly increasing the number of spacers up to 18-fold. PAMPHLET is validated on 20 CRISPR-Cas systems and successfully predicts PAM sequences for 18 protospacers. These predictions are further validated using the DocMF platform, which characterizes protein-DNA recognition patterns via next-generation sequencing. The high consistency between PAMPHLET predictions and DocMF results for novel Cas proteins demonstrates potential of PAMPHLET to enhance PAM sequence prediction accuracy, expedite the discovery process, and accelerate the development of CRISPR tools.

Arabidopsis Actin-Binding Protein WLIM2A Links PAMP-Triggered Immunity and Cytoskeleton Organization.

Arabidopsis LIM proteins are named after the initials of three proteins Lin-11, Isl-1, and MEC-3, which belong to a class of transcription factors that play an important role in the developmental regulation of eukaryotes and are also involved in a variety of life processes, including gene transcription, the construction of the cytoskeleton, signal transduction, and metabolic regulation. Plant LIM proteins have been shown to regulate actin bundling in different cells, but their role in immunity remains elusive. Mitogen-activated protein kinases (MAPKs) are a family of conserved serine/threonine protein kinases that link upstream receptors to their downstream targets. Pathogens produce pathogen-associated molecular patterns (PAMPs) that trigger the activation of MAPK cascades in plants. Recently, we conducted a large-scale phosphoproteomic analysis of PAMP-induced Arabidopsis plants to identify putative MAPK targets. One of the identified phospho-proteins was WLIM2A, an Arabidopsis LIM protein. In this study, we investigated the role of WLIM2A in plant immunity. We employed a reverse-genetics approach and generated wlim2a knockout lines using CRISPR-Cas9 technology. We also generated complementation and phosphosite-mutated WLIM2A expression lines in the wlim2a background. The wlim2a lines were compromised in their response to Pseudomonas syringae Pst DC3000 but showed enhanced resistance to the necrotrophic fungus Botrytis cinereae. Transcriptome analyses of wlim2a mutants revealed the deregulation of immune hormone biosynthesis and signaling of salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) pathways. The wlim2a mutants also exhibited altered stomatal phenotypes. Analysis of plants expressing WLIM2A variants of the phospho-dead or phospho-mimicking MAPK phosphorylation site showed opposing stomatal behavior and resistance phenotypes in response to Pst DC3000 infection, proving that phosphorylation of WLIM2A plays a crucial role in plant immunity. Overall, these data demonstrate that phosphorylation of WLIM2A by MAPKs regulates Arabidopsis responses to plant pathogens.

Opportunities for CRISPR-Cas9 application in farm animal genetic improvement.

CRISPR-Cas9 has emerged as a powerful tool in livestock breeding, enabling precise genetic modifications to address genetic diseases, enhance productivity, and develop disease-resistant animal breeds. A thorough analysis of previous research highlights the potential of CRISPR-Cas9 in overcoming genetic disorders by targeting specific mutations in genes. Furthermore, its integration with reproductive biotechnologies and genomic selection facilitates the production of gene-edited animals with high genomic value, contributing to genetic enhancement and improved productivity. Additionally, CRISPR-Cas9 opens new avenues for developing disease-resistant livestock and creating innovative breeding models for high-quality production. A key trend in the field is the development of multi-sgRNA vectors to correct mutations in various genes linked to productivity traits or certain diseases within individual genomes, thereby increasing resistance in animals. However, despite the potential advantages of CRISPR-Cas9, public acceptance of genetically modified agricultural products remains uncertain. Would consumers be willing to purchase such products? It is essential to advocate for bold and innovative research into genetically edited animals, with a focus on safety, careful promotion, and strict regulatory oversight to align with long-term goals and public acceptance. Continued advancements in this technology and its underlying mechanisms promise to improve poultry products and genetically modified livestock. Overall, CRISPR-Cas9 technology offers a promising pathway for advancing livestock breeding practices, with opportunities for genetic improvement, enhanced disease resistance, and greater productivity.

Unlocking Fungal Potential: The CRISPR-Cas System as a Strategy for Secondary Metabolite Discovery.

Natural products (NPs) are crucial for the development of novel antibiotics, anticancer agents, and immunosuppressants. To highlight the ability of fungi to produce structurally diverse NPs, this article focuses on the impact of genome mining and CRISPR-Cas9 technology in uncovering and manipulating the biosynthetic gene clusters (BGCs) responsible for NP synthesis. The CRISPR-Cas9 system, originally identified as a bacterial adaptive immune mechanism, has been adapted for precise genome editing in fungi, enabling targeted modifications, such as gene deletions, insertions, and transcription modulation, without altering the genomic sequence. This review elaborates on various CRISPR-Cas9 systems used in fungi, notably the Streptococcus pyogenes type II Cas9 system, and explores advancements in different Cas proteins for fungal genome editing. This review discusses the methodologies employed in CRISPR-Cas9 genome editing of fungi, including guide RNA design, delivery methods, and verification of edited strains. The application of CRISPR-Cas9 has led to enhanced production of secondary metabolites in filamentous fungi, showcasing the potential of this system in biotechnology, medical mycology, and plant pathology. Moreover, this article emphasizes the integration of multi-omics data (genomics, transcriptomics, proteomics, and metabolomics) to validate CRISPR-Cas9 editing effects in fungi. This comprehensive approach aids in understanding molecular changes, identifying off-target effects, and optimizing the editing protocols. Statistical and machine learning techniques are also crucial for analyzing multi-omics data, enabling the development of predictive models and identification of key molecular pathways affected by CRISPR-Cas9 editing. In conclusion, CRISPR-Cas9 technology is a powerful tool for exploring fungal NPs with the potential to accelerate the discovery of novel bioactive compounds. The integration of CRISPR-Cas9 with multi-omics approaches significantly enhances our ability to understand and manipulate fungal genomes for the production of valuable secondary metabolites and for promising new applications in medicine and industry.

Advances in CRISPR-Cas9 Technology for Tumor Therapy

Genome editing technology is an emerging toolbox that can specifically target genes. At present, the existing gene editing technologies include ZFN, TALEN, and CRISPR system editing technology with clusters of regularly spaced short palindromic repeats. However, ZFN and TALEN are no longer the preferred tools for gene editing due to some of their problems, and their complex protein design, high cost, and high difficulty are the main reasons that limit their use. The CRISPR system, on the other hand, is able to achieve specific binding by base complementary pairing of sgRNA to the target sequence. Traditional cancer treatment methods have limited efficacy and high recurrence rates, and the disease can be fundamentally treated by inactivating oncogenes or activating tumor suppressor genes through gene editing technology and then changing downstream signaling pathways for cancer treatment. With CRISPR-Cas9 technology, it is possible to have gene knockouts, insertions, and mutations. Here, we will investigate and explore the feasibility of the CRISPR system for tumor treatment, including the following three topics: oncogene knockout, augmentation of tumor suppressor gene expression, and enhancement of engineered T cell viability for effective tumor Through these methods, the CRISPR system is of great help and promising tumor therapy.