Exploring the Function of Gene Promoter Regulatory Elements Using CRISPR Tools.

Gene promoters serve as pivotal regulators of transcription, orchestrating the initiation, rate, and specificity of gene expression, resulting in cellular diversity found among distinct cell types within multicellular organisms. Identification of the sequence and function of promoters' regulatory elements and their complex interaction with transcription factors, enhancers, silencers, and insulators is fundamental to coordinated transcriptional processes within cells. Identifying these regulatory elements and scrutinizing their functions and interactions through the use of synthetic promoters can pave the way for researchers in various fields ranging from uncovering the origins of diseases associated with promoter mutations to harnessing these regulatory components in biotechnological applications.In this chapter, we describe the manipulation of regulatory elements within promoters, with a specific focus on the use of CRISPR technology on enhancers and silencer elements of the Ovalbumin gene promoter. We explain and discuss processes for the deletion of/interference with regulatory elements within the promoter, employing CRISPR-based approaches. Furthermore, we demonstrate that a CRISPR/Cas-manipulated promoter can activate gene transcription in cell types where it is normally inactive. This confirms that CRISPR technology can be effectively used to engineer synthetic promoters with desired characteristics, such as inducibility, tissue-specificity, or enhanced transcriptional strength. Such an approach provides valuable insights into the mechanisms and dynamics of gene expression, thereby offering new opportunities in the fields of biotechnology and medicine.

G-Protein Signaling Modulator 2 as a Potential Biomarker in Colorectal Cancer: Integrative Analysis Using Genetic Profiling and Pan-Cancer Studies.

Colorectal cancer (CRC) imposes a significant healthcare burden globally, prompting the quest for innovative biomarkers to enhance diagnostic and therapeutic strategies. This study investigates the G-protein signaling modulator (GPSM) family across several cancers and presents a comprehensive pan-cancer analysis of the GPSM2 gene across several gastrointestinal (GI) cancers. Leveraging bioinformatics methodologies, we investigated GPSM2 expression patterns, protein interactions, functional enrichments, prognostic implications, genetic alterations, and immune infiltration associations. Furthermore, the expression of the GPSM2 gene was analyzed using real-time analysis. Our findings reveal a consistent upregulation of GPSM2 expression in all GI cancer datasets analyzed, suggesting its potential as a universal biomarker in GI cancers. Functional enrichment analysis underscores the involvement of GPSM2 in vital pathways, indicating its role in tumor progression. The prognostic assessment indicates that elevated GPSM2 expression correlates with adverse overall and disease-free survival outcomes across multiple GI cancer types. Genetic alteration analysis highlights the prevalence of mutations, particularly missense mutations, in GPSM2. Furthermore, significant correlations between GPSM2 expression and immune cell infiltration are observed, suggesting its involvement in tumor immune evasion mechanisms. Collectively, our study underscores the multifaceted role of GPSM2 in GI cancers, particularly in CRC, emphasizing its potential as a promising biomarker for prognosis and therapeutic targeting. Further functional investigations are warranted to elucidate its clinical utility and therapeutic implications in CRC management.

The Prognostic Value of ASPHD1 and ZBTB12 in Colorectal Cancer: A Machine Learning-Based Integrated Bioinformatics Approach.

Introduction: Colorectal cancer (CRC) is a common cancer associated with poor outcomes, underscoring a need for the identification of novel prognostic and therapeutic targets to improve outcomes. This study aimed to identify genetic variants and differentially expressed genes (DEGs) using genome-wide DNA and RNA sequencing followed by validation in a large cohort of patients with CRC. Methods: Whole genome and gene expression profiling were used to identify DEGs and genetic alterations in 146 patients with CRC. Gene Ontology, Reactom, GSEA, and Human Disease Ontology were employed to study the biological process and pathways involved in CRC. Survival analysis on dysregulated genes in patients with CRC was conducted using Cox regression and Kaplan-Meier analysis. The STRING database was used to construct a protein-protein interaction (PPI) network. Moreover, candidate genes were subjected to ML-based analysis and the Receiver operating characteristic (ROC) curve. Subsequently, the expression of the identified genes was evaluated by Real-time PCR (RT-PCR) in another cohort of 64 patients with CRC. Gene variants affecting the regulation of candidate gene expressions were further validated followed by Whole Exome Sequencing (WES) in 15 patients with CRC. Results: A total of 3576 DEGs in the early stages of CRC and 2985 DEGs in the advanced stages of CRC were identified. ASPHD1 and ZBTB12 genes were identified as potential prognostic markers. Moreover, the combination of ASPHD and ZBTB12 genes was sensitive, and the two were considered specific markers, with an area under the curve (AUC) of 0.934, 1.00, and 0.986, respectively. The expression levels of these two genes were higher in patients with CRC. Moreover, our data identified two novel genetic variants-the rs925939730 variant in ASPHD1 and the rs1428982750 variant in ZBTB1-as being potentially involved in the regulation of gene expression. Conclusions: Our findings provide a proof of concept for the prognostic values of two novel genes-ASPHD1 and ZBTB12-and their associated variants (rs925939730 and rs1428982750) in CRC, supporting further functional analyses to evaluate the value of emerging biomarkers in colorectal cancer.

Study of the regulatory elements of the Ovalbumin gene promoter using CRISPR technology in chicken cells.

BACKGROUND: Hormone-dependent promoters are very efficient in transgene expression. Plasmid-based reporter assays have identified regulatory sequences of the Ovalbumin promoter that are involved in response to estrogen and have shown that the deletion of the steroid-dependent regulatory element (SDRE) and negative regulatory element (NRE) leads to a steroid-independent expression of a reporter. However, the functional roles of these regulatory elements within the native genomic context of the Ovalbumin promoter have not been evaluated. RESULTS: In this study, we show that the negative effects of the NRE element on the Ovalbumin gene can be counteracted by CRISPR interference. We also show that the CRISPR-mediated deletion of SDRE and NRE promoter elements in a non-oviduct cell can lead to the significant expression of the Ovalbumin gene. In addition, the targeted knock-in of a transgene reporter in the Ovalbumin coding region and its expression confirms that the truncated promoter of the Ovalbumin gene can be efficiently used for an estrogen-independent expression of a foreign gene. CONCLUSIONS: The methodology applied in this paper allowed the study of promoter regulatory sequences in their native nuclear organization.

Enhanced cultivation of chicken primordial germ cells.

The cultivation and expansion of chicken primordial germ cells (cPGCs) are of critical importance for both biotechnological applications and the management of poultry genetic biodiversity. The feeder-free culture system has become the most popular approach for the cultivation and expansion of cPGCs. However, despite some success in the cultivation of cPGCs, the reproducibility of culture conditions across different laboratories remains a challenge. This study aimed to compare two defined and enriched media for the growth of cPGCs originating from the Hubbard JA57 broiler. To this end, cPGCs were isolated from the embryonic blood of Hamburger-Hamilton (HH) stages 14-16 and cultured at various time points. The Growth properties and characteristics of these cells were evaluated in two different culture conditions (the defined or enriched medium) and their migratory properties were assessed after genetic engineering and injection into the vasculature of 2.5-day-old chicken embryos. The main finding of this study was that the use of an enriched medium (the defined medium with Knock-Out Serum Replacement; KOSR) resulted in improved growth properties of cPGCs originating from the Hubbard JA57 broiler compared to a defined medium. The ability to cultivate and expand cPGCs is crucial for the generation of both genetically engineered birds and breeds of interest from local or commercial origins. Therefore, these results highlight the importance of choosing an appropriate culture medium for cPGCs growth and expansion.

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

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

PTEN hamartoma tumour syndrome: case report based on data from the Iranian hereditary colorectal cancer registry and literature review.

BACKGROUND: PTEN hamartoma tumour syndrome (PHTS) is a rare hereditary disorder caused by germline pathogenic mutations in the PTEN gene. This study presents a case of PHTS referred for genetic evaluation due to multiple polyps in the rectosigmoid area, and provides a literature review of PHTS case reports published between March 2010 and March 2022. CASE PRESENTATION: A 39-year-old Iranian female with a family history of gastric cancer in a first-degree relative presented with minimal bright red blood per rectum and resistant dyspepsia. Colonoscopy revealed the presence of over 20 polyps in the rectosigmoid area, while the rest of the colon appeared normal. Further upper endoscopy showed multiple small polyps in the stomach and duodenum, leading to a referral for genetic evaluation of hereditary colorectal polyposis. Whole-exome sequencing led to a PHTS diagnosis, even though the patient displayed no clinical or skin symptoms of the condition. Further screenings identified early-stage breast cancer and benign thyroid nodules through mammography and thyroid ultrasound. METHOD AND RESULTS OF LITERATURE REVIEW: A search of PubMed using the search terms "Hamartoma syndrome, Multiple" [Mesh] AND "case report" OR "case series" yielded 43 case reports, predominantly in women with a median age of 39 years. The literature suggests that patients with PHTS often have a family history of breast, thyroid and endometrial neoplasms along with pathogenic variants in the PTEN/MMAC1 gene. Gastrointestinal polyps are one of the most common signs reported in the literature, and the presence of acral keratosis, trichilemmomas and mucocutaneous papillomas are pathognomonic characteristics of PHTS. CONCLUSION: When a patient presents with more than 20 rectosigmoid polyps, PHTS should be considered. In such cases, it is recommended to conduct further investigations to identify other potential manifestations and the phenotype of PHTS. Women with PHTS should undergo annual mammography and magnetic resonance testing for breast cancer screening from the age of 30, in addition to annual transvaginal ultrasounds and blind suction endometrial biopsies.

Genetically engineered birds; pre-CRISPR and CRISPR era†.

Generating biopharmaceuticals in genetically engineered bioreactors continues to reign supreme. Hence, genetically engineered birds have attracted considerable attention from the biopharmaceutical industry. Fairly recent genome engineering methods have made genome manipulation an easy and affordable task. In this review, we first provide a broad overview of the approaches and main impediments ahead of generating efficient and reliable genetically engineered birds, and various factors that affect the fate of a transgene. This section provides an essential background for the rest of the review, in which we discuss and compare different genome manipulation methods in the pre-clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR era in the field of avian genome engineering.

CRISPR/dCas9-mediated transposition with specificity and efficiency of site-directed genomic insertions.

The ability and efficiency of targeted nucleases to perform sequence replacements or insertions into the genome are limited. This limited efficiency for sequence replacements or insertions can be explained by the dependency on DNA repair pathways, the possibility of cellular toxicity, and unwanted activation of proto-oncogenes. The piggyBac (PB) transposase uses a very efficient enzymatic mechanism to integrate DNA fragments into the genome in a random manner. In this study, we fused an RNA-guided catalytically inactive Cas9 (dCas9) to the PB transposase and used dual sgRNAs to localize this molecule to specific genomic targets. We designed and used a promoter/reporter complementation assay to register and recover cells harboring-specific integrations, where only by complementation upon correct genomic integration, the reporter can be activated. Using an RNA-guided piggyBac transposase and dual sgRNAs, we were able to achieve site-directed integrations in the human ROSA26 safe harbor region in 0.32% of cells. These findings show that the methodology used in this study can be used for targeting genomic regions. An application for this finding could be in cancer cells to insert sequences into specific target regions that are intended to be destroyed, or to place promoter cargos behind the tumor suppressor genes to activate them.

CRISPR/Cas9 mediated GFP-human dentin matrix protein 1 (DMP1) promoter knock-in at the ROSA26 locus in mesenchymal stem cell for monitoring osteoblast differentiation.

BACKGROUND: Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone, playing a critical role in mineralization and phosphate metabolism. One important role for the expression of DMP1 in the nucleus of preosteoblasts is the up-regulation of osteoblast-specific genes such as osteocalcin and alkaline phosphatase1 . The present study aimed to investigate the potential application of human DMP1 promoter as an indicator marker of osteoblastic differentiation. METHODS: In the present study, we developed DMP1 promoter-DsRed-GFP knock-in mesenchymal stem cell (MSCs) via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system that enabled automatic detection of osteoblast differentiation. With the application of a homology-directed knock-in strategy, a 2-kb fragment of DMP1 promoter, which was inserted upstream of the GFP and DsRed reporter cassette, was integrated into the human ROSA locus to generate double fluorescent cells. We further differentiated MSCs under osteogenic media to monitor the fate of MSCs. First, cells were transfected using CRISPR/Cas9 plasmids, which culminated in MSCs with a green fluorescence intensity, then GFP-positive cells were selected using puromycin. Second, the GFP-positive MSCs were differentiated toward osteoblasts, which demonstrated an increased red fluorescence intensity. The osteoblast differentiation of MSCs was also verified by performing alkaline phosphatase and Alizarin Red assays. RESULTS: We have exploited the DMP1 promoter as a predictive marker of MSC differentiation toward osteoblasts. Using the CRISPR/Cas9 technology, we have identified a distinctive change in the fluorescence intensities of GFP knock-in (green) and osteoblast differentiated MSCs 2 . CONCLUSIONS: The data show that DMP1-DsRed-GFP knock-in MSCs through CRISPR/Cas9 technology provide a valuable indicator for osteoblast differentiation. Moreover, The DMP1 promoter might be used as a predictive marker of MSCs differentiated toward osteoblasts.

Screening for Lynch Syndrome in Cases with Colorectal Carcinoma from Mashhad.

INTRODUCTION: Lynch Syndrome (LS) is a genetically inherited autosomal disorder that increases the risk of many types of cancer, especially colorectal cancer (CRC). Identifying these subjects improves morbidity and mortality. We aimed to assess the prevalence of LS with both clinical criteria and universal strategy in Mashhad, Iran. METHODS: In this retrospective study, we screened 322 patients with CRC between 2013 and 2016 in Mashhad, Iran. CRCs were screened based on Amsterdam II criteria, revised Bethesda guideline, and universal strategy. Information regarding the clinical criteria was obtained by interviewing the patients or, their families. Tumors were screened by pathologists with IHC staining of four Mismatch repair (MMR) proteins (MLH1, MSH2, MSH6, and PMS2). Tumors with absent IHC staining of MLH1 were tested for BRAF mutations to exclude sporadic CRCs. RESULTS: Of 322 CRCs, 33 cases were found to be deficient-MMR; 22 of these had concurrent loss of MLH1 and PMS2, followed by concurrent loss of MSH2 and MSH6 in 8 CRCs. Twenty-two cases with a loss of MLH1 underwent testing for the BRAF mutation, 4 of which were recognized as a positive BRAF mutation. Finally, 29 CRCs were found as being positive screen for LS. Poor sensitivity (21.74%) was found for the Amsterdam II criteria and a poor positive predictive value (15.39%) for the revised Bethesda. CONCLUSION: Application of clinical criteria may not be effective enough to identify LS and at least 2-antibody panel (PMS2, MSH6) should be conducted for newly diagnosed CRCs.

Association between metabolic syndrome, BMI, and serum vitamin D concentrations in rheumatoid arthritis.

Rheumatoid arthritis (RA) is the most common autoimmune arthritis. The impact of chronic inflammation on atherosclerosis and insulin resistance has been observed in several autoimmune diseases. On the other hand, metabolic syndrome (MetS); a cluster of traditional risk factors for atherosclerosis and diabetes seems to be prevalent in RA patients. It is reasonable to think that protective factors against inflammation can protect patients against atherosclerosis and diabetes, too. Vitamin D (Vit D), a novel immunomodulator, is recently considered to play a protective role against cardiovascular diseases, insulin resistance, and obesity. This cross-sectional study was designed to evaluate the impact of serum Vit D on MetS and body mass index (BMI). One hundred twenty RA patients were enrolled. MetS was assessed according to Adult Treatment Panel III criteria. All patients with known confounders influencing Vit D serum levels were excluded. Serum value of 25-hydroxyvitamin D (25(OH)D) was measured using a commercial ELISA kit. Data were analyzed by SPSS software. A logistic regression analysis stated that prednisolone dosage [p = 0.028, ß = 0.177, odds ratio (OR) = 1.194, confidence interval (CI, 1.09-1.32)], age [p = 0.002, ß = 0.146, OR = 1.57, CI (1.05-1.27)] and Vit D serum levels [p = 0.049, ß = -3.766, OR = 0.023, CI (0.001-0.978)] are all significant predictors of MetS occurrence in RA patients. It was shown that 25(OH)D is a protective factor against MetS. It was also shown that there is a negative correlation between BMI and 25(OH)D serum levels (P = 0.037, r (s) = -0.266). In summary, this study suggested that 25(OH)D plays a protective role against MetS in RA patients. However, this cross-sectional study did not permit a power calculation on the causal relationship between Vit D and metabolic syndrome. On the other hand, Vit D has a negative correlation with BMI in these patients.