Multiplex Genome Editing in Chinese Hamster Ovary Cell Line Using All-in-One and HITI CRISPR Technology.

Purpose: CRISPR/Cas9 gene editing technology has revolutionized gene manipulation by providing the opportunity of gene knock out/in, transcriptional modification and base editing. The application of this system extended into different eras of biology, from cell development to animal modeling. Various generations of CRISPR technology have been developed to make genome editing easy which resulted in rapid protocols for amelioration of a large genome. Methods: We established a simple protocol for gene manipulation in Chinese hamster ovary (CHO) cells to achieve a Caspase 7 deficient cell line by using combination of all-in-one CRISPR technology and CRISPR/Cas9 homology-independent targeted integration (CRISPR HITI). Results: the findings of this study indicated that using CRISPR knocking in/out technology facilitates genomic manipulation in CHO cells. Integration of EGFP in target locus of caspase 7 gene made the selection of knockout CHO cell line easy which achieved by cell sorting and single-cell cloning. Conclusion: this system introduces an effective targeting strategy for multiplex genome engineering, coinciding gene integration which simplified the selection of desired genomic characteristics.

CRISPR systems: Novel approaches for detection and combating COVID-19.

Type V and VI CRISPR enzymes are RNA-guided, DNA and RNA-targeting effectors that allow specific gene knockdown. Cas12 and Cas13 are CRISPR proteins that are efficient agents for diagnosis and combating single-stranded RNA (ssRNA) viruses. The programmability of these proteins paves the way for the detection and degradation of RNA viruses by targeting RNAs complementary to its CRISPR RNA (crRNA). Approximately two-thirds of viruses causing diseases contain ssRNA genomes. The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has caused the outbreak of the coronavirus disease 2019 (COVID-19), which has infected more than 88 million people worldwide with near 2 million deaths since December 2019. Thus, accurate and rapid diagnostic and therapeutic tools are essential for early detection and treatment of this widespread infectious disease. For us, the CRISPR based platforms seem to be a plausible new approach for an accurate detection and treatment of SARS-CoV-2. In this review, we talk about Cas12 and Cas13 CRISPR systems and their applications in diagnosis and treatment of RNA virus mediated diseases. In continue, the SARS-CoV-2 pathogenicity, and its conventional diagnostics and antivirals will be discussed. Moreover, we highlight novel CRISPR based diagnostic platforms and therapies for COVID-19. We also discuss the challenges of diagnostic CRISPR based platforms as well as clarifying the proposed solution for high efficient selective in vivo delivery of CRISPR components into SARS-CoV-2-infected cells.

Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability.

BACKGROUND: Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell in the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. RESULTS: Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. CONCLUSION: These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.

CRISPR System: A High-throughput Toolbox for Research and Treatment of Parkinson's Disease.

In recent years, the innovation of gene-editing tools such as the CRISPR/Cas9 system improves the translational gap of treatments mediated by gene therapy. The privileges of CRISPR/Cas9 such as working in living cells and organs candidate this technology for using in research and treatment of the central nervous system (CNS) disorders. Parkinson's disease (PD) is a common, debilitating, neurodegenerative disorder which occurs due to loss of dopaminergic neurons and is associated with progressive motor dysfunction. Knowledge about the pathophysiological basis of PD has altered the classification system of PD, which manifests in familial and sporadic forms. The first genetic linkage studies in PD demonstrated the involvement of Synuclein alpha (SNCA) mutations and SNCA genomic duplications in the pathogenesis of PD familial forms. Subsequent studies have also insinuated mutations in leucine repeat kinase-2 (LRRK2), Parkin, PTEN-induced putative kinase 1 (PINK1), as well as DJ-1 causing familial forms of PD. This review will attempt to discuss the structure, function, and development in genome editing mediated by CRISP/Cas9 system. Further, it describes the genes involved in the pathogenesis of PD and the pertinent alterations to them. We will pursue this line by delineating the PD linkage studies in which CRISPR system was employed. Finally, we will discuss the pros and cons of CRISPR employment vis-à-vis the process of genome editing in PD patients' iPSCs.

Caspase-7 deficiency in Chinese hamster ovary cells reduces cell proliferation and viability

BACKGROUND: Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell In the commercial-scale production of biopharmaceutical proteins. Modification of genes involved in apoptosis may improve the productivity of CHO cells. Executive caspases, including caspases 3 and 7, play critical roles in apoptosis. The effects of the ablation of the caspase 7 gene on proliferation and viability of CHO cells remains unknown. In this study, we applied clustered regularly interspaced short palindromic repeat (CRISPR/Cas9) to target caspase 7 gene of CHO K1 cell via all in one and homology targeted integration strategies. Consequently, the effect of caspase 7 deficiency on cell proliferation, viability, and apoptosis was studied by MTT assay and flow cytometry. RESULTS: Findings of gel electrophoresis, western blotting, and sequencing confirmed the caspase 7 gene silencing in CHO cells (CHO-KO). Proliferation assay revealed that caspase 7 deficiency in CHO cells resulted in the reduction of proliferation in various CHO-KO clones. Besides, the disruption of caspase 7 had negative effects on cell viability in exposure with NaBu which confirmed by MTT assay. Results of flow cytometry using Anexin V/PI demonstrated that Nabu treatment (11 mM) declined the percentage of live CHO-K1 and CHO-KO cells to 70.3% and 5.79%. These results verified that the CHO-K1 cells were more resistant to apoptosis than CHO-KO, however most of CHO-KO cells undergone early apoptosis (91.9%) which seems to be a fascinating finding. CONCLUSION: These results reveal that caspase 7 may be involved in the cell cycle progression of CHO cells. Furthermore, it seems that targeting caspase 7 is not the ideal route as it had previously been imagined within the prevention of apoptosis but the relation between caspase 7 deficiency, cell cycle arrest, and the occurrence of early apoptosis will require more investigation.

CRISPR Cpf1 proteins: structure, function and implications for genome editing.

CRISPR and CRISPR-associated (Cas) protein, as components of microbial adaptive immune system, allows biologists to edit genomic DNA in a precise and specific way. CRISPR-Cas systems are classified into two main classes and six types. Cpf1 is a putative type V (class II) CRISPR effector, which can be programmed with a CRISPR RNA to bind and cleave complementary DNA targets. Cpf1 has recently emerged as an alternative for Cas9, due to its distinct features such as the ability to target T-rich motifs, no need for trans-activating crRNA, inducing a staggered double-strand break and potential for both RNA processing and DNA nuclease activity. In this review, we attempt to discuss the evolutionary origins, basic architectures, and molecular mechanisms of Cpf1 family proteins, as well as crRNA designing and delivery strategies. We will also describe the novel Cpf1 variants, which have broadened the versatility and feasibility of this system in genome editing, transcription regulation, epigenetic modulation, and base editing. Finally, we will be reviewing the recent studies on utilization of Cpf1as a molecular tool for genome editing.

Targeted cancer therapy through 17-DMAG as an Hsp90 inhibitor: Overview and current state of the art.

Heat shock protein 90 (Hsp90) is an evolutionary preserved molecular chaperone which mediates many cellular processes such as cell transformation, proliferation, and survival in normal and stress conditions. Hsp90 plays an important role in folding, maturation, stabilization and activation of Hsp90 client proteins which all contribute to the development, and proliferation of cancer as well as other inflammatory diseases. Functional inhibition of Hsp90 can have a massive effect on various oncogenic and inflammatory pathways, and will result in the degradation of their client proteins. This turns it into an interesting target in the treatment of different malignancies. 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) as a semi-synthetic derivative of geldanamycin, has several advantages over 17-Allylamino-17-demethoxygeldanamycin (17-AAG) such as higher water solubility, good bioavailability, reduced metabolism, and greater anti-tumour capability. 17-DMAG binds to the Hsp90, and inhibits its function which eventually results in the degradation of Hsp90 client proteins. Here, we reviewed the pre-clinical data and clinical trial data on 17-DMAG as a single agent, in combination with other agents and loaded on nanomaterials in various cancers and inflammatory diseases.

Copper sulfate pentahydrate reduced epithelial cytotoxicity induced by lipopolysaccharide from enterogenic bacteria.

The over usage of multiple antibiotics contributes to the emergence of a whole range of antibiotic-resistant strains of bacteria causing enterogenic infections in poultry science. Therefore, finding an appropriate alternative natural substance carrying an antibacterial capacity would be immensely beneficial. It has been previously discovered that the different types of cupric salts, especially copper sulfate pentahydrate (CuSO4·5H2O), to carry a potent bactericidal capacity. We investigated the neutralizing effect of CuSO4·5H2O (6.25µg/ml) on the reactive oxygen species generation, and expression of MyD88, an essential adaptor protein of Toll-like receptor, and NF-κB in three intestinal epithelial cell lines exposed to 50ng/ml lipopolysaccharide. In order to find the optimal cupric sulfate concentration without enteritis-inducing toxicity, broiler chickens were initially fed with water containing 0.4, 0.5, and 1mg/l during a period of 4days. After determination of appropriate dosage, two broiler chickens and turkey flocks with enteritis were fed with cupric compound for 4days. We found that cupric sulfate can lessen the cytotoxic effect of lipopolysaccharide by reducing the reactive oxygen species content (p<0.05). Additionally, the expression of MyD88 and NF-κB was remarkably down-regulated in the presence of lipopolysaccharide and cupric sulfate. The copper sulfate in doses lower than 0.4mg/ml expressed no cytotoxic effect on the liver, kidney, and the intestinal tract while a concentration of 0.5 and 1mg/ml contributed to a moderate to severe tissue injuries. Pearson Chi-Square analysis revealed the copper cation significantly diminished the rate of mortality during 4-day feeding of broiler chicken and turkey with enteritis (p=0.000). Thus, the results briefed above all confirm the potent anti-bactericidal feature of cupric sulfate during the course of enteritis.