Displaying publications 1 - 20 of 38 in total

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  1. A potential paradigm in CRISPR/Cas systems delivery: at the crossroad of microalgal gene editing and algal-mediated nanoparticles
    Feng S, Xie X, Liu J, Li A, Wang Q, Guo D, et al.
    J Nanobiotechnology, 2023 Oct 10;21(1):370.
    PMID: 37817254 DOI: 10.1186/s12951-023-02139-z
    Microalgae as the photosynthetic organisms offer enormous promise in a variety of industries, such as the generation of high-value byproducts, biofuels, pharmaceuticals, environmental remediation, and others. With the rapid advancement of gene editing technology, CRISPR/Cas system has evolved into an effective tool that revolutionised the genetic engineering of microalgae due to its robustness, high target specificity, and programmability. However, due to the lack of robust delivery system, the efficacy of gene editing is significantly impaired, limiting its application in microalgae. Nanomaterials have become a potential delivery platform for CRISPR/Cas systems due to their advantages of precise targeting, high stability, safety, and improved immune system. Notably, algal-mediated nanoparticles (AMNPs), especially the microalgae-derived nanoparticles, are appealing as a sustainable delivery platform because of their biocompatibility and low toxicity in a homologous relationship. In addition, living microalgae demonstrated effective and regulated distribution into specified areas as the biohybrid microrobots. This review extensively summarised the uses of CRISPR/Cas systems in microalgae and the recent developments of nanoparticle-based CRISPR/Cas delivery systems. A systematic description of the properties and uses of AMNPs, microalgae-derived nanoparticles, and microalgae microrobots has also been discussed. Finally, this review highlights the challenges and future research directions for the development of gene-edited microalgae.
    Matched MeSH terms: CRISPR-Cas Systems/genetics
  2. dCas9 Tells Tales: Probing Gene Function and Transcription Regulation in Cancer
    Mohamad Zamberi NN, Abuhamad AY, Low TY, Mohtar MA, Syafruddin SE
    CRISPR J, 2024 Apr;7(2):73-87.
    PMID: 38635328 DOI: 10.1089/crispr.2023.0078
    Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing is evolving into an essential tool in the field of biological and medical research. Notably, the development of catalytically deactivated Cas9 (dCas9) enzyme has substantially broadened its traditional boundaries in gene editing or perturbation. The conjugation of dCas9 with various molecular effectors allows precise control over transcriptional processes, epigenetic modifications, visualization of chromosomal dynamics, and several other applications. This expanded repertoire of CRISPR-Cas9 applications has emerged as an invaluable molecular tool kit that empowers researchers to comprehensively interrogate and gain insights into health and diseases. This review delves into the advancements in Cas9 protein engineering, specifically on the generation of various dCas9 tools that have significantly enhanced the CRISPR-based technology capability and versatility. We subsequently discuss the multifaceted applications of dCas9, especially in interrogating the regulation and function of genes that involve in supporting cancer pathogenesis. In addition, we also delineate the designing and utilization of dCas9-based tools as well as highlighting its current constraints and transformative potentials in cancer research.
    Matched MeSH terms: CRISPR-Cas Systems/genetics
  3. CRISPR-Cas9 Genome Editing Tool for the Production of Industrial Biopharmaceuticals
    Khan AH, Tye GJ, Noordin R
    Mol Biotechnol, 2020 Sep;62(9):401-411.
    PMID: 32749657 DOI: 10.1007/s12033-020-00265-9
    A broad range of cell lines with characteristic features are used as bio-factories to produce recombinant proteins for basic research and therapeutic purposes. Genetic engineering strategies have been used to manipulate the genome of mammalian cells, insects, and yeasts for heterologous expression. One reason is that the glycosylation pattern of the expression hosts differs somehow from mammalian cells, which may cause immunogenic reactions upon administration in humans. CRISPR-Cas9 is a simple, efficient, and versatile genome engineering tool that can be programmed to precisely make double-stranded breaks at the desired loci. Compared to the classical genome editing methods, a CRISPR-Cas9 system is an ideal tool, providing the opportunity to integrate or delete genes from the target organisms. Besides broadened applications, limited studies have used CRISPR-Cas9 for editing the endogenous pathways in expression systems for biopharmaceutical applications. In the present review, we discuss the use of CRISPR-Cas9 in expression systems to improve host cell lines, increase product yield, and humanize glycosylation pathways by targeting intrinsic genes.
    Matched MeSH terms: CRISPR-Cas Systems*
  4. Fulltext CRISPR/dCas9 platforms in plants: strategies and applications beyond genome editing
    Moradpour M, Abdulah SNA
    Plant Biotechnol J, 2020 Jan;18(1):32-44.
    PMID: 31392820 DOI: 10.1111/pbi.13232
    Clustered regularly interspaced short palindromic repeat (CRISPR) and Cas9-associated protein systems provide a powerful genetic manipulation tool that can drive plant research forward. Nuclease-dead Cas9 (dCas9) is an enzymatically inactive mutant of Cas9 in which its endonuclease activity is non-functional. The applications of CRISPR/dCas9 have expanded and diversified in recent years. Originally, dCas9 was used as a CRISPR/Cas9 re-engineering tool that enables targeted expression of any gene or multiple genes through recruitment of transcriptional effector domains without introducing irreversible DNA-damaging mutations. Subsequent applications have made use of its ability to recruit modifying enzymes and reporter proteins to DNA target sites. In this paper, the most recent progress in the applications of CRISPR/dCas9 in plants, which include gene activation and repression, epigenome editing, modulation of chromatin topology, live-cell chromatin imaging and DNA-free genetic modification, will be reviewed. The associated strategies for exploiting the CRISPR/dCas9 system for crop improvement with a dimer of the future of the CRISPR/dCas9 system in the functional genomics of crops and the development of traits will be briefly discussed.
    Matched MeSH terms: CRISPR-Cas Systems*
  5. A review of CRISPR-Cas and PCR-based methods for the detection of animal species in the food chain-current challenges and future prospects
    Sultana S, Azlan A, Mohd Desa MN, Mahyudin NA, Anburaj A
    Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 2024 Mar;41(3):213-227.
    PMID: 38284970 DOI: 10.1080/19440049.2024.2304577
    Regular testing and systematic investigation play a vital role to ensure product safety. Until now, the existing food authentication techniques have been based on proteins, lipids, and nucleic acid-based assays. Among various deoxyribonucleic acid (DNA)-based methods, the recently developed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based bio-sensing is an innovative and fast-expanding technology. The CRISPR/Cas-9 is known as Clustered Regularly Interspaced Short Palindromic Repeats due to the flexibility and simplicity of the CRISPR/Cas9 site-specific editing tool has been applied in many biological research areas such as Gene therapy, cell line development, discovering mechanisms of disease, and drug discovery. Nowadays, the CRISPR-Cas system has also been introduced into food authentication via detecting DNA barcodes of poultry and livestock both in processed and unprocessed food samples. This review documents various DNA based approaches, in an accessible format. Future CRISPR technologies are forecast while challenges are outlined.
    Matched MeSH terms: CRISPR-Cas Systems*
  6. Recent trends in cancer therapy: A review on the current state of gene delivery
    Yahya EB, Alqadhi AM
    Life Sci, 2021 Mar 15;269:119087.
    PMID: 33476633 DOI: 10.1016/j.lfs.2021.119087
    Cancer treatment has been always considered one of the most critical and vital themes of clinical issues. Many approaches have been developed, depending on the type and the stage of tumor. Gene therapy has the potential to revolutionize different cancer therapy. With the advent of recent bioinformatics technologies and genetic science, it become possible to identify, diagnose and determine the potential treatment using the technology of gene delivery. Several approaches have been developed and experimented in vitro and vivo for cancer therapy including: naked nucleic acids based therapy, targeting micro RNAs, oncolytic virotherapy, suicide gene based therapy, targeting telomerase, cell mediated gene therapy, and CRISPR/Cas9 based therapy. In this review, we present a straightforward introduction to cancer biology and occurrence, highlighting different viral and non-viral gene delivery systems for gene therapy and critically discussed the current and various strategies for cancer gene therapy.
    Matched MeSH terms: CRISPR-Cas Systems*
  7. The Promises and Pitfalls of CRISPR-Mediated Base Editing in Stem Cells
    Wong PK, Mohamad Zamberi NN, Syafruddin SE, Cheah FC, Azmi N, Law JX, et al.
    CRISPR J, 2023 Jun;6(3):196-215.
    PMID: 37219623 DOI: 10.1089/crispr.2023.0013
    Stem cells such as induced pluripotent stem cells, embryonic stem cells, and hematopoietic stem and progenitor cells are growing in importance in disease modeling and regenerative medicine. The applications of CRISPR-based gene editing to create a mélange of disease and nondisease stem cell lines have further enhanced the utility of this innately versatile group of cells in the studies of human genetic disorders. Precise base edits can be achieved using a variety of CRISPR-centric approaches, particularly homology-directed repair and the recently developed base editors and prime editors. Despite its much-touted potential, editing single DNA bases is technically challenging. In this review, we discuss the strategies for achieving exact base edits in the creation of various stem cell-based models for use in elucidating disease mechanisms and assessing drug efficacy, and the unique characteristics of stem cells that warrant special considerations.
    Matched MeSH terms: CRISPR-Cas Systems/genetics
  8. Dual-Functional Tetrahedron Multivalent Aptamer Assisted Amplification-Free CRISPR/Cas12a Assay for Sensitive Detection of Salmonella
    Qiao Z, Xue L, Sun M, Ma N, Shi H, Yang W, et al.
    J Agric Food Chem, 2024 Jan 10;72(1):857-864.
    PMID: 38134022 DOI: 10.1021/acs.jafc.3c07582
    Salmonellosis continues to impose a significant economic burden globally. Rapid and sensitive detection of Salmonella is crucial to preventing the outbreaks of foodborne illnesses, yet it remains a formidable challenge. Herein, a dual-functional tetrahedron multivalent aptamer assisted amplification-free CRISPR/Cas12a assay was developed for Salmonella detection. In the system, the aptamer was programmatically assembled on the tetrahedral DNA nanostructure to fabricate a multivalent aptamer (TDN-multiApt), which displayed a 3.5-fold enhanced avidity over the monovalent aptamer and possessed four CRISPR/Cas12a targeting fragments to amplify signal. Therefore, TDN-multiApt could directly activate Cas12a to achieve the second signal amplification without any nucleic acid amplification. By virtue of the synergism of high avidity and cascaded signal amplifications, the proposed method allowed the ultrasensitive detection of Salmonella as low as 7 cfu mL-1. Meanwhile, this novel platform also exhibited excellent specificity against target bacteria and performed well in the detection of various samples, indicating its potential application in real samples.
    Matched MeSH terms: CRISPR-Cas Systems*
  9. The Smart Programmable CRISPR Technology: A Next Generation Genome Editing Tool for Investigators
    Chakraborty C, Teoh SL, Das S
    Curr Drug Targets, 2017;18(14):1653-1663.
    PMID: 27231109 DOI: 10.2174/1389450117666160527142321
    BACKGROUND: The present era is fast experiencing rapid innovation in the genome-editing technology. CRISPR Cas9-mediated targeted genetic manipulation is an easy, cost-effective and scalable method. As a result, it can be used for a broad range of targeted genome engineering.

    OBJECTIVE: The main objective of the present review is to highlight the structural signature, classification, its mechanism and application from basic science to medicine and future challenges for this genome editing tool kit.

    RESULTS: The present review provides a brief description of the recent development of CRISPR-Cas9 genome editing technology. We discuss the paradigms shift for this next generation genome editing technology, CRISPR. The CRISPR structural significance, classification and its different applications are also being discussed. We portray the future challenges for this extraordinary genome in vivo editing tool. We also highlight the role of CRISPR genome editing in curing many diseases.

    CONCLUSION: Scientists and researchers are constantly looking one genome editing tool that is competent, simple and low-cost assembly of nucleases. It can target any particular site without any off-target mutations in the genome. The CRISPR-Cas9 has all of the above characteristics. The genome engineering technology may be a strong and inspiring technology meant for the next generation of drug development.

    Matched MeSH terms: CRISPR-Cas Systems*
  10. Fulltext The phylogenomics of CRISPR-Cas system and revelation of its features in Salmonella
    Kushwaha SK, Bhavesh NLS, Abdella B, Lahiri C, Marathe SA
    Sci Rep, 2020 12 03;10(1):21156.
    PMID: 33273523 DOI: 10.1038/s41598-020-77890-6
    Salmonellae display intricate evolutionary patterns comprising over 2500 serovars having diverse pathogenic profiles. The acquisition and/or exchange of various virulence factors influences the evolutionary framework. To gain insights into evolution of Salmonella in association with the CRISPR-Cas genes we performed phylogenetic surveillance across strains of 22 Salmonella serovars. The strains differed in their CRISPR1-leader and cas operon features assorting into two main clades, CRISPR1-STY/cas-STY and CRISPR1-STM/cas-STM, comprising majorly typhoidal and non-typhoidal Salmonella serovars respectively. Serovars of these two clades displayed better relatedness, concerning CRISPR1-leader and cas operon, across genera than between themselves. This signifies the acquisition of CRISPR1/Cas region could be through a horizontal gene transfer event owing to the presence of mobile genetic elements flanking CRISPR1 array. Comparison of CRISPR and cas phenograms with that of multilocus sequence typing (MLST) suggests differential evolution of CRISPR/Cas system. As opposed to broad-host-range, the host-specific serovars harbor fewer spacers. Mapping of protospacer sources suggested a partial correlation of spacer content with habitat diversity of the serovars. Some serovars like serovar Enteritidis and Typhimurium that inhabit similar environment/infect similar hosts hardly shared their protospacer sources.
    Matched MeSH terms: CRISPR-Cas Systems/genetics*
  11. Knockout of CTNNB1 by CRISPR-Cas9 technology inhibits cell proliferation through the Wnt/β-catenin signaling pathway
    Guan L, Zhu S, Han Y, Yang C, Liu Y, Qiao L, et al.
    Biotechnol Lett, 2018 Mar;40(3):501-508.
    PMID: 29249062 DOI: 10.1007/s10529-017-2491-2
    OBJECTIVE: To study the effects of CTNNB1 gene knockout by CRISPR-Cas9 technology on cell adhesion, proliferation, apoptosis, and Wnt/β-catenin signaling pathway.

    RESULTS: CTNNB1 gene of HEK 293T cells was knocked out by CRISPR-Cas9. This was confirmed by sequencing and western blotting. Methylthiazolyl-tetrazolium bromide assays indicated that deletion of β-catenin significantly weakened adhesion ability and inhibited proliferation rate (P 

    Matched MeSH terms: CRISPR-Cas Systems/genetics*
  12. Fulltext Therapeutic potentials of CRISPR-Cas genome editing technology in human viral infections
    Najafi S, Tan SC, Aghamiri S, Raee P, Ebrahimi Z, Jahromi ZK, et al.
    Biomed Pharmacother, 2022 Apr;148:112743.
    PMID: 35228065 DOI: 10.1016/j.biopha.2022.112743
    Viral infections are a common cause of morbidity worldwide. The emergence of Coronavirus Disease 2019 (COVID-19) has led to more attention to viral infections and finding novel therapeutics. The CRISPR-Cas9 system has been recently proposed as a potential therapeutic tool for the treatment of viral diseases. Here, we review the research progress in the use of CRISPR-Cas technology for treating viral infections, as well as the strategies for improving the delivery of this gene-editing tool in vivo. Key challenges that hinder the widespread clinical application of CRISPR-Cas9 technology are also discussed, and several possible directions for future research are proposed.
    Matched MeSH terms: CRISPR-Cas Systems*
  13. Fulltext Harnessing the Potential of CRISPR/Cas in Atherosclerosis: Disease Modeling and Therapeutic Applications
    Siew WS, Tang YQ, Kong CK, Goh BH, Zacchigna S, Dua K, et al.
    Int J Mol Sci, 2021 Aug 05;22(16).
    PMID: 34445123 DOI: 10.3390/ijms22168422
    Atherosclerosis represents one of the major causes of death globally. The high mortality rates and limitations of current therapeutic modalities have urged researchers to explore potential alternative therapies. The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system is commonly deployed for investigating the genetic aspects of Atherosclerosis. Besides, advances in CRISPR/Cas system has led to extensive options for researchers to study the pathogenesis of this disease. The recent discovery of Cas9 variants, such as dCas9, Cas9n, and xCas9 have been established for various applications, including single base editing, regulation of gene expression, live-cell imaging, epigenetic modification, and genome landscaping. Meanwhile, other Cas proteins, such as Cas12 and Cas13, are gaining popularity for their applications in nucleic acid detection and single-base DNA/RNA modifications. To date, many studies have utilized the CRISPR/Cas9 system to generate disease models of atherosclerosis and identify potential molecular targets that are associated with atherosclerosis. These studies provided proof-of-concept evidence which have established the feasibility of implementing the CRISPR/Cas system in correcting disease-causing alleles. The CRISPR/Cas system holds great potential to be developed as a targeted treatment for patients who are suffering from atherosclerosis. This review highlights the advances in CRISPR/Cas systems and their applications in establishing pathogenetic and therapeutic role of specific genes in atherosclerosis.
    Matched MeSH terms: CRISPR-Cas Systems/genetics*
  14. Fulltext The Impact of CRISPR-Cas System on Antiviral Therapy
    Bayat H, Naderi F, Khan AH, Memarnejadian A, Rahimpour A
    Adv Pharm Bull, 2018 Nov;8(4):591-597.
    PMID: 30607331 DOI: 10.15171/apb.2018.067
    Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein nuclease (Cas) is identified as an adaptive immune system in archaea and bacteria. Type II of this system, CRISPR-Cas9, is the most versatile form that has enabled facile and efficient targeted genome editing. Viral infections have serious impacts on global health and conventional antiviral therapies have not yielded a successful solution hitherto. The CRISPR-Cas9 system represents a promising tool for eliminating viral infections. In this review, we highlight 1) the recent progress of CRISPR-Cas technology in decoding and diagnosis of viral outbreaks, 2) its applications to eliminate viral infections in both pre-integration and provirus stages, and 3) various delivery systems that are employed to introduce the platform into target cells.
    Matched MeSH terms: CRISPR-Cas Systems
  15. Fulltext Mutant Allele-Specific CRISPR Disruption in DYT1 Dystonia Fibroblasts Restores Cell Function
    Cruz L, György B, Cheah PS, Kleinstiver BP, Eimer WA, Garcia SP, et al.
    Mol Ther Nucleic Acids, 2020 Sep 04;21:1-12.
    PMID: 32502938 DOI: 10.1016/j.omtn.2020.05.009
    Most individuals affected with DYT1 dystonia have a heterozygous 3-bp deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant-negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity. In this study, we used an engineered Cas9 variant from Streptococcus pyogenes (SpCas9-VRQR) to target the mutation in the TOR1A gene in order to disrupt mutant torsinA in DYT1 patient fibroblasts. Selective targeting of the DYT1 allele was highly efficient with most common non-homologous end joining (NHEJ) edits, leading to a predicted premature stop codon with loss of the torsinA C terminus (delta 302-332 aa). Structural analysis predicted a functionally inactive status of this truncated torsinA due to the loss of residues associated with ATPase activity and binding to LULL1. Immunoblotting showed a reduction of the torsinA protein level in Cas9-edited DYT1 fibroblasts, and a functional assay using HSV infection indicated a phenotypic recovery toward that observed in control fibroblasts. These findings suggest that the selective disruption of the mutant TOR1A allele using CRISPR-Cas9 inactivates mutant torsinA, allowing the remaining wild-type torsinA to exert normal function.
    Matched MeSH terms: CRISPR-Cas Systems
  16. Fulltext The CRISPR Growth Spurt: from Bench to Clinic on Versatile Small RNAs
    Bayat H, Omidi M, Rajabibazl M, Sabri S, Rahimpour A
    J Microbiol Biotechnol, 2017 Feb 28;27(2):207-218.
    PMID: 27840399 DOI: 10.4014/jmb.1607.07005
    Clustered regulatory interspaced short palindromic repeats (CRISPR) in association with CRISPR-associated protein (Cas) is an adaptive immune system, playing a pivotal role in the defense of bacteria and archaea. Ease of handling and cost effectiveness make the CRISPR-Cas system an ideal programmable nuclease tool. Recent advances in understanding the CRISPR-Cas system have tremendously improved its efficiency. For instance, it is possible to recapitulate the chronicle CRISPR-Cas from its infancy and inaugurate a developed version by generating novel variants of Cas proteins, subduing off-target effects, and optimizing of innovative strategies. In summary, the CRISPR-Cas system could be employed in a number of applications, including providing model systems, rectification of detrimental mutations, and antiviral therapies.
    Matched MeSH terms: CRISPR-Cas Systems
  17. Investigative study on the role of the Toxo 5699 gene in the Toxoplasma gondii lytic cycle using the CRISPR/Cas9 system
    De Silva JR, Ching XT, Lau YL
    Trop Biomed, 2020 Jun 01;37(2):324-332.
    PMID: 33612802
    The focus of the current study was to disrupt the Toxo 5699 gene via CRISPR/Cas9 to evaluate the effects of gene disruption on the parasite lytic cycle. In the present work, a single plasmid expressing both the guide RNA and Cas9 nuclease together with a selectable marker of human dihydrofolate reductase (DHFR) was introduced into Toxoplasma gondii. Targeted disruption of the Toxo 5699 gene was carried out via the CRISPR/Cas9 system and confirmed by PCR, sequencing, and immunofluorescence microscopy. Disrupted and nondisrupted control parasites were allowed to invade HS27 cell monolayers and plaques were counted. The average number of plaques from three replicates per group was obtained between the disrupted and non-disrupted T. gondii RH strain and was compared using a onetailed t-test. It was observed that there was a significant decrease in number and size of plaque formation in the Toxo 5699 gene disrupted parasite line. This is an indication that the Toxo 5699 gene may play a role in the lytic cycle of the parasite, particularly during the replication phase and thus would be a novel target for disruption or silencing. The Toxo 5699 gene presented in the current work is an important part of the T. gondii lytic cycle, therefore meriting further inquiry into its potential as a target for further genetic-silencing or disruption studies.
    Matched MeSH terms: CRISPR-Cas Systems
  18. Fulltext CNPase, a 2',3'-Cyclic-nucleotide 3'-phosphodiesterase, as a Therapeutic Target to Attenuate Cardiac Hypertrophy by Enhancing Mitochondrial Energy Production
    Tan KS, Wang D, Lu Z, Zhang Y, Li S, Lin Y, et al.
    Int J Mol Sci, 2021 Oct 06;22(19).
    PMID: 34639145 DOI: 10.3390/ijms221910806
    Heart failure is the end-stage of all cardiovascular diseases with a ~25% 5-year survival rate, and insufficient mitochondrial energy production to meet myocardial demand is the hallmark of heart failure. Mitochondrial components involved in the regulation of ATP production remain to be fully elucidated. Recently, roles of 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) in the pathophysiological processes of heart diseases have emerged, implicated by evidence that mitochondrial CNPase proteins are associated with mitochondrial integrity under metabolic stress. In this study, a zebrafish heart failure model was established, by employing antisense morpholino oligonucleotides and the CRISPR-Cas9 gene-editing system, which recapitulates heart failure phenotypes including heart dysfunction, pericardial edema, ventricular enlargement, bradycardia, and premature death. The translational implications of CNPase in the pathophysiological process of heart failure were tested in a pressure overload-induced heart hypertrophy model, which was carried out in rats through transverse abdominal aorta constriction (TAAC). AAV9-mediated myocardial delivery of CNPase mitigated the hypertrophic response through the specific hydrolysis of 2'-3'-cyclic nucleotides, supported by the decrease of cardiac hypertrophy and fibrosis, the integrity of mitochondrial ultrastructure, and indicators of heart contractility in the AAV9-TAAC group. Finally, the biometrics of a mitochondrial respiration assay carried out on a Seahorse cellular energy analyzer demonstrated that CNPase protects mitochondrial respiration and ATP production from AngII-induced metabolic stress. In summary, this study provides mechanistic insights into CNPase-2',3'-cyclic nucleotide metabolism that protects the heart from energy starvation and suggests novel therapeutic approaches to treat heart failure by targeting CNPase activity.
    Matched MeSH terms: CRISPR-Cas Systems*
  19. Fulltext Lung development, repair and cancer: A study on the role of MMP20 gene in adenocarcinoma
    Mok PL, Anandasayanam ANK, Oscar David HM, Tong J, Farhana A, Khan MSA, et al.
    PLoS One, 2021;16(4):e0250552.
    PMID: 33914777 DOI: 10.1371/journal.pone.0250552
    Multiple matrix metalloproteinases have significant roles in tissue organization during lung development, and repair. Imbalance of proteinases may lead to chronic inflammation, changes in tissue structure, and are also highly associated to cancer development. The role of MMP20 is not well studied in lung organogenesis, however, it was previously shown to be present at high level in lung adenocarcinoma. The current study aimed to identify the functional properties of MMP20 on cell proliferation and motility in a lung adenocarcinoma in vitro cell model, and relate the interaction of MMP20 with other molecular signalling pathways in the lung cells after gaining tumoral properties. In this study, two different single guide RNA (sgRNAs) that specifically targeted on MMP20 sites were transfected into human lung adenocarcinoma A549 cells by using CRISPR-Cas method. Following that, the changes of PI3-K, survivin, and MAP-K mRNA gene expression were determined by Real-Time Polymerase Chain Reaction (RT-PCR). The occurrence of cell death was also examined by Acridine Orange/Propidium Iodide double staining. Meanwhile, the motility of the transfected cells was evaluated by wound healing assay. All the data were compared with non-transfected cells as a control group. Our results demonstrated that the transfection of the individual sgRNAs significantly disrupted the proliferation of the A549 cell line through suppression in the gene expression of PI3-K, survivin, and MAP-K. When compared to non-transfected cells, both experimental cell groups showed reduction in the migration rate, as reflected by the wider gaps in the wound healing assay. The current study provided preliminary evidence that MMP20 could have regulatory role on stemness and proliferative genes in the lung tissues and affect the cell motility. It also supports the notion that targeting MMP20 could be a potential treatment mode for halting cancer progression.
    Matched MeSH terms: CRISPR-Cas Systems/genetics
  20. Fulltext Endogenous HIF2A reporter systems for high-throughput functional screening
    Zaini MN, Patel SA, Syafruddin SE, Rodrigues P, Vanharanta S
    Sci Rep, 2018 08 13;8(1):12063.
    PMID: 30104738 DOI: 10.1038/s41598-018-30499-2
    Tissue-specific transcriptional programs control most biological phenotypes, including disease states such as cancer. However, the molecular details underlying transcriptional specificity is largely unknown, hindering the development of therapeutic approaches. Here, we describe novel experimental reporter systems that allow interrogation of the endogenous expression of HIF2A, a critical driver of renal oncogenesis. Using a focused CRISPR-Cas9 library targeting chromatin regulators, we provide evidence that these reporter systems are compatible with high-throughput screening. Our data also suggests redundancy in the control of cancer type-specific transcriptional traits. Reporter systems such as those described here could facilitate large-scale mechanistic dissection of transcriptional programmes underlying cancer phenotypes, thus paving the way for novel therapeutic approaches.
    Matched MeSH terms: CRISPR-Cas Systems/genetics
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