Displaying publications 21 - 40 of 43 in total

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  1. 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
  2. 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
  3. 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*
  4. 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
  5. 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
  6. Fulltext Characterization of CRISPR Mutants Targeting Genes Modulating Pectin Degradation in Ripening Tomato
    Wang D, Samsulrizal NH, Yan C, Allcock NS, Craigon J, Blanco-Ulate B, et al.
    Plant Physiol, 2019 02;179(2):544-557.
    PMID: 30459263 DOI: 10.1104/pp.18.01187
    Tomato (Solanum lycopersicum) is a globally important crop with an economic value in the tens of billions of dollars, and a significant supplier of essential vitamins, minerals, and phytochemicals in the human diet. Shelf life is a key quality trait related to alterations in cuticle properties and remodeling of the fruit cell walls. Studies with transgenic tomato plants undertaken over the last 20 years have indicated that a range of pectin-degrading enzymes are involved in cell wall remodeling. These studies usually involved silencing of only a single gene and it has proved difficult to compare the effects of silencing these genes across the different experimental systems. Here we report the generation of CRISPR-based mutants in the ripening-related genes encoding the pectin-degrading enzymes pectate lyase (PL), polygalacturonase 2a (PG2a), and β-galactanase (TBG4). Comparison of the physiochemical properties of the fruits from a range of PL, PG2a, and TBG4 CRISPR lines demonstrated that only mutations in PL resulted in firmer fruits, although mutations in PG2a and TBG4 influenced fruit color and weight. Pectin localization, distribution, and solubility in the pericarp cells of the CRISPR mutant fruits were investigated using the monoclonal antibody probes LM19 to deesterified homogalacturonan, INRA-RU1 to rhamnogalacturonan I, LM5 to β-1,4-galactan, and LM6 to arabinan epitopes, respectively. The data indicate that PL, PG2a, and TBG4 act on separate cell wall domains and the importance of cellulose microfibril-associated pectin is reflected in its increased occurrence in the different mutant lines.
    Matched MeSH terms: CRISPR-Cas Systems*
  7. Fulltext Evolutionary study of Yersinia genomes deciphers emergence of human pathogenic species
    Tan SY, Tan IK, Tan MF, Dutta A, Choo SW
    Sci Rep, 2016 10 31;6:36116.
    PMID: 27796355 DOI: 10.1038/srep36116
    On record, there are 17 species in the Yersinia genus, of which three are known to be pathogenic to human. While the chromosomal and pYV (or pCD1) plasmid-borne virulence genes as well as pathogenesis of these three species are well studied, their genomic evolution is poorly understood. Our study aims to predict the key evolutionary events that led to the emergence of pathogenic Yersinia species by analyzing gene gain-and-loss, virulence genes, and "Clustered regularly-interspaced short palindromic repeats". Our results suggest that the most recent ancestor shared by the human pathogenic Yersinia was most probably an environmental species that had adapted to the human body. This might have led to ecological specialization that diverged Yersinia into ecotypes and distinct lineages based on differential gene gain-and-loss in different niches. Our data also suggest that Y. pseudotuberculosis group might be the donor of the ail virulence gene to Y. enterocolitica. Hence, we postulate that evolution of human pathogenic Yersinia might not be totally in parallel, but instead, there were lateral gene transfer events. Furthermore, the presence of virulence genes seems to be important for the positive selection of virulence plasmid. Our studies provide better insights into the evolutionary biology of these bacteria.
    Matched MeSH terms: CRISPR-Cas Systems/genetics
  8. Fulltext Deep Brain Photoreceptor (val-opsin) Gene Knockout Using CRISPR/Cas Affects Chorion Formation and Embryonic Hatching in the Zebrafish
    Hang CY, Moriya S, Ogawa S, Parhar IS
    PLoS One, 2016;11(10):e0165535.
    PMID: 27792783 DOI: 10.1371/journal.pone.0165535
    Non-rod non-cone photopigments in the eyes and the brain can directly mediate non-visual functions of light in non-mammals. This was supported by our recent findings on vertebrate ancient long (VAL)-opsin photopigments encoded by the val-opsinA (valopa) and val-opsinB (valopb) genes in zebrafish. However, the physiological functions of valop isoforms remain unknown. Here, we generated valop-mutant zebrafish using CRISPR/Cas genome editing, and examined the phenotypes of loss-of-function mutants. F0 mosaic mutations and germline transmission were confirmed via targeted insertions and/or deletions in the valopa or valopb gene in F1 mutants. Based on in silico analysis, frameshift mutations converted VAL-opsin proteins to non-functional truncated forms with pre-mature stop codons. Most F1 eggs or embryos from F0 female valopa/b mutants showed either no or only partial chorion elevation, and the eggs or embryos died within 26 hour-post-fertilization. However, most F1 embryos from F0 male valopa mutant developed but hatched late compared to wild-type embryos, which hatched at 4 day-post-fertilization. Late-hatched F1 offspring included wild-type and mutants, indicating the parental effects of valop knockout. This study shows valop gene knockout affects chorion formation and embryonic hatching in the zebrafish.
    Matched MeSH terms: CRISPR-Cas Systems/genetics*
  9. Therapeutic potential of combined viral transduction and CRISPR/Cas9 gene editing in treating neurodegenerative diseases
    Kuruvilla J, Sasmita AO, Ling APK
    Neurol Sci, 2018 Nov;39(11):1827-1835.
    PMID: 30076486 DOI: 10.1007/s10072-018-3521-0
    BACKGROUND AND PURPOSE: The central nervous system (CNS) faces unique difficulties in attaining permanent therapy for neurodegenerative disorder (ND). Genomic level forms of therapy have garnered interest in the recent decade, with the novel CRISPR/Cas9 gene editing tool continuing to be explored due to its efficiency, safety, and adaptability to varying conditions. With the aid of viral vectors as transport vectors, the gene editing tool has produced promising in vitro and in vivo findings in study models. Thus, this review focuses on the recent advancements and update of CRISPR/Cas9 to combat neurodegenerative diseases.

    METHODS: Articles detailing potential applications of CRISPR/Cas9 in neurodegenerative settings were retrieved from PubMed and Google Scholar with the keywords "CRISPR," "gene editing," and "neurodegenerative diseases." Relevant information was collected and critically reviewed.

    RESULTS: The utility of CRISPR/Cas9 coupled with viral transduction ranges from the disruption of amyloid precursor protein (APP) production at a genomic level in Alzheimer's disease (AD) to the deletion of varying exon portions of the Dmd gene in Duchenne muscular dystrophy (DMD) which would increase dystrophin expression. This usage of CRISPR/Cas9 also extends to experimentally ameliorate the neurodegenerative effects caused by viral infections.

    CONCLUSION: The CRISPR/Cas9 gene editing tool is a powerful arsenal in the field of gene therapy and molecular medicine; hence, more research should be called to focus on the ample potential this tool has to offer in the field of neurodegenerative diseases.

    Matched MeSH terms: CRISPR-Cas Systems
  10. Identification of biomarkers and genetic approaches toward chronic obstructive pulmonary disease
    Wadhwa R, Aggarwal T, Malyla V, Kumar N, Gupta G, Chellappan DK, et al.
    J Cell Physiol, 2019 08;234(10):16703-16723.
    PMID: 30912142 DOI: 10.1002/jcp.28482
    Chronic obstructive pulmonary disease accounts as the leading cause of mortality worldwide prominently affected by genetic and environmental factors. The disease is characterized by persistent coughing, breathlessness airways inflammation followed by a decrease in forced expiratory volume1 and exacerbations, which affect the quality of life. Determination of genetic, epigenetic, and oxidant biomarkers to evaluate the progression of disease has proved complicated and challenging. Approaches including exome sequencing, genome-wide association studies, linkage studies, and inheritance and segregation studies played a crucial role in the identification of genes, their pathways and variation in genes. This review highlights multiple approaches for biomarker and gene identification, which can be used for differential diagnosis along with the genome editing tools to study genes associated with the development of disease and models their function. Further, we have discussed the approaches to rectify the abnormal gene functioning of respiratory tissues and various novel gene editing techniques like Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9).
    Matched MeSH terms: CRISPR-Cas Systems
  11. Fulltext Genome sequencing and analysis of Salmonella enterica subsp. enterica serovar Stanley UPM 517: Insights on its virulence-associated elements and their potentials as vaccine candidates
    Ashari KS, Roslan NS, Omar AR, Bejo MH, Ideris A, Mat Isa N
    PeerJ, 2019;7:e6948.
    PMID: 31293824 DOI: 10.7717/peerj.6948
    Salmonella enterica subsp. enterica serovar Stanley (S. Stanley) is a pathogen that contaminates food, and is related to Salmonella outbreaks in a variety of hosts such as humans and farm animals through products like dairy items and vegetables. Despite the fact that several vaccines of Salmonella strains had been constructed, none of them were developed according to serovar Stanley up to this day. This study presents results of genome sequencing and analysis on our S. Stanley UPM 517 strain taken from fecal swabs of 21-day-old healthy commercial chickens in Perak, Malaysia and used Salmonella enterica subsp. enterica serovar Typhimurium LT2 (S. Typhimurium LT2) as a reference to be compared with. First, sequencing and assembling of the Salmonella Stanley UPM 517 genome into a contiguous form were done. The work was then continued with scaffolding and gap filling. Annotation and alignment of the draft genome was performed with S. Typhimurium LT2. The other elements of virulence estimated in this study included Salmonella pathogenicity islands, resistance genes, prophages, virulence factors, plasmid regions, restriction-modification sites and the CRISPR-Cas system. The S. Stanley UPM 517 draft genome had a length of 4,736,817 bp with 4,730 coding sequence and 58 RNAs. It was discovered via genomic analysis on this strain that there were antimicrobial resistance properties toward a wide variety of antibiotics. Tcf and ste, the two fimbrial virulence clusters related with human and broiler intestinal colonizations which were not found in S. Typhimurium LT2, were atypically discovered in the S. Stanley UPM 517 genome. These clusters are involved in the intestinal colonization of human and broilers, respectively. There were seven Salmonella pathogenicity islands (SPIs) within the draft genome, which contained the virulence factors associated with Salmonella infection (except SPI-14). Five intact prophage regions, mostly comprising of the protein encoding Gifsy-1, Fels-1, RE-2010 and SEN34 prophages, were also encoded in the draft genome. Also identified were Type I-III restriction-modification sites and the CRISPR-Cas system of the Type I-E subtype. As this strain exhibited resistance toward numerous antibiotics, we distinguished several genes that had the potential for removal in the construction of a possible vaccine candidate to restrain and lessen the pervasiveness of salmonellosis and to function as an alternative to antibiotics.
    Matched MeSH terms: CRISPR-Cas Systems
  12. Fulltext Electrosprayed Alginate Nanoparticles as CRISPR Plasmid DNA Delivery Carrier: Preparation, Optimization, and Characterization
    Alallam B, Altahhan S, Taher M, Mohd Nasir MH, Doolaanea AA
    Pharmaceuticals (Basel), 2020 Jul 22;13(8).
    PMID: 32707857 DOI: 10.3390/ph13080158
    Therapeutic gene editing is becoming more feasible with the emergence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system. However, the successful implementation of CRISPR/Cas9-based therapeutics requires a safe and efficient in vivo delivery of the CRISPR components, which remains challenging. This study presents successful preparation, optimization, and characterization of alginate nanoparticles (ALG NPs), loaded with two CRISPR plasmids, using electrospray technique. The aim of this delivery system is to edit a target gene in another plasmid (green fluorescent protein (GFP)). The effect of formulation and process variables were evaluated. CRISPR ALG NPs showed mean size and zeta potential of 228 nm and -4.42 mV, respectively. Over 99.0% encapsulation efficiency was achieved while preserving payload integrity. The presence of CRISPR plasmids in the ALG NPs was confirmed by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy. The tests revealed that the nanoparticles were cytocompatible and successfully introduced the Cas9 transgene in HepG2 cells. Nanoparticles-transfected HepG2 was able to edit its target plasmid by introducing double-strand break (DSB) in GFP gene, indicating the bioactivity of CRISPR plasmids encapsulated in alginate nanoparticles. This suggests that this method is suitable for biomedical application in vitro or ex vivo. Future investigation of theses nanoparticles might result in nanocarrier suitable for in vivo delivery of CRISPR/Cas9 system.
    Matched MeSH terms: CRISPR-Cas Systems
  13. Fulltext Genome-wide identification of Aedes albopictus long noncoding RNAs and their association with dengue and Zika virus infection
    Azlan A, Obeidat SM, Theva Das K, Yunus MA, Azzam G
    PLoS Negl Trop Dis, 2021 01;15(1):e0008351.
    PMID: 33481791 DOI: 10.1371/journal.pntd.0008351
    The Asian tiger mosquito, Aedes albopictus (Ae. albopictus), is an important vector that transmits arboviruses such as dengue (DENV), Zika (ZIKV) and Chikungunya virus (CHIKV). Long noncoding RNAs (lncRNAs) are known to regulate various biological processes. Knowledge on Ae. albopictus lncRNAs and their functional role in virus-host interactions are still limited. Here, we identified and characterized the lncRNAs in the genome of an arbovirus vector, Ae. albopictus, and evaluated their potential involvement in DENV and ZIKV infection. We used 148 public datasets, and identified a total of 10, 867 novel lncRNA transcripts, of which 5,809, 4,139, and 919 were intergenic, intronic and antisense respectively. The Ae. albopictus lncRNAs shared many characteristics with other species such as short length, low GC content, and low sequence conservation. RNA-sequencing of Ae. albopictus cells infected with DENV and ZIKV showed that the expression of lncRNAs was altered upon virus infection. Target prediction analysis revealed that Ae. albopictus lncRNAs may regulate the expression of genes involved in immunity and other metabolic and cellular processes. To verify the role of lncRNAs in virus infection, we generated mutations in lncRNA loci using CRISPR-Cas9, and discovered that two lncRNA loci mutations, namely XLOC_029733 (novel lncRNA transcript id: lncRNA_27639.2) and LOC115270134 (known lncRNA transcript id: XR_003899061.1) resulted in enhancement of DENV and ZIKV replication. The results presented here provide an important foundation for future studies of lncRNAs and their relationship with virus infection in Ae. albopictus.
    Matched MeSH terms: CRISPR-Cas Systems
  14. Genoproteomics-assisted improvement of Andrographis paniculata: toward a promising molecular and conventional breeding platform for autogamous plants affecting the pharmaceutical industry
    Valdiani A, Talei D, Lattoo SK, Ortiz R, Rasmussen SK, Batley J, et al.
    Crit Rev Biotechnol, 2017 Sep;37(6):803-816.
    PMID: 28049346 DOI: 10.1080/07388551.2016.1260525
    Andrographis paniculata (Burm. f.) Wall. ex Nees. (AP) is a hermaphroditic, self-compatible, and habitual inbreeding plant. Its main bioactive component is andrographolide, which is capable of inducing autophagic cell death in some human cancer cells and helps fight HIV/AIDS. Increasing the andrographolide content by investigating the genetic mechanisms controlling its biosynthesis in order to improve and develop high-yielding cultivars are the main breeding targets for AP. However, there might exist some limitations or barriers for crossability within AP accessions. Recently, this problem was addressed in AP by using a combination of crossbreeding and biotechnology-aided genetic methods. This review emphasizes that development of a breeding platform in a hard-to-breed plant, such as AP, requires the involvement of a broad range of methods from classical genetics to molecular breeding. To this end, a phenological stage (for example, flowering and stigma development) can be simplified to a quantitative morphological trait (for example, bud or stigma length) to be used as an index to express the highest level of receptivity in order to manage outcrossing. The outcomes of the basic crossability research can be then employed in diallel mating and crossbreeding. This review explains how genomic data could produce useful information regarding genetic distance and its influence on the crossability of AP accessions. Our review indicates that co-dominant DNA markers, such as microsatellites, are also capable of resolving the evolutionary pathway and cryptic features of plant populations and such information can be used to select the best breeding strategy. This review also highlights the importance of proteomic analysis as a breeding tool. In this regard, protein diversification, as well as the impact of normal and stress-responsive proteins on morphometric and physiological behaviors, could be used in breeding programs. These findings have immense potential for improving plant production and, therefore, can be regarded as prospective breeding platforms for medicinal plants that have an autogamous mode of reproduction. Finally, this review suggests that novel site-directed genome editing approaches such as TALENs (Transcription Activator-Like Effector Nucleases) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein-9 nuclease) systems together with other new plant breeding technologies (NPBT) should simultaneously be taken into consideration for improvement of pharmaceutical plants.
    Matched MeSH terms: CRISPR-Cas Systems
  15. Fulltext CRISPR Gene-Editing Models Geared Toward Therapy for Hereditary and Developmental Neurological Disorders
    Wong PK, Cheah FC, Syafruddin SE, Mohtar MA, Azmi N, Ng PY, et al.
    Front Pediatr, 2021;9:592571.
    PMID: 33791256 DOI: 10.3389/fped.2021.592571
    Hereditary or developmental neurological disorders (HNDs or DNDs) affect the quality of life and contribute to the high mortality rates among neonates. Most HNDs are incurable, and the search for new and effective treatments is hampered by challenges peculiar to the human brain, which is guarded by the near-impervious blood-brain barrier. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR), a gene-editing tool repurposed from bacterial defense systems against viruses, has been touted by some as a panacea for genetic diseases. CRISPR has expedited the research into HNDs, enabling the generation of in vitro and in vivo models to simulate the changes in human physiology caused by genetic variation. In this review, we describe the basic principles and workings of CRISPR and the modifications that have been made to broaden its applications. Then, we review important CRISPR-based studies that have opened new doors to the treatment of HNDs such as fragile X syndrome and Down syndrome. We also discuss how CRISPR can be used to generate research models to examine the effects of genetic variation and caffeine therapy on the developing brain. Several drawbacks of CRISPR may preclude its use at the clinics, particularly the vulnerability of neuronal cells to the adverse effect of gene editing, and the inefficiency of CRISPR delivery into the brain. In concluding the review, we offer some suggestions for enhancing the gene-editing efficacy of CRISPR and how it may be morphed into safe and effective therapy for HNDs and other brain disorders.
    Matched MeSH terms: CRISPR-Cas Systems
  16. Fulltext Small Interfering RNA (siRNA) and Clustered regularly interspaced short palindromic repeats (CRISPR): emerging molecular tools for genetic manipulation
    Simon I. Okekpa, Rabiatul Basria S.M.N. Mydin, Munirah Mohd Nor, Emmanuel Jairaj Moses
    Malaysian Journal of Medicine and Health Sciences, 2020;16(3):300-308.
    MyJurnal
    Gene manipulation tools have transformed biomedical research and improved the possibilities of their uses for therapeutic purposes. These tools have aided effective genomic modification in many organisms and have been successfully applied in biomedical engineering, biotechnology and biomedicine. They also shown a potential for therapeutic applications to alleviate genetic and non-genetic diseases. Small interfering RNA (siRNA) and clustered regularly inter-spaced short-palindromic repeat/associated-protein system (CRISPR/Cas) are two of the tools applied in genetic manipulation. This review aims to evaluate the molecular influence of siRNA and CRISPR/Cas as novel tools for genetic manipulations. This review discusses the molecular mechanism of siRNA and CRISPR/Cas, and the advantages and disadvantages of siRNA and CRISPR/Cas. This review also presents comparison between siRNA and CRISPR/Cas as potential tools for gene therapy. siRNA therapeutic applications occur through protein knockout with- out causing damage to cells. siRNA knocks down gene expression at the mRNA level, whereas CRISPR/Cas knocks out gene permanently at the DNA level. Inconclusion, gene manipulation tools have potential for applications that improve therapeutic strategies and plant-derived products, but ethical standards must be established before the clin- ical application of gene editing.

    Matched MeSH terms: CRISPR-Cas Systems
  17. Plant-pathogen interactions: toward development of next-generation disease-resistant plants
    Nejat N, Rookes J, Mantri NL, Cahill DM
    Crit Rev Biotechnol, 2017 Mar;37(2):229-237.
    PMID: 26796880 DOI: 10.3109/07388551.2015.1134437
    Briskly evolving phytopathogens are dire threats to our food supplies and threaten global food security. From the recent advances made toward high-throughput sequencing technologies, understanding of pathogenesis and effector biology, and plant innate immunity, translation of these means into new control tools is being introduced to develop durable disease resistance. Effectoromics as a powerful genetic tool for uncovering effector-target genes, both susceptibility genes and executor resistance genes in effector-assisted breeding, open up new avenues to improve resistance. TALENs (Transcription Activator-Like Effector Nucleases), engineered nucleases and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems are breakthrough and powerful techniques for genome editing, providing efficient mechanisms for targeted crop protection strategies in disease resistance programs. In this review, major advances in plant disease management to confer durable disease resistance and novel strategies for boosting plant innate immunity are highlighted.
    Matched MeSH terms: CRISPR-Cas Systems
  18. Fulltext A novel Pseudorabies virus vaccine developed using HDR-CRISPR/Cas9 induces strong humoral and cellular immune response in mice
    Luo C, Wang Q, Guo R, Zhang J, Zhang J, Zhang R, et al.
    Virus Res, 2022 Dec;322:198937.
    PMID: 36174845 DOI: 10.1016/j.virusres.2022.198937
    Outbreaks of Pseudorabies (PR) by numerous highly virulent and antigenic variant Pseudorabies virus (PRV) strains have been causing severe economic losses to the pig industry in China since 2011. However, current commercial vaccines are often unable to induce thorough protective immunity. In this study, a TK/gI/gE deleted recombinant PRV expressing GM-CSF was developed by using the HDR-CRISPR/Cas9 system. Here, a four-sgRNA along with the Cas9D10A targeting system was utilized for TK/gI/gE gene deletion and GM-CSF insertion. Our study showed that the four-sgRNA targeting system appeared to have higher knock-in efficiency for PRVs editing. The replication of the recombinant PRVs were slightly lower than that of the parental strain, but they appeared to have similar properties in terms of growth curves and plaque morphology. The mice vaccinated with the recombinant PRV expressing GM-CSF via intramuscular injection showed no obvious clinical symptoms, milder pathological lesions, and were completely protected against wild-type PRV challenge. When compared to the triple gene-deleted PRV, the gB antibodies and neutralizing antibody titers were improved and the immunized mice appeared to have lower viral load and higher mRNA levels of IL-2, IL-4, IL-6, and IFN-γ in spleens. Our study offers a novel approach for recombinant PRV construction, and the triple gene-deleted PRV expressing GM-CSF could serve as a promising vaccine candidate for PR control.
    Matched MeSH terms: CRISPR-Cas Systems
  19. Comparative genomic and phylogenetic analysis of a toxigenic clinical isolate of Corynebacterium diphtheriae strain B-D-16-78 from Malaysia
    Hong KW, Asmah Hani AW, Nurul Aina Murni CA, Pusparani RR, Chong CK, Verasahib K, et al.
    Infect Genet Evol, 2017 Oct;54:263-270.
    PMID: 28711373 DOI: 10.1016/j.meegid.2017.07.015
    In this study, we report the comparative genomics and phylogenetic analysis of Corynebacterium diphtheriae strain B-D-16-78 that was isolated from a clinical specimen in 2016. The complete genome of C. diphtheriae strain B-D-16-78 was sequenced using PacBio Single Molecule, Real-Time sequencing technology and consists of a 2,474,151-bp circular chromosome with an average GC content of 53.56%. The core genome of C. diphtheriae was also deduced from a total of 74 strains with complete or draft genome sequences and the core genome-based phylogenetic analysis revealed close genetic relationship among strains that shared the same MLST allelic profile. In the context of CRISPR-Cas system, which confers adaptive immunity against re-invading DNA, 73 out of 86 spacer sequences were found to be unique to Malaysian strains which harboured only type-II-C and/or type-I-E-a systems. A total of 48 tox genes which code for the diphtheria toxin were retrieved from the 74 genomes and with the exception of one truncated gene, only nucleotide substitutions were detected when compared to the tox gene sequence of PW8. More than half were synonymous substitution and only two were nonsynonymous substitutions whereby H24Y was predicted to have a damaging effect on the protein function whilst T262V was predicted to be tolerated. Both toxigenic and non-toxigenic toxin-gene bearing strains have been isolated in Malaysia but the repeated isolation of toxigenic strains with the same MLST profile suggests the possibility of some of these strains may be circulating in the population. Hence, efforts to increase herd immunity should be continued and supported by an effective monitoring and surveillance system to track, manage and control outbreak of cases.
    Matched MeSH terms: CRISPR-Cas Systems
  20. Fulltext Functional annotation of the 2q35 breast cancer risk locus implicates a structural variant in influencing activity of a long-range enhancer element
    Baxter JS, Johnson N, Tomczyk K, Gillespie A, Maguire S, Brough R, et al.
    Am J Hum Genet, 2021 Jul 01;108(7):1190-1203.
    PMID: 34146516 DOI: 10.1016/j.ajhg.2021.05.013
    A combination of genetic and functional approaches has identified three independent breast cancer risk loci at 2q35. A recent fine-scale mapping analysis to refine these associations resulted in 1 (signal 1), 5 (signal 2), and 42 (signal 3) credible causal variants at these loci. We used publicly available in silico DNase I and ChIP-seq data with in vitro reporter gene and CRISPR assays to annotate signals 2 and 3. We identified putative regulatory elements that enhanced cell-type-specific transcription from the IGFBP5 promoter at both signals (30- to 40-fold increased expression by the putative regulatory element at signal 2, 2- to 3-fold by the putative regulatory element at signal 3). We further identified one of the five credible causal variants at signal 2, a 1.4 kb deletion (esv3594306), as the likely causal variant; the deletion allele of this variant was associated with an average additional increase in IGFBP5 expression of 1.3-fold (MCF-7) and 2.2-fold (T-47D). We propose a model in which the deletion allele of esv3594306 juxtaposes two transcription factor binding regions (annotated by estrogen receptor alpha ChIP-seq peaks) to generate a single extended regulatory element. This regulatory element increases cell-type-specific expression of the tumor suppressor gene IGFBP5 and, thereby, reduces risk of estrogen receptor-positive breast cancer (odds ratio = 0.77, 95% CI 0.74-0.81, p = 3.1 × 10-31).
    Matched MeSH terms: CRISPR-Cas Systems
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