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The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated sequence (CRISPR/Cas) system is a cutting-edge genome-editing tool employed to explore the functions of normal and disease-related genes. The CRISPR/Cas system has a remarkable diversity in the composition and architecture of genomic loci and Cas protein sequences. Owing to its excellent efficiency and specificity, this system adds an outstanding dimension to biomedical research on genetic manipulation of eukaryotic cells. However, safe, efficient, and specific delivery of this system to target cells and tissues and their off-target effects are considered critical bottlenecks for the therapeutic applications. Recently discovered anti-CRISPR proteins (Acr) play a significant role in limiting the effects of this system. Acrs are relatively small proteins that are highly specific to CRISPR variants and exhibit remarkable structural diversity. The in silico approaches, crystallography, and cryo-electron microscopy play significant roles in elucidating the mechanisms of action of Acrs. Acrs block the CRISPR/Cas system mainly by employing four mechanisms: CRISPR/Cas complex assembly interruption, target-binding interference, target cleavage prevention, and degradation of cyclic oligonucleotide signaling molecules. Engineered CRISPR/Cas systems are frequently used in gene therapy, diagnostics, and functional genomics. Understanding the molecular mechanisms underlying Acr action may help in the safe and effective use of CRISPR/Cas tools for genetic modification, particularly in the context of medicine. Thus, attempts to regulate prokaryotic CRISPR/Cas surveillance complexes will advance the development of antimicrobial drugs and treatment of human diseases. In this review, recent updates on CRISPR/Cas systems, especially CRISPR/Cas9 and Acrs, and their novel mechanistic insights are elaborated. In addition, the role of Acrs in the novel applications of CRISPP/Cas biotechnology for precise genome editing and other applications is discussed., Competing Interests: The authors declare no competing interests in this work., (© 2024 Allemailem et al.)

The genome editing approach by clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) is a revolutionary advancement in genetic engineering. Owing to its simple design and powerful genome-editing capability, it offers a promising strategy for the treatment of different infectious, metabolic, and genetic diseases. The crystal structure of Streptococcus pyogenes Cas9 (SpCas9) in complex with sgRNA and its target DNA at 2.5 Å resolution reveals a groove accommodating sgRNA:DNA heteroduplex within a bilobate architecture with target recognition (REC) and nuclease (NUC) domains. The presence of a PAM is significantly required for target recognition, R-loop formation, and strand scission. Recently, the spatiotemporal control of CRISPR/Cas9 genome editing has been considerably improved by genetic, chemical, and physical regulatory strategies. The use of genetic modifiers anti-CRISPR proteins, cell-specific promoters, and histone acetyl transferases has uplifted the application of CRISPR/Cas9 as a future-generation genome editing tool. In addition, interventions by chemical control, small-molecule activators, oligonucleotide conjugates and bioresponsive delivery carriers have improved its application in other areas of biological fields. Furthermore, the intermediation of physical control by using heat-, light-, magnetism-, and ultrasound-responsive elements attached to this molecular tool has revolutionized genome editing further. These strategies significantly reduce CRISPR/Cas9's undesirable off-target effects. However, other undesirable effects still offer some challenges for comprehensive clinical translation using this genome-editing approach. In this review, we summarize recent advances in CRISPR/Cas9 structure, mechanistic action, and the role of small-molecule activators, inhibitors, promoters, and physical approaches. Finally, off-target measurement approaches, challenges, future prospects, and clinical applications are discussed., Competing Interests: The authors report no conflicts of interest in this work., (© 2024 Allemailem et al.)

The recent developments in the study of clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) system have revolutionized the art of genome-editing and its applications for cellular differentiation and immune response behavior. This technology has further helped in understanding the mysteries of cancer progression and possible designing of novel antitumor immunotherapies. CRISPR/Cas9-based genome-editing is now often used to engineer universal T-cells, equipped with recombinant T-cell receptor (TCR) or chimeric antigen receptor (CAR). In addition, this technology is used in cytokine stimulation, antibody designing, natural killer (NK) cell transfer, and to overcome immune checkpoints. The innovative potential of CRISPR/Cas9 in preparing the building blocks of adoptive cell transfer (ACT) immunotherapy has opened a new window of antitumor immunotherapy and some of them have gained FDA approval. The manipulation of immunogenetic regulators has opened a new interface for designing, implementation and interpretation of CRISPR/Cas9-based screening in immuno-oncology. Several cancers like lymphoma, melanoma, lung, and liver malignancies have been treated with this strategy, once thought to be impossible. The safe and efficient delivery of CRISPR/Cas9 system within the immune cells for the genome-editing strategy is a challenging task which needs to be sorted out for efficient immunotherapy. Several targeting approaches like virus-mediated, electroporation, microinjection and nanoformulation-based methods have been used, but each procedure offers some limitations. Here, we elaborate the recent updates of cancer management through immunotherapy in partnership with CRISPR/Cas9 technology. Further, some innovative methods of targeting this genome-editing system within the immune system cells for reprogramming them, as a novel strategy of anticancer immunotherapy is elaborated. In addition, future prospects and clinical trials are also discussed., Competing Interests: The authors declare no conflicts of interest in this work., (© 2023 Allemailem et al.)

The smart strategy of cancer cells to bypass the caspase-dependent apoptotic pathway has led to the discovery of novel anti-cancer approaches including the targeting of lysosomes. Recent discoveries observed that lysosomes perform far beyond just recycling of cellular waste, as these organelles are metabolically very active and mediate several signalling pathways to sense the cellular metabolic status. These organelles also play a significant role in mediating the immune system functions. Thus, direct or indirect lysosome-targeting with different drugs can be considered a novel therapeutic approach in different disease including cancer. Recently, some anticancer lysosomotropic drugs (eg, nortriptyline, siramesine, desipramine) and their nanoformulations have been engineered to specifically accumulate within these organelles. These drugs can enhance lysosome membrane permeabilization (LMP) or disrupt the activity of resident enzymes and protein complexes, like v-ATPase and mTORC1. Other anticancer drugs like doxorubicin, quinacrine, chloroquine and DQ661 have also been used which act through multi-target points. In addition, autophagy inhibitors, ferroptosis inducers and fluorescent probes have also been used as novel theranostic agents. Several lysosome-specific drug nanoformulations like mixed charge and peptide conjugated gold nanoparticles (AuNPs), Au-ZnO hybrid NPs, TPP-PEG-biotin NPs, octadecyl-rhodamine-B and cationic liposomes, etc. have been synthesized by diverse methods. These nanoformulations can target cathepsins, glucose-regulated protein 78, or other lysosome specific proteins in different cancers. The specific targeting of cancer cell lysosomes with drug nanoformulations is quite recent and faces tremendous challenges like toxicity concerns to normal tissues, which may be resolved in future research. The anticancer applications of these nanoformulations have led them up to various stages of clinical trials. Here in this review article, we present the recent updates about the lysosome ultrastructure, its cross-talk with other organelles, and the novel strategies of targeting this organelle in tumor cells as a recent innovative approach of cancer management., Competing Interests: The authors declare no conflicts of interest for this work., (© 2021 Allemailem et al.)

Any variation in normal cellular function results in mitochondrial dysregulation that occurs in several diseases, including cancer. Such processes as oxidative stress, metabolism, signaling, and biogenesis play significant roles in cancer initiation and progression. Due to their central role in cellular metabolism, mitochondria are favorable therapeutic targets for the prevention and treatment of conditions like neurodegenerative diseases, diabetes, and cancer. Subcellular mitochondria-specific theranostic nanoformulations for simultaneous targeting, drug delivery, and imaging of these organelles are of immense interest in cancer therapy. It is a challenging task to cross multiple barriers to target mitochondria in diseased cells. To overcome these multiple barriers, several mitochondriotropic nanoformulations have been engineered for the transportation of mitochondria-specific drugs. These nanoformulations include liposomes, dendrimers, carbon nanotubes, polymeric nanoparticles (NPs), and inorganic NPs. These nanoformulations are made mitochondriotropic by conjugating them with moieties like dequalinium, Mito-Porter, triphenylphosphonium, and Mitochondria-penetrating peptides. Most of these nanoformulations are meticulously tailored to control their size, charge, shape, mitochondriotropic drug loading, and specific cell-membrane interactions. Recently, some novel mitochondria-selective antitumor compounds known as mitocans have shown high toxicity against cancer cells. These selective compounds form vicious oxidative stress and reactive oxygen species cycles within cancer cells and ultimately push them to cell death. Nanoformulations approved by the FDA and EMA for clinical applications in cancer patients include Doxil, NK105, and Abraxane. The novel use of these NPs still faces tremendous challenges and an immense amount of research is needed to understand the proper mechanisms of cancer progression and control by these NPs. Here in this review, we summarize current advancements and novel strategies of delivering different anticancer therapeutic agents to mitochondria with the help of various nanoformulations., Competing Interests: The authors declare no conflicts of interest., (© 2021 S Allemailem et al.)

Purpose: The study aimed to find an effective method for fungal-mediated synthesis of zinc oxide nanoparticles using endophytic fungal extracts and to evaluate the efficiency of synthesized ZnO NPs as antimicrobial and anticancerous agents., Methods: Zinc oxide nanoparticles (ZnO NPs) were produced from zinc nitrate hexahydrate with fungal filtrate by the combustion method. The spectroscopy and microscopy techniques, such as ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy (FT-IR), powder X-ray diffraction (PXRD), scanning electron microscopy (SEM) with energy-dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS), and transmission electron microscopy (TEM) with selected area electron diffraction (SAED), were used to characterize the obtained product. Antibacterial activity on Gram-positive ( Staphylococcus aureus and Bacillus cereus ) and Gram-negative ( Pseudomonas aeruginosa and Escherichia coli ) samples was tested by broth microplate dilution technique. ZnO NPs antifungal activity was determined against plant pathogenic and regular contaminating fungi using the food-poison method. The anticancerous assay of the synthesized ZnO NPs was also investigated by cell uptake, MTT assay, and apoptosis assay., Results: The fungal synthesized ZnO NPs were pure, mainly hexagonal in shape and size range of 34-55 nm. The biosynthesized ZnO NPs could proficiently inhibit both Gram-positive and Gram-negative bacteria. ZnO NPs synthesized from fungal extract exhibited antifungal activity in a dose-dependent manner with a high percentage of mycelial inhibition. The cell uptake analysis of ZnO NPs suggests that a significant amount of ZnO NPs (1 μg/mL) was internalized without disturbing cancer cells' morphology. As a result, the synthesized ZnO NPs showed significant anticancer activity against cancer cells at 1 μg/mL concentration., Conclusion: This fungus-mediated synthesis of ZnO NPs is a simple, eco-friendly, and non-toxic method. Our results show that the synthesized ZnO NPs are an excellent novel antimicrobial and anticancer agent. Further studies are required to understand the mechanism of the antimicrobial, anticancerous action of ZnO NPs and their possible genotoxicity., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Sumanth et al.)

Background: Fungal infections are becoming more prevalent and threatening because of the continuous emergence of azole-resistant fungal infections. The present study was aimed to assess the activity of free Methylglyoxal (MG) or MG-conjugated chitosan nanoparticles (MGCN) against fluconazole-resistant Candida albicans ., Materials and Methods: A novel formulation of MGCN was prepared and characterized to determine their size, shape and polydispersity index. Moreover, the efficacy of fluconazole or MG or MGCN was determined against intracellular C. albicans in macrophages and the systematic candidiasis in a murine model. The safety of MG or MGCN was tested in mice by analyzing the levels of hepatic and renal toxicity parameters., Results: Candida albicans did not respond to fluconazole, even at the highest dose of 20 mg/kg, whereas MG and MGCN effectively eliminated C. albicans from the macrophages and infected mice. Mice in the group treated with MGCN at a dose of 10 mg/kg exhibited a 90% survival rate and showed the lowest fungal load in the kidney, whereas the mice treated with free MG at the same dose exhibited 50% survival rate. Moreover, the administration of MG or MGCN did not induce any liver and kidney toxicity in the treated mice., Conclusion: The findings of the present work suggest that MGCN may be proved a promising therapeutic formulation to treat azole-resistant C. albicans infections., Competing Interests: The authors report no conflicts of interest in this work., (© 2020 Khan et al.)

Purpose: The pathogenicity in Candida spp was attributed by several virulence factors such as production of tissue damaging extracellular enzymes, germ tube formation, hyphal morphogenesis and establishment of drug resistant biofilm. The objective of present study was to investigate the effects of silver nanoparticles (AgNPs) on growth, cell morphology and key virulence attributes of Candida species. Methods: AgNPs were synthesized by the using seed extract of Syzygium cumini (Sc), and were characterized by UV-Vis spectrophotometer, Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray (EDX), and transmission electron microscopy (TEM). ScAgNPs were used to evaluate their antifungal and antibacterial activity as well as their potent inhibitory effects on germ tube and biofilm formation and extracellular enzymes viz. phospholipases, proteinases, lipases and hemolysin secreted by Candida spp. Results: The MICs values of ScAgNPs were ranged from 0.125-0.250 mg/ml, whereas the MBCs and MFCs were 0.250 and 0.500 mg/ml, respectively. ScAgNPs significantly inhibit the production of phospholipases by 82.2, 75.7, 78.7, 62.5, and 65.8%; proteinases by 82.0, 72.0, 77.5, 67.0, and 83.7%; lipase by 69.4, 58.8, 60.0, 42.9, and 65.0%; and hemolysin by 62.8, 69.7, 67.2, 73.1, and 70.2% in C. albicans , C. tropicalis , C. dubliniensis , C. parapsilosis and C. krusei , respectively, at 500 μg/ml. ScAgNPs inhibit germ tube formation in C. albicans up to 97.1% at 0.25 mg/ml. LIVE/DEAD staining results showed that ScAgNPs almost completely inhibit biofilm formation in C. albicans. TEM analysis shows that ScAgNPs not only anchored onto the cell surface but also penetrated and accumulated in the cytoplasm that causes severe damage to the cell wall and cytoplasmic membrane. Conclusion: To summarize, the biosynthesized ScAgNPs strongly suppressed the multiplication, germ tube and biofilm formation and most importantly secretion of hydrolytic enzymes (viz. phospholipases, proteinases, lipases and hemolysin) by Candia spp. The present research work open several avenues of further study, such as to explore the molecular mechanism of inhibition of germ tubes and biofilm formation and suppression of production of various hydrolytic enzymes by Candida spp., Competing Interests: The authors declare that they have no conflicts of interest in this work.