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Recognizing CRISPR as the new age disease-modifying drug: Strategies to bioengineer CRISPR/Cas for direct in vivo delivery

Thevendran R, Maheswaran S

Biotechnol J, 2023 Sep;18(9):e2300077.
PMID: 37179485 DOI: 10.1002/biot.202300077

Clustered regularly interspaced short palindromic repeats (CRISPR) have established itself as a frontier technology in genetic engineering. Researchers have successfully used the CRISPR/Cas system as precise gene editing tools and have further expanded their scope beyond both imaging and diagnostic applications. The most prominent utility of CRISPR is its capacity for gene therapy, serving as the contemporary, disease-modifying drug at the genetic level of human medical disorders. Correcting these diseases using CRISPR-based gene editing has developed to the extent of preclinical trials and possible patient treatments. A major impediment in actualizing this is the complications associated with in vivo delivery of the CRISPR/Cas complex. Currently, only the viral vectors (e.g., lentivirus) and non-viral encapsulation (e.g., lipid particles, polymer-based, and gold nanoparticles) techniques have been extensively reviewed, neglecting the efficiency of direct delivery. However, the direct delivery of CRISPR/Cas for in vivo gene editing therapies is an intricate process with numerous drawbacks. Hence, this paper discusses in detail both the need and the strategies that can potentially improve the direct delivery aspects of CRISPR/Cas biomolecules for gene therapy of human diseases. Here, we focus on enhancing the molecular and functional features of the CRISPR/Cas system for targeted in vivo delivery such as on-site localization, internalization, reduced immunogenicity, and better in vivo stability. We additionally emphasize the CRISPR/Cas complex as a multifaceted, biomolecular vehicle for co-delivery with therapeutic agents in targeted disease treatments. The delivery formats of efficient CRISPR/Cas systems for human gene editing are also briefly elaborated.
Spiked nanorosette: A novel zinc oxide-gold textured nanostructure for impedance biosensing of target DNA signatures

Haarindraprasad RP, Thurga Devi N, Thevendran R, Maheswaran S

Biotechnol J, 2023 Aug;18(8):e2300092.
PMID: 37139895 DOI: 10.1002/biot.202300092

The creation of nanostructure is profound for the generation of nanobiosensors in several medical diagnosis. Here, we employed an aqueous hydrothermal route using Zinc-oxide (ZnO) and Gold (Au), which under optimal conditions formed an ultra-crystalline rose-like nanostructure textured with nanowires on the surface, coined as "spiked nanorosette." The spiked nanorosette structures was further characterized to possess crystallites of ZnO and Au grains with average sizes of 27.60 and 32.33 nm, respectively. The intensity for both ZnO (002) and Au (111) planes of the nanocomposite was inferred to be controlled by fine-tuning the percentage of Au nanoparticles doped in the ZnO/Au matrix, as referred by X-ray diffraction analysis. The formation of ZnO/Au-hybrid nanorosettes were additionally verified by the distinct corresponding peaks from photoluminescence and X-ray photoelectron spectroscopy, supported by electrical validations. The biorecognition properties of the spiked nanorosettes were also examined using custom targeted and non-target DNA sequences. The DNA targeting capabilities of the nanostructures were analyzed by Fourier Transform Infrared and electrochemical impedance spectroscopy. The fabricated nanowire-embedded nanorosette exhibited a detection limit at the lower picomolar range of 1 × 10-12 M, with high selectivity, stability and reproducibility and good linearity, under optimal conditions. Impedance-based techniques are more sensitive to the detection of nucleic acid molecule whereas this novel spiked nanorosette demonstrate promising attributes as excellent nanostructures for nanobiosensor developments and their potential future application for nucleic-acids or disease diagnostics.
Studying the cellular efficacy and tolerability of using CRISPR-gRNA ribonucleoprotein (RNP) complex for in-vitro knockdown of TRIB2 in acute myeloid leukaemia (AML) cells as preliminary clinical evaluations

Tan JYP, Thevendran R, Quek YS, Maheswaran S

Mol Biol Rep, 2025 Apr 09;52(1):379.
PMID: 40205014 DOI: 10.1007/s11033-025-10487-2

BACKGROUND: Acute Myeloid Leukemia (AML) is a complex, genetically driven cancer disease with several mutations that complicate therapy regimes. The Tribbles gene family, specifically the TRIB2 gene, has garnered substantial interest as a crucial oncogenic factor of AML progression. Studying cancer through gene expression studies and focusing on the primary oncogenes provide accurate information for future therapies. Here, CRISPR stands as the most exceptional tool used to explore both oncogene functionalities and therapeutic utilities. The ribonucleoprotein (RNP) mode of CRISPR formation and usage are preferred compared to plasmid-mediated CRISPR systems, however, it can lead to complications post-transfection to sensitive cellular entities such as human cancer cells in-vitro, transcribing to similar outcomes in-vivo as well. Therefore, this study describes the use of in-house designed CRISPR-RNP systems targeting the TRIB2 oncogene and evaluates their post-transfection cellular safety and efficacy aspects for future clinical applications.
METHODS: This study uses a designed guide RNA targeting the TRIB2 gene assembled via In-vitro synthesis. The gRNA with Cas9 protein leads to the formation of CRISPR-RNP structures, which target and cleave the TRIB2 gene. The assembled CRISPR-RNP system is transfected into target AML cell and control cell lines (i.e. HEK cells), and the subsequent gene cleavage and resulting changes to the AML cells in terms of cellular safety/tolerability and gene knockdown efficacy were studied via RT-qPCR, flow cytometry, and cell viability analysis.
RESULTS: The outcome demonstrates the well-tolerated transfection of the in-vitro assembled CRISPR RNP system with no signs of cellular toxicity and disruptions towards the AML cell's metabolic activities, promoting the safety aspects of CRISPR RNP post-transfection to human cells. The study further highlights the in-vitro efficacy of the CRISPR RNP in targeting the TRIB2 oncogene, where a statistically significant gene knockdown of more than 80% was detected via qPCR analysis of TRIB2 gene expression with minimal to no background effects from individual RNP components, equating to their targeted gene cleavage effects. In addition, the CRISPR TRIB2 gene knockdown also indicated the possibilities of induced AML cell death measured via flow cytometry markers, translating to favourable outcomes in eliminating cancerous growths.
CONCLUSIONS: This study research contributes to the refinement of the CRISPR-RNP strategy and preliminary evaluation for future clinical uses.
Prospects of Innovative Therapeutics in Combating the COVID-19 Pandemic

Mahendran TR, Cynthia B, Thevendran R, Maheswaran S

Mol Biotechnol, 2025 Jul;67(7):2598-2606.
PMID: 39085563 DOI: 10.1007/s12033-024-01240-4

The sudden global crisis of COVID-19, driven by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), demands swift containment measures due to its rapid spread and numerous problematic mutations, which complicate the establishment of herd immunity. With escalating fatalities across various nations no foreseeable end in sight, there is a pressing need to create swiftly deployable, rapid, cost-effective detection, and treatment methods. While various steps are taken to mitigate the transmission and severity of the disease, vaccination is proven throughout mankind history as the best method to acquire immunity and circumvent the spread of infectious diseases. Nonetheless, relying solely on vaccination might not be adequate to match the relentless viral mutations observed in emerging variants of SARS-CoV-2, including alterations to their RBD domain, acquisition of escape mutations, and potential resistance to antibody binding. Beyond the immune system activation achieved through vaccination, it is crucial to develop new medications or treatment methods to either impede the infection or enhance existing treatment modalities. This review emphasizes innovative treatment strategies that aim to directly disrupt the virus's ability to replicate and spread, which could play a role in ending the SARS-CoV-2 pandemic.