The current developments in engineering of Cas effector protein, optimizing guide RNA designs, developing advanced reporter probes, and utilizing chemical additives are summarized and their significant contribution to increasing the fidelity of editing and diagnostic sensitivity is highlighted.
Abstract
CRISPR‐Cas systems have far‐reaching implications for genome editing and molecular diagnostics, and are emerging as important tools in biomedical research and clinical practice. These systems enable precise genetic manipulation and highly sensitive nucleic acid detection, capabilities that are invaluable for therapeutic development and pathogen surveillance. Nevertheless, native CRISPR‐Cas systems possess inherent limitations that constrain their broader utility. These limitations in genome editing include insufficient editing efficiency, a restricted range of targets due to protospacer adjacent motif constraints and non‐negligible off‐target effects. In molecular diagnostics, the remaining problems include detecting low‐abundance targets, achieving high specificity, and ensuring effective compatibility with isothermal amplification. To address these limitations, researchers have proposed many enhancement strategies that mainly center around four aspects: engineering of Cas effector protein, optimizing guide RNA designs, developing advanced reporter probes, and utilizing chemical additives. This review summarizes the current developments in these areas and highlights their significant contribution to increasing the fidelity of editing and diagnostic sensitivity. In addition, we discuss future prospects for enhanced CRISPR‐Cas systems, focusing on their accelerating clinical translation and expanding biomedical applications.
CRISPR–Cas9 has revolutionised genome editing by enabling efficient and programmable modification of defined DNA sequences, with guide RNAs (gRNAs) serving as indispensable elements that direct Cas9 to specific genomic loci. Initially regarded as auxiliary components, gRNAs are now recognized as critical determinants of editing efficiency and specificity and have attracted growing attention as independent targets for engineering. Chemical modification, sequence optimisation, and structural alteration of gRNAs have been shown to enhance on‐target activity, suppress off‐target effects and cytotoxicity, and even achieve allele‐selective precision editing in a programmable manner. Moreover, advances in artificial intelligence and machine learning have markedly improved the predictive accuracy of gRNA design through large‐scale data analysis. Despite rapid progress, a consolidated review that integrates chemical, structural, and computational advances in gRNA engineering and highlights their translational potential for therapeutic genome editing has been lacking. This review uniquely addresses that gap by presenting an integrated framework that connects molecular design principles with clinical applicability.
Masaki Kawamata, S. Niwa, Atsushi Suzuki· Chemical Biology and Drug De...· 0 citations
CRISPR has emerged as a next-generation gene-editing tool with the potential to target the molecular pathways associated with ageing and related disorders. It functions through RNA-guided Cas nucleases, directing DNA cleavage and utilizing the native DNA repair machinery for genetic manipulations. Advances in CRISPR technology have significantly enhanced the precision and flexibility of techniques for genome editing. The enzyme Cas9's ability to cut DNA at exact site has revolutionized genome editing by enabling accurate modifications within living eukaryotic cells. This review critically examines recent developments in CRISPR-based technologies, including Cas9, Cas12, base editing, prime editing, and CRISPR-mediated gene regulation. It highlights their rising applications in ageing research, with more emphasis on neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. The review also discusses the major pharmacological and translational challenges that currently limit clinical applications, including inefficient tissue-specific delivery, off-target genome editing, immunogenicity, manufacturing complexity, and long-term safety concerns. Also, recent progress in both, viral and non-viral delivery methods are critically evaluated, including adeno-associated viruses, lentivirus vectors, lipid nanoparticles, gold nanoparticles, exosomes, electroporation, and microinjection, is thoroughly discussed to highlight their therapeutic potential and translational limitations. Current studies indicate that CRISPR-based approaches have preclinical potential for targeting important hallmarks of ageing, particularly genomic instability, telomere attrition, and mitochondrial dysfunction. Other hallmarks of ageing, such as stem cell exhaustion, epigenetic modifications, and microbiome changes, are at earlier stages of development. Overall, this review describes future strategies for developing safe, precise, and clinically translatable CRISPR-based treatments to promote healthy ageing.
Sakshi Rathore, Akash Gupta, Kamal Shah et al.· Ageing Research Reviews· 0 citations
A comprehensive review of delivery modalities of CRISPR systems solely in vivo that underpin their therapeutic translation and outlines the remaining barriers to durable, tissue-selective, and broadly deployable CRISPR therapeutics is provided.
L. Martin, Jure Bohinc, Alessandra Recchia et al.· International Journal on Bio...· 0 citations
The potential application of CRISPR technology for the possible management of geneticbased conditions, including sickle-cell anemia, β-thalassemia, cystic fibrosis, and Duchenne muscular dystrophy is described.
M. Veer, Poonam Nikam, Omkar More et al.· International Journal of Dru...· 0 citations
This review focuses on analytical and translational frameworks for CRISPR fidelity assessment, with emphasis on the strengths and limitations of current bioanalytical platforms.
Arpita Mukherjee· Journal of Rare Diseases· 1 citation
A consolidated guide for selecting suitable CRISPR-Cas technologies and underscoring important considerations for their continued development in leishmaniasis research is offered, highlighting the transition of CRISPR-Cas systems from proof-of-concept tools to versatile platforms for functional genomics, target validation and translational research in Leishmania.
A. Ata, Derya Topuz Ata· Molecular Biology Reports· 0 citations