Masaki Kawamata, Satoshi Niwa, Atsushi Suzuki
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.