DOI: https://doi.org/10.36347/sajb.2025.v13i07.001

Pages: 867-876

By offering a precise, effective, and adaptable method for genome editing, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has completely transformed genetic biotechnology. Through a biochemical lens that highlights the molecular complexities behind its methods and discoveries, this review explores the complex field of CRISPR cloning and its emerging frontiers. We examine the molecular underpinnings of target identification, protospacer adjacent motif (PAM) specificity, and Cas nuclease activation, starting with the groundbreaking discovery of CRISPR-Cas systems in bacterial adaptive immunity. Advances in CRISPR-based cloning techniques, such as the creation of synthetic guide RNAs, Cas variants with specific functions, and high-fidelity and base-editing systems that reduce off-target effects, are also examined in the study. The molecular dynamics of DNA-RNA-protein interactions during gene targeting, activation of DNA repair pathways, and epigenetic state regulation are given special attention. We investigate cutting-edge uses beyond traditional genome editing, including RNA-targeting Cas proteins (like Cas13), CRISPR-based diagnostics and treatments, CRISPR interference (CRISPRi), and CRISPR activation (CRISPRa). Additionally covered is the incorporation of CRISPR with other biochemical technologies, such as gene drives, programmable transcription factors, and synthetic biology circuits. The field's increasing biochemical sophistication is highlighted by new developments like CRISPR-nanoparticle conjugates for targeted delivery and CRISPR screening platforms for functional genomics. In the end, this review emphasizes how a thorough biochemical comprehension of CRISPR systems is essential to maximizing their potential in environmental engineering, biotechnology, medicine, and agriculture.