Volodin Sergey¹, Nakamura Yuka², Khalid Sana³, Pires Eduardo⁴, Schmitt Julian⁵, Dubois Anaëlle⁶
ABSTRACT:
Background: The advent of CRISPR-based technologies has revolutionized functional biochemistry by enabling precise, programmable manipulation of genomic and epigenomic landscapes. Originally characterized as a prokaryotic adaptive immune system, CRISPR-Cas systems have evolved into versatile molecular tools for genome editing, transcriptional modulation, epigenetic reprogramming, base editing, and live-cell imaging. These tools are transforming how biochemical functions are interrogated, modified, and controlled in living systems. Methods and Results: This review outlines the key innovations and expanding applications of CRISPR-based technologies in biochemical research. CRISPR-Cas9 and its derivatives allow for targeted gene knockouts, knock-ins, and allele-specific editing with high precision and efficiency. Catalytically inactive Cas variants (dCas9) fused to effector domains facilitate transcriptional activation (CRISPRa) or repression (CRISPRi), enabling temporal and tissue-specific control of gene expression without altering DNA sequence. Base editors and prime editing systems offer programmable single-nucleotide conversions with minimized double-strand break risk. CRISPR-based epigenetic editors can deposit or erase histone modifications and DNA methylation at defined loci, allowing dissection of chromatin regulatory elements and noncoding genome function. Additionally, CRISPR tools are increasingly integrated with biosensors, protein labeling systems, and high-throughput screens to map biochemical pathways and synthetic gene networks. Despite remarkable progress, challenges such as off-target effects, delivery efficiency, and immune responses remain active areas of investigation. Conclusion: CRISPR-based systems represent a transformative platform in functional biochemistry, offering unprecedented capabilities to manipulate and monitor biological systems at the molecular level.