CRISPR: A Revolution of Genome Editing Technology

Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Endonucleases (TALENs) are early programmable DNA nucleases that advanced genome engineering. More recently, CRISPR-Cas9 has emerged as a highly popular and efficient genome-editing tool. Discovered by Yoshizumi Ishino during microbial studies, CRISPR-Cas9 quickly gained favor due to its simplicity, precision, and ease of design, surpassing TALENs and ZFNs in widespread use. The system consists of the Cas9 enzyme and guide RNA (gRNA), which together form a complex to target and introduce a DNA break at a specific sequence. CRISPR systems are classified into two main types, Class I and Class II, based on the Cas proteins involved. Class II, which includes types II and V, is most commonly used in biotechnology. The Cas9 protein in Class II has two main lobes: the nuclease (NUC) lobe, which contains critical domains for DNA recognition and cleavage (RuvC, HNH, and PAM interaction), and the recognition (REC) lobe, which binds to the gRNA. A breakthrough by Nobel laureates Jennifer Doudna and Emmanuelle Charpentier streamlined the system by combining crRNA and tracrRNA into a single guide RNA (sgRNA), enhancing the precision and efficiency of CRISPR-Cas9. Various delivery methods for CRISPR components include physical (microinjection, electroporation), viral vectors (AAV, lentivirus, adenovirus), and non-viral approaches (lipid nanoparticles, gold nanoparticles). Physical delivery methods remain the most commonly used.