CRISPR-Cas9 كأداة لتحرير الجينات قابلة للبرمجة

The transformation of the CRISPR-Cas9 bacterial immune system into a universal tool for genome editing was a landmark achievement in molecular biology. The key insight was recognizing its potential for being reprogrammed. In its natural form, the Type II system uses three components: the Cas9 protein, a crRNA that contains the targeting sequence, and a tracrRNA that is crucial for crRNA maturation and Cas9 activation. The Doudna and Charpentier labs demonstrated that this system could be simplified. They engineered a single chimeric RNA, which they termed a single-guide RNA (sgRNA), by linking the 3′ end of the crRNA to the 5′ end of the tracrRNA with a synthetic hairpin loop. This sgRNA retained all the necessary functions of the dual-RNA system.

This simplification was revolutionary because it meant that to retarget the Cas9 nuclease to a new DNA site, one only needed to synthesize a new sgRNA with a different 20-nucleotide guide sequence. This made the technology remarkably easy to use, cheap, and scalable compared to previous editing methods like Zinc Finger Nucleases (ZFNs) and TALENs, which required complex and costly protein engineering for each new target. When the Cas9-sgRNA complex is introduced into a cell, it locates its target DNA sequence and creates a double-strand break (DSB). The cell’s natural DNA repair machinery then takes over. The error-prone Non-Homologous End Joining (NHEJ) pathway often introduces small insertions or deletions (indels), effectively knocking out the gene. Alternatively, if a donor DNA template is supplied, the more precise Homology-Directed Repair (HDR) pathway can be used to insert new sequences or correct mutations.