Final Answer:
The combined use of tracr and crRNA to create a guide RNA (gRNA) that associates directly with Cas9 facilitates DNA cleavage, triggering non-homologous end joining (NHEJ) repair. This repair process often introduces deletions or insertions, leading to the formation of stop codons and ultimately resulting in the knockout (KO) of the targeted gene.
Step-by-step explanation:
The synthesis of a guide RNA (gRNA) through the fusion of tracr and crRNA is a key step in the CRISPR-Cas9 gene editing system. This gRNA acts as a molecular guide, directing the Cas9 enzyme to the specific target sequence on the DNA. Upon binding to the target site, Cas9 induces a double-strand break in the DNA. This break triggers the cellular repair mechanism known as non-homologous end joining (NHEJ). In this repair process, the broken DNA ends are rejoined, often leading to the introduction of random deletions or insertions. These genetic alterations can cause frameshift mutations that result in the generation of premature stop codons within the targeted gene.
The introduction of stop codons due to NHEJ repair disrupts the normal reading frame of the gene, resulting in a non-functional or truncated protein product. This intentional disruption of gene function is a powerful strategy for achieving gene knockout (KO). By preventing the synthesis of a functional protein, the targeted gene's normal biological activity is abolished. The CRISPR-Cas9 system's precision in targeting specific genes, coupled with the cell's own repair mechanisms, enables researchers to effectively induce gene knockouts for a variety of applications in biological research and therapeutic development.