This study proposes the implementation of clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) technology for gene therapy targeting genetic mutations in human lymphocytes affected by chronic lymphocytic leukemia (CLL), offering new opportunities for effective treatment of this heterogeneous disease. It focuses on the application of CRISPR–Cas9-mediated targeted sequencing to systematically characterize the biological effects of monoallelic and biallelic TP53 gene lesions, aiming to replace mutant TP53 genes in CLL cells through this technology. CRISPR–Cas9 technology employs a specific enzyme guided by a designed guide RNA (gRNA) to a DNA target. The enzyme first introduces a cut at the target site, and following this cleavage event, it can further disrupt the TP53 gene. The gRNA plays a crucial role by directing the Cas9 protein to the DNA sequence of interest. The gRNA consists of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) sequences, responsible for target recognition and Cas9 binding, respectively. Examination of the predicted secondary structure of the tracrRNA–crRNA duplex suggests that the features required for Cas9-catalyzed DNA cleavage at specific sites can be captured within a single chimeric RNA. Although the natural tracrRNA–crRNA mechanism operates efficiently, the use of a single RNA-guided Cas9 system is particularly attractive due to its potential for programmed DNA cleavage and genome editing. Importantly, Cas9 can bind and cleave a target sequence only if it is adjacent to a protospacer adjacent motif. Once the gRNA–Cas9 complex binds to the target DNA, Cas9 induces a double-strand break at the specified site. In conclusion, CRISPR–Cas9 technology represents a powerful genetic engineering tool capable of inserting, deleting, or replacing DNA within an organism’s genome using these “molecular scissors.”