Background

Drug development requires not only identifying genes associated with a disease but also verifying whether these genes are the actual cause of the disease or influence drug response. Traditional methods such as gene overexpression or RNA interference often fail to completely eliminate gene function or induce non-specific effects, reducing the accuracy of target validation. Additionally, cell lines derived from patients and preclinical models often fail to fully recapitulate the genetic background of the actual disease.

CRISPR-associated protein 9 (Cas9) enables genome editing by cutting the DNA sequence specified by a guide RNA, thereby removing or correcting the function of a specific gene. Researchers can not only edit candidate genes individually but also perform genome-wide functional screening to investigate thousands of genes simultaneously. This review categorizes the applications of CRISPR-Cas9 into drug target discovery, personalized therapy, cancer drug resistance, genetic disorders, and antimicrobial resistance, summarizing its clinical translational potential and limitations.

Key Findings

CRISPR-Cas9 allows the direct introduction of disease-related mutations into cells or animals, enabling the creation of preclinical models that closely mimic the molecular characteristics of patients. By comparing drug responses between normal and edited cells under the same genetic background, it becomes easier to determine whether observed differences are attributable to specific mutations. Combining patient genomic data with edited models can lead to precision medicine strategies that distinguish patient groups with expected drug efficacy from those at risk of toxicity.

In cancer research, CRISPR screening compares genetic changes in cells that survive before and after chemotherapy to identify targets involved in drug resistance. Key targets include genes related to drug uptake and efflux, DNA damage repair, apoptosis, and bypass signaling of target proteins. A major advantage is the ability to confirm whether drug sensitivity is actually restored after removing candidate genes, rather than relying on simple correlation analysis.

In genetic disorders, therapeutic approaches involving the correction of pathogenic mutations or modulation of compensatory pathways are being explored. The most clinically advanced applications are in hemoglobinopathies such as sickle cell disease and beta-thalassemia requiring frequent blood transfusions. A representative approach involves editing the BCL11A erythroid enhancer in patients' autologous CD34-positive hematopoietic stem cells to reactivate fetal hemoglobin production, which is then reinfused into the body. In bacteria, strategies targeting resistance genes or resistance plasmids to restore antibiotic susceptibility have been proposed. However, this paper is a review that synthesizes multiple applications and clinical evidence, not a study reporting new experimental results.

Implications and Outlook

CRISPR-Cas9 is becoming both a therapeutic candidate and a research platform for validating the mechanisms of action of new drug candidates. By directly modifying the function of target genes and measuring drug responses, it is possible to filter out high-risk candidates at an early stage and identify combination targets for resistant patients. Clinical successes in hemoglobinopathies demonstrate that the current most feasible pathway is to edit, screen, and then administer cells ex vivo.

To expand its application, several challenges remain. Unintended off-target effects, large deletions or rearrangements at target sites, and long-term tracking are necessary. Immune responses to Cas9 or delivery vectors must also be evaluated. While delivery to organs such as the liver is relatively straightforward, delivering editing tools efficiently and selectively to the brain, lungs, or solid tumors remains difficult. Strategies targeting antibiotic-resistant bacteria also face barriers such as strain-specific sequence differences, delivery efficiency, and the potential for microbiome disruption. For broad clinical application, manufacturing costs, institutional cell-processing capabilities, and ethical and regulatory standards for germline editing must also be addressed.