CRISPR/Cas9 is a powerful genome-editing technology used for studying genetic elements, creating genetically modified organisms, and preclinical research of genetic disorders. However, off-target effects—mutations at sites other than the intended target—are a major concern, especially for therapeutic applications. This review discusses the mechanisms of off-target cutting, methods for detecting off-target mutations, and strategies to minimize off-target cleavage. Improving off-target specificity in CRISPR/Cas9 will enhance genotype-phenotype correlations, enabling accurate interpretation of genome-editing data and facilitating its clinical application. The CRISPR/Cas9 system functions as an RNA-based adaptive immune system in bacteria and archaea. It uses a guide RNA (gRNA) to target specific DNA sequences, with the PAM (protospacer adjacent motif) sequence (typically NGG) essential for binding. The seed sequence, located near the PAM, plays a critical role in determining Cas9 specificity. However, off-target effects can occur even with minor mismatches in the gRNA sequence. Strategies to reduce off-target effects include modifying sgRNA sequences, using purified Cas9 protein, and employing paired nickases or dCas9-fokI fusions. These approaches enhance specificity and reduce unintended mutations. Off-target detection methods include T7 endonuclease I assay, deep sequencing, ChIP-seq, and Digenome-seq. Digenome-seq is considered the gold standard for detecting off-target effects in genome-wide studies. Other methods, such as GUIDE-seq and HTGTS, also provide high-resolution data on off-target cleavage. These methods help identify rare and harmful off-target sites before clinical applications. To minimize off-target effects, researchers have developed strategies such as using double nicking, which reduces off-target activity by 50–1,500-fold. Additionally, fusions of dCas9 with FokI nuclease improve specificity. These approaches, along with optimized sgRNA design and improved delivery methods, enhance the precision and safety of CRISPR/Cas9 technology. The review highlights the importance of understanding and minimizing off-target effects to ensure the safe and effective use of CRISPR/Cas9 in both basic research and clinical applications. Future advancements in CRISPR/Cas9 technology, including the development of new nucleases like Cpf1, may further improve genome editing capabilities. Overall, ongoing research aims to enhance the specificity, efficiency, and safety of CRISPR/Cas9 for therapeutic use.CRISPR/Cas9 is a powerful genome-editing technology used for studying genetic elements, creating genetically modified organisms, and preclinical research of genetic disorders. However, off-target effects—mutations at sites other than the intended target—are a major concern, especially for therapeutic applications. This review discusses the mechanisms of off-target cutting, methods for detecting off-target mutations, and strategies to minimize off-target cleavage. Improving off-target specificity in CRISPR/Cas9 will enhance genotype-phenotype correlations, enabling accurate interpretation of genome-editing data and facilitating its clinical application. The CRISPR/Cas9 system functions as an RNA-based adaptive immune system in bacteria and archaea. It uses a guide RNA (gRNA) to target specific DNA sequences, with the PAM (protospacer adjacent motif) sequence (typically NGG) essential for binding. The seed sequence, located near the PAM, plays a critical role in determining Cas9 specificity. However, off-target effects can occur even with minor mismatches in the gRNA sequence. Strategies to reduce off-target effects include modifying sgRNA sequences, using purified Cas9 protein, and employing paired nickases or dCas9-fokI fusions. These approaches enhance specificity and reduce unintended mutations. Off-target detection methods include T7 endonuclease I assay, deep sequencing, ChIP-seq, and Digenome-seq. Digenome-seq is considered the gold standard for detecting off-target effects in genome-wide studies. Other methods, such as GUIDE-seq and HTGTS, also provide high-resolution data on off-target cleavage. These methods help identify rare and harmful off-target sites before clinical applications. To minimize off-target effects, researchers have developed strategies such as using double nicking, which reduces off-target activity by 50–1,500-fold. Additionally, fusions of dCas9 with FokI nuclease improve specificity. These approaches, along with optimized sgRNA design and improved delivery methods, enhance the precision and safety of CRISPR/Cas9 technology. The review highlights the importance of understanding and minimizing off-target effects to ensure the safe and effective use of CRISPR/Cas9 in both basic research and clinical applications. Future advancements in CRISPR/Cas9 technology, including the development of new nucleases like Cpf1, may further improve genome editing capabilities. Overall, ongoing research aims to enhance the specificity, efficiency, and safety of CRISPR/Cas9 for therapeutic use.