CRISPR–Cas9 is the state-of-the-art programmable genome-editing tool widely used in many areas. For safe therapeutic applications in clinical medicine, its off-target effect must be dramatically minimized. In recent years, extensive studies have been conducted to improve the gene-editing specificity of the most popular CRISPR–Cas9 nucleases using different strategies. In this review, we summarize and discuss these strategies and achievements, with a major focus on improving the gene-editing specificity through Cas9 protein engineering.
