The CRISPR/Cas9 system provides a robust and multiplexable genome editing tool, enabling researchers to precisely manipulate specific genomic elements and facilitating the elucidation of target gene function in biology and diseases. CRISPR/Cas9 consists of a nonspecific Cas9 nuclease and a set of programmable sequence-specific CRISPR RNA (crRNA), which can guide Cas9 to cleave DNA and generate double-strand breaks at target sites. Subsequent cellular DNA repair processes lead to desired insertions, deletions, or substitutions at target sites. The particularity of CRISPR/Cas9-mediated DNA cleavage requires target sequences matching crRNA and a protospacer adjacent motif located downstream of target sequences. Here, we review the molecular mechanism, applications, and challenges of CRISPR/Cas9-mediated genome editing and the clinical therapeutic potential of CRISPR/Cas9 in the future.