CRISPR-Cas9-mediated homology-directed repair for precise gene editing

CRISPR-Cas9-mediated homology-directed repair (HDR) is a versatile platform for creating precise site-specific DNA insertions, deletions, and substitutions. These precise edits are made possible through the use of exogenous donor templates that carry the desired sequence. CRISPR-Cas9-mediated HDR can be widely used to study protein functions, disease modeling, and gene therapy. However, HDR is limited by its low efficiency, especially in postmitotic cells. Here, we review CRISPR-Cas9-mediated HDR, with a focus on methodologies for boosting HDR efficiency, and applications of precise editing via HDR. First, we describe two common mechanisms of DNA repair, non-homologous end joining (NHEJ), and HDR, and discuss their impact on CRISPR-Cas9-mediated precise genome editing. Second, we discuss approaches for improving HDR efficiency through inhibition of the NHEJ pathway, activation of the HDR pathway, modification of donor templates, and delivery of Cas9/sgRNA reagents. Third, we summarize the applications of HDR for protein labeling in functional studies, disease modeling, and ex vivo and in vivo gene therapies. Finally, we discuss alternative precise editing platforms and their limitations, and describe potential avenues to improving CRISPR-Cas9-mediated HDR efficiency and fidelity in future research.