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.

The rapid improvement of descriptive genomic technologies has fueled a dramatic increase in hypothesized connections between cardiovascular gene expression and phenotypes. However, in vivo testing of these hypotheses has predominantly been relegated to slow, expensive, and linear generation of genetically modified mice. In the study of genomic cis-regulatory elements, generation of mice featuring transgenic reporters or cis-regulatory element knockout remains the standard approach. While the data obtained is of high quality, the approach is insufficient to keep pace with candidate identification and therefore results in biases introduced during the selection of candidates for validation. However, recent advances across a range of disciplines are converging to enable functional genomic assays that can be conducted in a high-throughput manner. Here, we review one such method, massively parallel reporter assays (MPRAs), in which the activities of thousands of candidate genomic regulatory elements are simultaneously assessed via the next-generation sequencing of a barcoded reporter transcript. We discuss best practices for MPRA design and use, with a focus on practical considerations, and review how this emerging technology has been successfully deployed in vivo. Finally, we discuss how MPRAs are likely to evolve and be used in future cardiovascular research.

Skeletal muscle, constituting ~40% of body mass, serves as a primary effector for movement and a key metabolic regulator through myokine secretion. Hereditary myopathies, including dystrophinopathies (DMD/BMD), limb–girdle muscular dystrophies (LGMD), and metabolic disorders like Pompe disease, arise from pathogenic mutations in structural, metabolic, or ion channel genes, leading to progressive weakness and multi-organ dysfunction. Gene therapy has emerged as a transformative strategy, leveraging viral and non-viral vectors to deliver therapeutic nucleic acids. Adeno-associated virus (AAV) vectors dominate clinical applications due to their efficient transduction of post-mitotic myofibers and sustained transgene expression. Innovations in AAV engineering, such as capsid modification (chemical conjugation, rational design, directed evolution), self-complementary genomes, and tissue-specific promoters (e.g., MHCK7), enhance muscle tropism while mitigating immunogenicity and off-target effects. Non-viral vectors (liposomes, polymers, exosomes) offer advantages in cargo capacity (delivering full-length dystrophin), biocompatibility, and scalable production but face challenges in transduction efficiency and endosomal escape. Clinically, AAV-based therapies (e.g., Elevidys^® for DMD, Zolgensma^® for SMA) demonstrate functional improvements, though immune responses and hepatotoxicity remain concerns. Future directions focus on AI-driven vector design, hybrid systems (AAV–exosomes), and standardized manufacturing to achieve “single-dose, lifelong cure” paradigms for muscular disorders.