Abstract Body: Introduction: Precise somatic genome editing could enable durable therapies for inherited cardiovascular disease, but efficient in vivo editing in cardiomyocytes remains limited. AAV-mediated homology-directed repair (AAV-HDR) is a promising strategy for permanent correction, yet its regulatory mechanisms are not well defined.

Hypothesis: High-throughput in vivo forward genetic screening will identify key regulators of AAV-HDR, which can be targeted to improve editing efficiency.

Aims: We sought to identify DNA-repair factors that regulate AAV-HDR efficiency.

Methods: We established an in vivo functional genomics platform in neonatal mouse cardiomyocytes using pooled AAV-delivered CRISPR libraries and high-throughput sequencing to quantify effects on editing performance. Top hits were prioritized for targeted genetic and pharmacologic follow-up.

Results: Screening identified the DNA Damage Tolerance (DDT) pathway as a central determinant of AAV-HDR efficiency. UBE2A, UBE2B, and SHPRH, key mediators of PCNA ubiquitination, emerged as strong candidate regulators. Pathway-level analyses support a model in which the template-switching branch of the DDT pathway is associated with improved precise editing, whereas the translesion synthesis branch is predicted to compete with HDR.

Conclusions: These findings link core DNA damage tolerance biology to genome editing performance in cardiomyocytes and identify ubiquitin-dependent DDT control as a targetable mechanism to enhance precise cardiac gene correction. How the template switching machinery of the DDT pathway facilitates AAV-HDR will be the subject of future studies.