| First Author | Puzzo F | Year | 2026 |
| Journal | Mol Ther Nucleic Acids | Volume | 37 |
| Issue | 2 | Pages | 102880 |
| PubMed ID | 41909468 | Mgi Jnum | J:386510 |
| Mgi Id | MGI:8332321 | Doi | 10.1016/j.omtn.2026.102880 |
| Citation | Puzzo F, et al. (2026) Potential effects of endogenous RNA/DNA hybrids on CRISPR-Cas9-mediated homology-directed repair. Mol Ther Nucleic Acids 37(2):102880 |
| abstractText | Recombinant adeno-associated virus (rAAV) vectors and CRISPR-Cas9 are widely used in gene therapy. However, how endogenous DNA secondary structures may potentially affect genome editing outcomes is not fully understood. RNA/DNA hybrids (R-loops), which form mostly during transcription, are dynamically regulated in cells and have been implicated in influencing DNA repair pathway choice. Here, we investigated whether genomic R-loops are associated with differences in Cas9-mediated genome editing outcomes in vitro and in vivo. By targeting regions with relatively low or high R-loop levels within the murine albumin (Alb) and actin (Actb) loci, we observed comparable insertion/deletion (indel) frequencies across sites with different R-loop abundance. In contrast, homology-directed repair (HDR) efficiency appeared reduced at R-loop-enriched sites in proliferating hepatocyte-derived cells (HEPA1-6) but not in quiescent hepatocytes in vivo. Manipulations resulting in reducing R-loop levels, including RNaseH1 overexpression or pharmacological induction of G1 arrest were associated with increased HDR at these genomic sites. In addition, T cell activation correlated with elevated R-loop accumulation suggesting they might influence ex vivo genome editing. Together, these observations suggest that endogenous R-loop levels might influence HDR efficiency during Cas9-mediated editing and is a parameter to consider when designing genome editing strategies. |
