Potential effects of endogenous RNA/DNA hybrids on CRISPR-Cas9-mediated homology-directed repair

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.