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

## TL;DR

The study shows that RNA/DNA hybrids (R-loops) can reduce the efficiency of CRISPR-Cas9 gene editing in certain cell types, and reducing R-loops can improve editing outcomes.

## Contribution

The study reveals that R-loop levels modulate HDR efficiency in Cas9-mediated genome editing, particularly in proliferating cells.

## Key findings

- HDR efficiency is reduced at R-loop-enriched sites in proliferating hepatocyte-derived cells.
- Reducing R-loop levels through RNaseH1 overexpression or G1 arrest increases HDR efficiency.
- T cell activation correlates with elevated R-loop accumulation, potentially affecting ex vivo genome editing.

## Abstract

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.

Kay and colleagues discovered how R-loops can modulate Cas9-mediated genome editing by suppressing homology-directed repair (HDR) at R-loop-rich regions in proliferating cells. Reducing R-loops levels restores HDR, highlighting R-loops as key regulators of repair outcomes and important considerations for optimizing therapeutic genome editing.