RNA Transcripts Serve as a Template for Double-Strand Break Repair in Human Cells

Amplicon sequencing to identify RNA templated double strand break repair (RT-DSBR) by analyzing the repair products generated following a site directed Cas9 break at the AAVS1 locus. The cells are co-transfected with a RNA containing donor template that, if utilized as a template for repair, provides an identifiable scar in the repair product validating that human cells can utilize RNA as a viable repair template. Additionally, RT-DSBR frequencies were measured across multiple samples, with the inactivation of different polymerase, with the aim of identifying the polymerase responsible for the reverse transcription step essential in RT-DSBR.