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.

本数据集通过扩增子测序（Amplicon sequencing）鉴定RNA模板依赖的双链断裂修复（RNA templated double strand break repair, RT-DSBR），具体方式为分析AAVS1位点经定向Cas9切割后产生的修复产物。实验中将细胞与携带供体模板的RNA共转染，若该RNA被用作修复模板，则会在修复产物中留下可识别的标记序列，以此验证人类细胞可将RNA作为可行的修复模板。此外，本数据集还在多组样本中通过灭活不同聚合酶（polymerase）检测了RT-DSBR的发生频率，旨在明确RT-DSBR过程中逆转录步骤所必需的聚合酶种类。