Gates Package — page 853

Fig.9. Off-target analysis after in vitro and in vivo base editing with HDAdEF1a.ABE8e (based on CIRCLE-seq prediction). Genomic DNA from naïve CD46/- YAC mice was cleaved with recombinant SpCas9 plus sgHBG#2 RNA. CIRCLE-seq identified 272 OTS from which the top 20 were subjected to NGS. Amplicons were prepared using genomic DNA template from a naïve mouse and an in vivo transduced mouse with the highest on-targeting editing rate. The middle two panels show A>G conversion frequencies in the targetable window (position 3 to 14) of the top 20 candidate OTSs. Note that for site mOTS11, the percentage of target site conversion was similar for untreated and HDAd-EF1a.ABE8e treated mice, making it unlikely that the conversions were triggered by ABE8e. The bottom panel shows the indel frequency in a quantification window of 140 bp.