Genome-wide Profiling of off-targets from Prime editor in vitro and in vivo using PE-Tag

Prime editors (PE) are robust genome-editing tools with a broad range of research and potential clinical applications. However, methods to delineate their genome-wide editing activities have relied on indirect genome-wide assays of the corresponding nuclease or computational prediction. In addition, the factors that affect off-target editing efficiency have not been extensively investigated. Herein we describe Tag-based PE off-target detection (PE-Tag) that relies on attachment or insertion of a unique tag by PE2 into the nicking site to allow genome-wide detection of off-target editing. Using extracted genomic DNA and PE RNP, we showed that in vitro PE-Tag can detect off-target nicking sites missed by DSB-based GUIDE-Seq. In cultured cells, PE-Tag detected functional off-target sites, which can harbor up to two mismatches in a 7bp 3 prime flap homology region. Based on the homology, we developed an algorithm to refine the in vitro PE -tag (filter out non-functional OT sites). PE-Tag enables genome-wide profiling of off-targets from prime editing in vitro using extracted genomic DNA, in mammalian cultured cell lines and in vivo in adult mice liver. PE-Tag components can be delivered as plasmid, ribonucleoprotein (RNP) complexes or mRNA for off-target detection. Application of PE-Tag in vitro and in vivo revealed wide variability in PE off-target activities. PE-Tag represents an accessible, rapid, and comprehensive approach for genome-wide identification of prime editing and provides an approach for evaluating the safety of prime editors.