Extended Data Fig. 1: Workflow of Detect-seq.
From: Mitochondrial base editor induces substantial nuclear off-target mutations
Endogenous 5fdC was protected by O-ethylhydroxylamine (EtONH2). Damage repair step eliminates endogenous DNA damages including abasic sites (AP), single strand breaks (SSB), etc. Deoxyuridine (dU) generated by DdCBE in vivo was labeled by the in vitro reconstituted base excision repair (BER) reaction: UDG specifically recognizes and cleaves dU, leaving abasic sites; Endo IV removes abasic sites, leaving 3’-OH remnant; Bst DNA polymerase initiates DNA strand replacement after the 3’-OH; ligase sews the final nicks. Through the so-called “nick translation” activity of Bst polymerase during this step, biotinylated dUTPs and 5fdCTPs were incorporated 3’ to dU. Malononitrile treatment marks the incorporated 5fdCs, generating a characteristic tandem C-to-T mutation pattern to trace DdCBE edits. Biotin pulldown followed by NaOH treatment enriches DdCBE edited DNA fragments and enhances Detect-seq signals.
