Amplicon sequencing of Ymu1-mediated genome editing in Phtheirospermum japonicum
Amplicon sequencing was used to quantify the extent of genome editing in the parasitic plant Phtheirospermum japonicum using virally delivered TnpB Ymu1 and seven different guide RNAs targeting phytoene desaturase (PDS) and 3,8-divinyl protochlorophyllide a 8-vinyl reductase (DVR). Point mutations in Ymu1 altered the transposon-associated motif requirement from TTGAT to TYTAT (K76A) and increased Ymu1 activity (H4Y, V3105R). A bipartite nuclear localization sequence was used to enhance nuclear localization efficiency. For some guides, a 103 bp deletion in the C-terminus of Ymu1 and the omegaRNA was included; this deletion does not alter the protein sequence.