Efficient transgene-free multiplexed genome editing via viral delivery of an engineered TnpB

Virus-induced genome editing (VIGE) using compact RNA-guided endonucleases is a transformational new approach in plant biotechnology, enabling tissue-culture-independent and transgene-free genome editing. We recently established a transgene-free VIGE approach for heritable editing at single loci in Arabidopsis by delivering ISYmu1 TnpB (Ymu1) and its guide RNA (gRNA) via Tobacco Rattle Virus (TRV). Here, we greatly improved this system by devising a multiple gRNA expression system and by utilizing an engineered high-activity Ymu1 variant (Ymu1-WFR) to develop an efficient multiplexed genome editing approach. Overall design: Bulk bacterial RNA sequencing was performed to characterize heterologous RNA expression from a defined plasmid construct in Escherichia coli. Cells were grown under identical conditions and induced with arabinose prior to RNA extraction. Each sample represents total RNA isolated from an induced bacterial culture processed through rRNA depletion and small-RNA-seq library preparation. Sequencing reads were aligned to the Ymu1 locus to assess RNA expression profiles.