Fig. 1

Gene targeting with geminivirus replicons. a Structure of the bean yellow dwarf virus (BeYDV) genome. The single-stranded DNA genome encodes three major functions: replicase proteins (Rep and RepA) mediate rolling circle replication, and movement and coat proteins are essential for viral movement. The long intergenic region (LIR) is the origin of replication and also functions as a bidirectional promoter that drives expression of viral genes. The short intergenic region (SIR) is the origin of C-strand synthesis and contains transcription termination and polyadenylation signals. b Structure of BeYDV genome modified for gene targeting. Coding sequences for movement and coat proteins were replaced with the site-specific nuclease and donor template for gene targeting. The modified virus is not capable of infection due to the lack of essential viral proteins. Further, the size exceeds the limit for successful packaging and cell-to-cell movement. The replication function is preserved, and the vector can replicate when delivered to plant cells by transformation. c Illustration of gene targeting with the modified BeYDV vector through Agrobacterium-mediated transformation. The BeYDV genome, containing the nuclease and donor template for gene targeting, is cloned into a transfer DNA (T-DNA) vector. One LIR is placed on each side of the viral genome to ensure release from the T-DNA in the plant cell. During Agrobacterium infection, linear T-DNA molecules are delivered to the nucleus of a plant cell, where the viral genome is replicationally released in a circular form and amplified into thousands of copies by rolling circle replication, mediated by the replicase proteins expressed from the LIR. The nuclease expressed from the viral genome induces DSBs at the target locus, and the donor template is copied into the target site by homology-directed repair. The high copy number of donor templates increases the frequency of gene targeting. LB left T-DNA border, SSN sequence-specific nuclease, RB right T-DNA border