FIGURE 1.

Geminivirus mediated plant genome engineering. The generalized molecular mechanism of geminivirus based replicons for delivering clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR associated9 (CRISPR/Cas9) reagents in plant cell. The deconstructed virus genome is cloned in T-DNA, transformed in Agrobacterium tumefaciens and delivered to plants, namely agroinfiltration. The DNA is inserted into the plant cell nucleus where the single stranded viral DNA is converted into the double stranded DNA replicative form and RNA is transcribed. The engineered viral transcript contains two long intergenic regions (LIRs) spanning the Cas9, guide RNA (gRNA), repair template, short intergenic region (SIR) and a replication associated protein (Rep). The Cas9 and gRNA combine to from a Cas9-gRNA complex that recognizes and binds to the target site adjacent to the protospacer adjacent motif (PAM), where Cas9 nuclease produces a double stranded break (DSB). In case of homolog directed repair (HDR), the DSB is repaired with the aid of a repair template that becomes the part of plant genome. Rep protein on the other hand starts the replication of viral DNA producing numerous transcripts that further follow the same pipeline and ensure efficient CRISPR/Cas9 mediated plant genome engineering.