Strategies for Precision Genome Editing in Plants

The most widely practiced genome editing involves generating DNA double stranded breaks (DSBs) and subsequent DNA repair through the non-homologous end joining pathway, leading to loss-of-function indel mutations in the target gene. Precise genome editing can be achieved using strategies such as base editing, prime editing and DSB-facilitated gene targeting. Gene targeting generates the most desired precision genome editing events, i.e., precise sequence insertions or replacements, but the success rate is very low, due to the extremely low activity of homology-directed repair (HDR) in plants. We recently developed the tandem repeat (TR)-facilitated HDR (TR-HDR) genome editing strategy for precise sequence insertions or replacements in plants. In TR-HDR, a double stranded donor DNA with 5’-phosphorylation and phosphorothioate-linkage modifications and sequence homology to the target locus is inserted via a DSB adjacent to the target sequence with high efficiency. Upon insertion, a target site for the same original gRNA is generated. This second DSB would trigger efficient HDR, causing precise replacement of the target sequence with the donor sequence or seamless insertion of the donor DNA. In this talk, I will also present various genome editing strategies for targeted and precise control of the levels of gene transcription or mRNA translation.