ORIGINAL ABSTRACT

Prime editing (PE) enables precise genome modifications to mammalian cells and monocot staple crops, but remains relatively challenging in dicot plants. Here, we develop a Flanking-Nicks Prime Editor (FLICK-PE) system that boosts editing efficiency in soybean and tobacco. We show that optimization for PE by adding a nicking sgRNA could dramatically enhance intended-editing efficiency in soybean. Inspired by this observation, we design a FLICK-PE strategy to confer a pair of nicks flanking the target site. In soybean, FLICK-PE achieves on average a 15.7-fold increase in intended-editing efficiency compared to PE2, and a 2.2-fold increase compared to PE3. Using FLICK-PE, we efficiently engineer glyphosate resistance in soybean by introducing TAP-IVS mutations in EPSPS1a, achieving three amino-acid substitutions and an intended editing efficiency of 21.1%. This approach yields stable edited soybean varieties with vigorous glyphosate tolerance and minimal growth penalties in a field trial. FLICK-PE also demonstrates efficacy in tobacco, underscoring its broad applicability and versatility for rapid, precision breeding in agriculturally vital crops.