Repeated High-Temperature Treatment Can Increase Prime Editing Efficiency in Dicot Model Species

Plant synthetic biology is an emerging and pioneering field for designing and manipulating genome information to modify metabolic pathways. Prime Editing (PE) has the advantage of being able to insert DNA segments into the genome. However, the low efficiency of PE in dicot plants has hindered its development and application. To address this issue, we have developed a method called Repeated High-Temperature Treatment (RHTT), which combines a repeated short period of heat stress with longer recovery periods in a cyclical manner. This approach maintains a balance between the contradictory effects of heat stress and efficiency enhancement. RHTT increased knockout efficiency (equivalent to cleavage ability of Cas9) 1.26 to 2.57-fold and precise PE efficiency 1.85 to 16.30-fold in Nicotiana benthamiana (Nb). When applied to Arabidopsis thaliana (At) for small segment insertion, RHTT improved PE efficiency by up to 15.67-fold.