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

Plant\nsynthetic biology is an emerging and pioneering field for\ndesigning and manipulating genome information to modify metabolic\npathways. Prime Editing (PE) has the advantage of being able to insert\nDNA segments into the genome. However, the low efficiency of PE in\ndicot plants has hindered its development and application. To address\nthis issue, we have developed a method called Repeated High-Temperature\nTreatment (RHTT), which combines a repeated short period of heat stress\nwith longer recovery periods in a cyclical manner. This approach maintains\na balance between the contradictory effects of heat stress and efficiency\nenhancement. RHTT increased knockout efficiency (equivalent to cleavage\nability of Cas9) 1.26 to 2.57-fold and precise PE efficiency 1.85\nto 16.30-fold in <i>Nicotiana benthamiana</i> (<i>Nb</i>). When applied to <i>Arabidopsis thaliana</i> (<i>At</i>) for small segment insertion, RHTT improved PE efficiency\nby up to 15.67-fold.