LAUSR

Efficient and precise gene editing is the gold standard of any reverse genetic study. The recently developed Prime Editing approach, a modified CRISPR-Cas9 editing method, has reached the precision goal but its editing rate can be improved. We present here an improved methodology that allows for routine Prime Editing in the model plant Physcomitrium patens, whilst exploring potential new Prime Editing improvements. Using a standardized protoplast transfection procedure, multiple pegRNA structural and Prime Editor variants were evaluated targeting the APT reporter gene through direct plant selection. Together, improvements of expression of the Prime Editor, modifications of the 3' extension of the pegRNA and the addition of synonymous mutation in the RT-template sequence of the pegRNA improve dramatically the editing rate without affecting the quality of the edits. Furthermore, based on the results obtained at PpAPT locus through direct selection, we show that the Prime Editing is amenable to edit a gene of interest through indirect selection as demonstrated by the generation of a Ppdek10 mutant. Additionally, we show that a plant retrotransposon RT enables Prime Editing. Finally, we show for the first time the possibility of performing Prime Editing with two independently coded peptides. This will facilitate the future test of new active domains of the Prime Editor in plants.