Université Paris-Saclay, INRAE, AgroParisTech, Institut Jean-Pierre Bourgin (IJPB), 78000 Versailles, France.
Centre for Research in Agricultural Genomics CSIC-IRTA-UAB-UB, Campus UAB, Edifici CRAG, Bellaterra, 08193 Barcelona, Spain.
J Exp Bot. 2023 Oct 13;74(19):6176-6187. doi: 10.1093/jxb/erad189.
Efficient and precise gene editing is the gold standard of any reverse genetic study. The recently developed prime editing approach, a modified CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein] editing method, has reached the precision goal but its editing rate can be improved. We present 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 prime editing guide RNA (pegRNA) structural and prime editor variants were evaluated targeting the APT reporter gene through direct plant selection. Together, enhancements of expression of the prime editor, modifications of the 3' extension of the pegRNA, and the addition of synonymous mutation in the reverse transcriptase template sequence of the pegRNA dramatically improve the editing rate without affecting the quality of the edits. Furthermore, we show that prime editing is amenable to edit a gene of interest through indirect selection, as demonstrated by the generation of a Ppdek10 mutant. Additionally, we determine that a plant retrotransposon reverse transcriptase enables prime editing. Finally, we show for the first time the possibility of performing prime editing with two independently coded peptides.
高效、精确的基因编辑是任何反向遗传学研究的金标准。最近开发的主导编辑方法(一种改良的 CRISPR/Cas9 [成簇规律间隔短回文重复序列 (CRISPR)/CRISPR 相关蛋白] 编辑方法)已经达到了精确的目标,但编辑效率可以提高。我们提出了一种改进的方法,允许在模式植物泡囊藻中进行常规的主导编辑,同时探索潜在的新的主导编辑改进。使用标准化的原生质体转染程序,通过直接植物选择,针对 APT 报告基因评估了多种主导编辑引导 RNA (pegRNA) 结构和主导编辑器变体。通过提高主导编辑器的表达、pegRNA 的 3' 延伸的修饰以及 pegRNA 的逆转录酶模板序列中的同义突变的添加,在不影响编辑质量的情况下,大大提高了编辑效率。此外,我们还表明,通过间接选择,主导编辑可用于编辑感兴趣的基因,这是通过生成 Ppdek10 突变体来证明的。此外,我们确定植物逆转录转座子逆转录酶能够进行主导编辑。最后,我们首次展示了使用两个独立编码肽进行主导编辑的可能性。