Institut Agro, University Angers, INRAE, IRHS, SFR 4207 QuaSaV, 49000 Angers, France.
IGEPP, INRAE, Institut Agro, University Rennes, 29260 Ploudaniel, France.
Int J Mol Sci. 2020 Dec 30;22(1):319. doi: 10.3390/ijms22010319.
Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the () gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for "regenerated T0" lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: - (conferring resistance to chlorsulfuron) and . The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing and genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.
尽管最近取得了进展，但 CRISPR/Cas9 在多年生植物中的应用仍然有许多障碍需要克服。我们之前在苹果和梨中使用 CRISPR/Cas9 的结果表明，在编辑导致白化表型的 () 基因后，经常会产生表型和基因型嵌合体。因此，我们的第一个目标是确定是否添加一个来自初级转基因植物（T0）叶片的不定芽再生步骤是否会减少嵌合体的产生。在一个斑驳的 T0 系再生的数百个不定芽中，有 89%是同质的白化体。此外，对这 12 个再生系的靶区序列进行分析（RT0 表示“再生 T0”系）表明，99%的 RT0 等位基因预计会产生截断的靶蛋白，并且 67%的 RT0 植物的编辑谱比 T0 更不均匀。碱基编辑器是基于 CRISPR/Cas9 的新型基因组编辑工具，可在不产生双链断裂的情况下实现精确的核苷酸替换。因此，我们的第二个目标是证明 CRISPR/Cas9 碱基编辑在苹果和梨中的可行性，使用两个易于评分的基因：-（赋予对氯磺隆的抗性）和 。在 MdU3 和 MdU6 启动子下的两个向导 RNA 与一个胞嘧啶碱基编辑器连接，该编辑器包含一个与切口酶融合的胞嘧啶脱氨酶。使用该载体，我们在靶基因中诱导了 C 到 T 的 DNA 替换，导致氨基酸序列的离散变化，并产生了新的等位基因。通过共编辑 和 基因，我们成功地在梨中获得了对氯磺隆抗性和白化系。总的来说，我们的工作表明，再生步骤可以有效地降低初始嵌合体的产生，并可以与碱基编辑的应用相结合，在多年生植物中创建精确的基因组编辑。
