Institute of Fruit Tree and Tea Science, National Agriculture and Food Research Organization, Tsukuba, Japan.
Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
PLoS One. 2023 Jun 8;18(6):e0286698. doi: 10.1371/journal.pone.0286698. eCollection 2023.
Transposition of transposable elements affect expression levels, splicing and epigenetic status, and function of genes located in, or near, the inserted/excised locus. For example, in grape, presence of the Gret1 retrotransposon in the promoter region of the VvMYBA1a allele at the VvMYBA1 locus suppress the expression of the VvMYBA1 transcription factor gene for the anthocyanin biosynthesis and this transposon insertion is responsible for the green berry skin color of Vitis labrascana, 'Shine Muscat', a major grape cultivar in Japan. To prove that transposons in grape genome can be removed by genome editing, we focused on Gret1 in the VvMYBA1a allele as a target of CRISPR/Cas9 mediated transposon removal. PCR amplification and sequencing detected Gret1 eliminated cells in 19 of 45 transgenic plants. Although we have not yet confirmed any effects on grape berry skin color, we were successful in demonstrating that cleaving the long terminal repeat (LTR) present at both ends of Gret1 can efficiently eliminate the transposon.
转座元件的转位会影响位于插入/切除位点或附近的基因的表达水平、剪接和表观遗传状态及功能。例如，在葡萄中，Gret1 逆转座子位于 VvMYBA1 基因座的 VvMYBA1a 等位基因的启动子区域，抑制了 VvMYBA1 转录因子基因对花色苷生物合成的表达，这种转座子插入是 Vitis labrascana（“Shine Muscat”）葡萄品种绿色果皮颜色的原因，“Shine Muscat”是日本主要的葡萄品种之一。为了证明葡萄基因组中的转座子可以通过基因组编辑去除，我们将重点放在 VvMYBA1a 等位基因中的 Gret1 作为 CRISPR/Cas9 介导的转座子去除的靶标。PCR 扩增和测序检测到在 45 个转基因植物中有 19 个植物的 Gret1 被消除。尽管我们尚未证实对葡萄浆果果皮颜色的任何影响，但我们成功地证明了切割存在于 Gret1 两端的长末端重复序列（LTR）可以有效地消除转座子。
