High-frequency, precise modification of the tomato genome.
High-frequency, precise modification of the tomato genome.
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DOI：
10.1186/s13059-015-0796-9
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发表时间：
2015-11-06
期刊：
影响因子：
12.3
通讯作者：
Voytas DF
中科院分区：
文献类型：
--
作者：
Čermák T;Baltes NJ;Čegan R;Zhang Y;Voytas DF
由于缺乏将DNA修复模板输送到植物细胞的有效方法，使用同源重组来精确修改植物基因组一直是具有挑战性的。尽管序列特异性核酸酶的出现，通过产生有针对性的DNA双链断裂来刺激预先定义的基因组位置的同源重组，但只有少数研究报告了作物内源基因的精确编辑。需要更有效的方法来通过同源重组来修改植物基因组，理想的情况是不随机整合外源DNA。在这里，我们使用双生病毒复制子对番茄基因组进行可遗传修改，其频率比传统的DNA传递方法(即农杆菌)高10倍。在控制花青素生物合成的基因上游插入了一个强启动子，导致番茄组织中色素的过度表达和异位积累。超过三分之二的插入是精确的，没有意外的序列修改。TALENS和CRISPR/CAS9都以相似的效率实现了基因靶向。此外，有针对性的修饰以孟德尔式的方式传递给后代。即使供体分子在载体中复制，也没有发现持续的染色体外复制子或T-DNA或复制子序列的脱靶整合的证据。利用双生病毒复制子实现了对番茄基因组的高频、精确修改，这表明这些载体可以克服使植物基因打靶具有挑战性的效率障碍。这项工作为作物基因组的高效编辑提供了基础，而不需要随机整合外源DNA。本文的在线版本(doi：10.1186/s13059-0150796-9)包含补充材料，授权用户可以使用。
The use of homologous recombination to precisely modify plant genomes has been challenging, due to the lack of efficient methods for delivering DNA repair templates to plant cells. Even with the advent of sequence-specific nucleases, which stimulate homologous recombination at predefined genomic sites by creating targeted DNA double-strand breaks, there are only a handful of studies that report precise editing of endogenous genes in crop plants. More efficient methods are needed to modify plant genomes through homologous recombination, ideally without randomly integrating foreign DNA. Here, we use geminivirus replicons to create heritable modifications to the tomato genome at frequencies tenfold higher than traditional methods of DNA delivery (i.e., Agrobacterium). A strong promoter was inserted upstream of a gene controlling anthocyanin biosynthesis, resulting in overexpression and ectopic accumulation of pigments in tomato tissues. More than two-thirds of the insertions were precise, and had no unanticipated sequence modifications. Both TALENs and CRISPR/Cas9 achieved gene targeting at similar efficiencies. Further, the targeted modification was transmitted to progeny in a Mendelian fashion. Even though donor molecules were replicated in the vectors, no evidence was found of persistent extra-chromosomal replicons or off-target integration of T-DNA or replicon sequences. High-frequency, precise modification of the tomato genome was achieved using geminivirus replicons, suggesting that these vectors can overcome the efficiency barrier that has made gene targeting in plants challenging. This work provides a foundation for efficient genome editing of crop genomes without the random integration of foreign DNA. The online version of this article (doi:10.1186/s13059-015-0796-9) contains supplementary material, which is available to authorized users.