Transcription Activator-Like Effector Nucleases Enable Efficient Plant Genome Engineering
Transcription Activator-Like Effector Nucleases Enable Efficient Plant Genome Engineering
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DOI：
10.1104/pp.112.205179
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发表时间：
2013-01-01
期刊：
影响因子：
7.4
通讯作者：
Voytas, Daniel F.
中科院分区：
文献类型：
--
作者：
Zhang, Yong;Zhang, Feng;Voytas, Daniel F.
精确设计植物基因组的能力为推进基础和应用植物生物学提供了巨大的潜力。在这里，我们描述了利用转录激活子样效应核酸酶（TALENs）靶向修饰植物基因组的方法。利用烟草（Nicotiana tabacum）原生质体和靶向乙酰乳酸合成酶（ALS）基因的TALENs对方法进行优化。通过原生质体单链退火实验确定了最佳的TALEN支架，其中TALEN裂解产生了一个功能性的黄色荧光蛋白基因，从而可以通过流式细胞术定量TALEN活性。通过对包含TALEN识别位点的聚合酶链反应产物进行高通量DNA测序，不同支架的TALENs单链退火活性数据与它们在内源性靶标上的活性高度相关。TALENs在30%的转化细胞中引入了ALS的靶向突变，靶向基因插入的频率约为14%。这些效率使得在没有选择或富集机制的情况下恢复基因组修饰成为可能：用talen编码构建体转化的原生质体产生的32%的烟草愈伤组织具有talen诱导的ALS突变，并且在详细描述的16个愈伤组织中，每个重复的ALS基因（SurA和SurB）都有一个等位基因突变。在用TALEN和与ALS编码序列相差6bp的322 bp供体分子处理的愈伤组织中，4%的愈伤组织显示出靶向基因替代的证据。在植物原生质体中实施的优化试剂应可用于多种植物物种细胞的靶向修饰，并使用多种试剂递送手段。
The ability to precisely engineer plant genomes offers much potential for advancing basic and applied plant biology. Here, we describe methods for the targeted modification of plant genomes using transcription activator-like effector nucleases (TALENs). Methods were optimized using tobacco (Nicotiana tabacum) protoplasts and TALENs targeting the acetolactate synthase (ALS) gene. Optimal TALEN scaffolds were identified using a protoplast-based single-strand annealing assay in which TALEN cleavage creates a functional yellow fluorescent protein gene, enabling quantification of TALEN activity by flow cytometry. Single-strand annealing activity data for TALENs with different scaffolds correlated highly with their activity at endogenous targets, as measured by high-throughput DNA sequencing of polymerase chain reaction products encompassing the TALEN recognition sites. TALENs introduced targeted mutations in ALS in 30% of transformed cells, and the frequencies of targeted gene insertion approximated 14%. These efficiencies made it possible to recover genome modifications without selection or enrichment regimes: 32% of tobacco calli generated from protoplasts transformed with TALEN-encoding constructs had TALEN-induced mutations in ALS, and of 16 calli characterized in detail, all had mutations in one allele each of the duplicate ALS genes (SurA and SurB). In calli derived from cells treated with a TALEN and a 322-bp donor molecule differing by 6 bp from the ALS coding sequence, 4% showed evidence of targeted gene replacement. The optimized reagents implemented in plant protoplasts should be useful for targeted modification of cells from diverse plant species and using a variety of means for reagent delivery.