Christian Michelle, Qi Yiping, Zhang Yong, Voytas Daniel F
Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, Minnesota 55455.
G3 (Bethesda). 2013 Oct 3;3(10):1697-705. doi: 10.1534/g3.113.007104.
Custom TAL effector nucleases (TALENs) are increasingly used as reagents to manipulate genomes in vivo. Here, we used TALENs to modify the genome of the model plant, Arabidopsis thaliana. We engineered seven TALENs targeting five Arabidopsis genes, namely ADH1, TT4, MAPKKK1, DSK2B, and NATA2. In pooled seedlings expressing the TALENs, we observed somatic mutagenesis frequencies ranging from 2-15% at the intended targets for all seven TALENs. Somatic mutagenesis frequencies as high as 41-73% were observed in individual transgenic plant lines expressing the TALENs. Additionally, a TALEN pair targeting a tandemly duplicated gene induced a 4.4-kb deletion in somatic cells. For the most active TALEN pairs, namely those targeting ADH1 and NATA2, we found that TALEN-induced mutations were transmitted to the next generation at frequencies of 1.5-12%. Our work demonstrates that TALENs are useful reagents for achieving targeted mutagenesis in this important plant model.
定制的转录激活样效应因子核酸酶（TALENs）越来越多地被用作在体内操纵基因组的试剂。在此，我们使用TALENs对模式植物拟南芥的基因组进行修饰。我们设计了七种靶向五个拟南芥基因的TALENs，这五个基因分别是ADH1、TT4、MAPKKK1、DSK2B和NATA2。在表达TALENs的混合幼苗中，我们观察到所有七种TALENs在预期靶点处的体细胞突变频率在2%至15%之间。在表达TALENs的单个转基因植株系中观察到高达41%至73%的体细胞突变频率。此外，一对靶向串联重复基因的TALENs在体细胞中诱导了一个4.4 kb的缺失。对于最活跃的TALEN对，即那些靶向ADH1和NATA2的TALEN对，我们发现TALEN诱导的突变以1.5%至12%的频率传递给下一代。我们的工作表明，TALENs是在这个重要植物模型中实现靶向诱变的有用试剂。
