Establishment of targeted mutagenesis system in higher plants

TAL效应核酸酶（TALENs）诱导的DNA双链断裂通常通过非同源末端连接（NHEJ）不精确修复，随后导致修复位点发生突变。在这里，我们研究了DNA连接酶4 （Lig4）的缺乏；NHEJ通路的关键因子之一；影响了TALENs诱导水稻细胞DSBs后NHEJ修复的保真度和效率。我们发现在Lig4突变体中突变频率增加。为了建立高效、通用的CRISPR/Cas介导水稻诱变系统，我们比较了不同的Cas9表达构建体，发现不同的Cas9表达构建体突变频率差异很大。我们还发现Cas9的表达水平、Cas9和引导RNA转化的愈伤组织的培养周期影响突变频率。

DNA double strand breaks induced by TAL effector nucleases (TALENs) are often imprecisely repaired by non-homologous end joining (NHEJ) and subsequently lead to mutations at repaired site. Here, we investigated whether the lack of DNA ligase 4 (Lig4); one of the key factor of NHEJ pathway ; affected the fidelity and efficiency of NHEJ repair after induction of DSBs by TALENs in rice cells. We found an enhanced mutation frequency in Lig4 mutant.For establishing an efficient and universal CRISPR/Cas mediated mutagenesis system in rice, we compared different Cas9 expression constructs and found that mutation frequency varied greatly depending on the Cas9 expression constructs. We also revealed that expression level of Cas9, culture period of calli transformed with Cas9 and guide RNA affected mutation frequency.