Application of CRISPR/Cas9 Nuclease in Amphioxus Genome Editing.
Application of CRISPR/Cas9 Nuclease in Amphioxus Genome Editing.
复制标题
CRISPR/Cas9核酸酶在文昌鱼基因组编辑中的应用
作者：
Su L;Shi C;Huang X;Wang Y;Li G
头索动物文昌鱼是研究脊椎动物起源的重要模式动物。虽然转录激活因子样效应核酸酶（TALEN）已被用于文昌鱼基因组编辑，但其TALEN蛋白的劳动密集型构建限制了其在许多实验室中的使用。在这里，我们报告了CRISPR/Cas9系统在这组动物中的应用，这是一种更适合的基因组编辑方法。我们的数据表明，虽然将Cas9 mRNA和sgRNA共注射到文昌鱼未受精卵中不会在靶位点引起可检测的突变，但在两细胞阶段注射Cas9 mRNA和sgRNA，或在受精前注射Cas9蛋白和sgRNA，可以有效破坏靶基因。在与Cas9蛋白共注射的9种测试的sgRNA（靶向5个基因）中，7种引入了突变，效率范围为18.4%至90%，4种在注射的胚胎中引起了特定的表型。我们还证明，通过热处理或修饰sgRNA结构使sgRNA单体化可以增加突变效率。我们的研究不仅将推动基因组编辑方法在文昌鱼研究中的应用，也将为CRISPR/Cas9系统尚未成功应用的其他生物提供宝贵的经验。
The cephalochordate amphioxus is a promising animal model for studying the origin of vertebrates due to its key phylogenetic position among chordates. Although transcription activator-like effector nucleases (TALENs) have been adopted in amphioxus genome editing, its labor-intensive construction of TALEN proteins limits its usage in many laboratories. Here we reported an application of the CRISPR/Cas9 system, a more amenable genome editing method, in this group of animals. Our data showed that while co-injection of Cas9 mRNAs and sgRNAs into amphioxus unfertilized eggs caused no detectable mutations at targeted loci, injections of Cas9 mRNAs and sgRNAs at the two-cell stage, or of Cas9 protein and sgRNAs before fertilization, can execute efficient disruptions of targeted genes. Among the nine tested sgRNAs (targeting five genes) co-injected with Cas9 protein, seven introduced mutations with efficiency ranging from 18.4% to 90% and four caused specific phenotypes in the injected embryos. We also demonstrated that monomerization of sgRNAs via thermal treatment or modifying the sgRNA structure could increase mutation efficacies. Our study will not only promote application of genome editing method in amphioxus research, but also provide valuable experiences for other organisms in which the CRISPR/Cas9 system has not been successfully applied.
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影响因子：
12.3
作者：
Dang Y;Jia G;Choi J;Ma H;Anaya E;Ye C;Shankar P;Wu H
通讯作者：
Wu H
利用准确的非同源末端连接在 CRISPR/Cas9 介导的基因组编辑中实现高效精确删除
影响因子：
12.3
作者：
Guo T;Feng YL;Xiao JJ;Liu Q;Sun XN;Xiang JF;Kong N;Liu SC;Chen GQ;Wang Y;Dong MM;Cai Z;Lin H;Cai XJ;Xie AY
通讯作者：
Xie AY
DOI：
10.1073/pnas.1308335110
发表时间：
2013-08-20
影响因子：
11.1
作者：
Jao, Li-En;Wente, Susan R.;Chen, Wenbiao
通讯作者：
Chen, Wenbiao
影响因子：
3.7
作者：
Li G;Yang X;Shu Z;Chen X;Wang Y
通讯作者：
Wang Y
中国文昌鱼（Branchiostoma belcheri）的实验室全年繁殖和诱导产卵。
影响因子：
3.7
作者：
Li G;Shu Z;Wang Y
通讯作者：
Wang Y