Germ cell mutations of the ascidian Ciona intestinalis with TALE nucleases

Summary: Targeted mutagenesis of genes‐of‐interest, or gene‐knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases (TALENs) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN‐mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN‐mediated germ cell mutagenesis. genesis 52:431–439, 2014. © 2014 Wiley Periodicals, Inc.     

Single-stranded DNA versus tailed duplex in sequence conversion of lacZα DNA

Abstract

Targeted DNA editing has great potential to cure some genetic diseases; however, the use of artificial nucleases such as CRISPR-Cas9 and TALEN in gene therapy can potentially cause severe side effects due to off-target DNA cleavages. Single-stranded (ss) DNAs and 5'-tailed duplexes (TDs) can achieve target base substitutions when introduced without artificial nucleases into cultured cells and mouse liver. In this study, ss DNA and TD were separately co-introduced into human U2OS cells, together with a target plasmid DNA bearing an inactivated lacZα gene, and the gene correction efficiencies were compared. Unlike the genes examined in previous studies, ss DNA and TD showed similar efficiencies. Therefore, ss DNAs might be as useful as TD for gene correction, depending on the target sequence.     

非人灵长类基因修饰模型研究进展

非人灵长类动物在生命科学基础研究和生物医药研究领域具有非常重要的地位。近年来随着慢病毒载体转染及靶向核酸酶(ZFN,TALEN,CRISPR/Cas9)等基因操作技术的出现,科学家们成功地获得了外源基因过表达的转基因猴和目的基因定点切割的基因编辑猴。文中对目前利用慢病毒载体获得转基因猴和利用靶向核酸酶获得基因编辑猴的研究进展进行了综述,并讨论了基因修饰猴的嵌合体现象、脱靶现象及非人灵长类动物较长性成熟时间这几个影响非人灵长类基因修饰模型推广应用的因素,最后展望了非人灵长类基因修饰模型构建技术的研究热点及发展趋势。     

基因修饰技术研究进展

基因修饰技术是用于基因组定点改造的分子工具,目前主要有锌指核酸酶(ZFN)技术、转录激活子样效应物核酸酶(TALEN)技术和CRISPR-Cas核酸酶(CRISPR-Cas)技术。这些核酸酶都可以在DNA靶位点产生双链断裂(DSB),诱发细胞内源性的修复机制,激活体内非同源末端连接(NHEJ)或同源重组(HR)两种不同的修复机制,从而实现内源基因的敲除或外源基因的定点敲入。近年来,基因修饰技术已成功应用到细菌、酵母、人类细胞、果蝇、斑马鱼、小鼠、大鼠、家畜、食蟹猴、拟南芥、水稻、烟草、玉米、高粱、小麦和大麦等多种生物,显示了其强大的基因编辑优势。特别是新近出现的CRISPR-Cas9技术,降低了成本,使基因编辑变得简洁、高效和易于操作,得到了很多研究人员的关注。本文系统介绍了以上3种技术的原理及最新研究进展,并对未来的研究和应用做出了展望。     

A large-scale in vivo analysis reveals that TALENs are significantly more mutagenic than ZFNs generated using context-dependent assembly

Zinc-finger nucleases (ZFNs) and TAL effector nucleases (TALENs) have been shown to induce targeted mutations, but they have not been extensively tested in any animal model. Here, we describe a large-scale comparison of ZFN and TALEN mutagenicity in zebrafish. Using deep sequencing, we found that TALENs are significantly more likely to be mutagenic and induce an average of 10-fold more mutations than ZFNs. We observed a strong correlation between somatic and germ-line mutagenicity, and identified germ line mutations using ZFNs whose somatic mutations rates are well below the commonly used threshold of 1%. Guidelines that have previously been proposed to predict optimal ZFN and TALEN target sites did not predict mutagenicity in vivo. However, we observed a significant negative correlation between TALEN mutagenicity and the number of CpG repeats in TALEN target sites, suggesting that target site methylation may explain the poor mutagenicity of some TALENs in vivo. The higher mutation rates and ability to target essentially any sequence make TALENs the superior technology for targeted mutagenesis in zebrafish, and likely other animal models.