锌指核酸酶在基因组定向修饰中的应用

同源重组和逆转录病毒介导转基因法是目前基因组修饰中常用的两种主要方法.由于这些传统方法效率低,特异性差等缺点,制约了其在研究中的应用.锌指核酸酶(zinc finger nuclease,ZFN）是一种人工合成酶,含有锌指蛋白DNA结合域和非特异性核酸酶FokI结构域. ZFN在对基因组的靶向修饰时,表现出高度特异性和高效性. 最新研究结果显示,锌指核酸酶在哺乳动物细胞和斑马鱼基因组靶向敲除的效率高达20%.这一技术的出现,将给基因组靶向修饰的研究和应用领域带来革命,特别是在基因治疗人类疾病方面有巨大的潜力和广阔的前景.     

人工锌指核酸酶介导的基因组定点修饰技术

锌指核酸酶(ZFN)由锌指蛋白(ZFP)结构域和Fok I核酸内切酶的切割结构域人工融合而成,是近年来发展起来的一种可用于基因组定点改造的分子工具。ZFN可识别并结合特定的DNA序列,并通过切割这一序列的特定位点造成DNA的双链断裂(DSB)。在此基础上,人们可以对基因组的特定位点进行各种遗传操作,包括基因打靶、基因定点插入、基因修复等,从而能够方便快捷地对基因组实现靶向遗传修饰。这种新的基因组定点修饰方法的突出优势是适用性好,对物种没有选择性,并且可以在细胞和个体水平进行遗传操作。文章综述了ZFN技术的研究进展及应用前景,重点介绍ZFN的结构与作用机制、现有的靶点评估及锌指蛋白库的构建与筛选方法、基因组定点修饰的策略,以及目前利用这一技术已成功实现突变的物种及内源基因,为开展这一领域的研究工作提供参考。     

锌指核酸内切酶:基因操作的有力工具

针对动植物进行基因靶向操作的技术,是解析基因功能、研究疾病,以及农业经济生产中一个有用的工具。至今,基因靶向操作主要是通过在胚胎干细胞(embryonic stem cell,ES cell)中进行同源重组或者是体细胞核转移的方法进行,但同源重组方法由于需要ES细胞而被限制在个别物种,而核转移方法存在核去分化、效率低、成本高的缺陷。近几年,一种基于锌指核酸内切酶(zinc-finger nuclease,ZFN)基因靶向修饰的新技术被应用于包括植物、果蝇、爪蟾、斑马鱼和大鼠等不同物种的基因操作。通过胚胎注射ZFN的质粒或是mRNA可以有效地定靶并迅速地在内源基因上引起可遗传的突变。ZFN介导基因靶向敲除的可行性,使得那些无法获得ES细胞和克隆技术支持的物种的基因靶向修饰成为可能。     

人工核酸酶介导的基因组编辑技术

传统的基因组编辑技术是基于胚胎干细胞和同源重组实现生物基因组定向改造,但是该技术打靶效率低,严重制约了生命科学以及医学的研究.因此,研究新的基因组编辑技术十分重要.人工核酸酶介导的基因组编辑技术是通过特异性识别靶位点造成DNA双链断裂,引起细胞内源性的修复机制实现靶基因的修饰.与传统的基因组编辑技术相比,人工核酸酶技术打靶效率高,这对于基因功能的研究、构建人类疾病动物模型以及探索新型疾病治疗方案有着重要的意义.人工核酸酶技术有3种类型：锌指核酸酶(ZFN)、类转录激活因子核酸酶(TALEN)及规律成簇的间隔短回文重复序列(CRISPR).本文将对以上3种人工核酸酶技术的原理以及在生命科学和医学研究的应用进行综述.     

基因组编辑技术中供体DNA类型及选择北大核心CSCD

基因组编辑技术能够实现基因组的精确修饰和改造,是后基因组时代研究基因功能和遗传信息的主要手段。传统的基因打靶技术通过低效率的细胞自发同源重组实现目的基因的定点修饰。真核细胞中DNA双链断裂介导的同源重组效率远高于自发同源重组,利用人工核酸内切酶特异性地在基因组靶序列处引入双链断裂,通过提供适当形式的、含有一定长度同源臂的供体DNA,能够实现相对高效的基因组靶向编辑。本文系统总结了环状质粒、线性化质粒、聚合酶链式反应产物及单链寡聚脱氧核苷酸4种类型的供体DNA在基因组精确编辑研究中的应用及候选原则,以期为以后相关研究中供体DNA的选择、设计提供参考和借鉴。     

基因组编辑技术中供体DNA类型及选择

基因组编辑技术能够实现基因组的精确修饰和改造,是后基因组时代研究基因功能和遗传信息的主要手段。传统的基因打靶技术通过低效率的细胞自发同源重组实现目的基因的定点修饰。真核细胞中DNA双链断裂介导的同源重组效率远高于自发同源重组,利用人工核酸内切酶特异性地在基因组靶序列处引入双链断裂,通过提供适当形式的、含有一定长度同源臂的供体DNA,能够实现相对高效的基因组靶向编辑。本文系统总结了环状质粒、线性化质粒、聚合酶链式反应产物及单链寡聚脱氧核苷酸4种类型的供体DNA在基因组精确编辑研究中的应用及候选原则,以期为以后相关研究中供体DNA的选择、设计提供参考和借鉴。     

锌指蛋白核酸酶的作用原理及其应用

锌指蛋白核酸酶(Zinc finger nucleases,ZFN)因其能特异性识别并切割DNA序列以及可设计性,被用于基因定点突变和外源基因定点整合。目前,ZFN技术以其准确的靶位点设计能力和诱发高效率基因打靶的优势,越来越受到基因改造研究者的重视,已经成功应用于动植物细胞、胚胎的基因改造。随着鉴定靶DNA高亲和力的锌指蛋白(Zinc finger protein,ZFP)实验技术日渐成熟,可以预见到不久的将来这项技术会在基因工程和育种中得到广泛应用。文章介绍了锌指蛋白识别DNA靶位点和ZFN介导的基因打靶(Double strand break gene targeting,DSB-GT)的原理,同时还综述了目前ZFN技术用于基因改造的研究进展。     

Evaluation of OPEN Zinc Finger Nucleases for Direct Gene Targeting of the ROSA26 Locus in Mouse Embryos

Zinc finger nucleases (ZFNs) enable precise genome modification in a variety of organisms and cell types. Commercial ZFNs were reported to enhance gene targeting directly in mouse zygotes, whereas similar approaches using publicly available resources have not yet been described. Here we report precise targeted mutagenesis of the mouse genome using Oligomerized Pool Engineering (OPEN) ZFNs. OPEN ZFN can be constructed using publicly available resources and therefore provide an attractive alternative for academic researchers. Two ZFN pairs specific to the mouse genomic locus gt(ROSA26)Sor were generated by OPEN selections and used for gene disruption and homology-mediated gene replacement in single cell mouse embryos. One specific ZFN pair facilitated non-homologous end joining (NHEJ)-mediated gene disruption when expressed in mouse zygotes. We also observed a single homologous recombination (HR)-driven gene replacement event when this ZFN pair was co-injected with a targeting vector. Our experiments demonstrate the feasibility of achieving both gene ablation through NHEJ and gene replacement by HR by using the OPEN ZFN technology directly in mouse zygotes.     

Gene editing in stem cells hits the target

Two recent reports describe promising, highly efficient methods to modify genes in pluripotent stem cells using zinc finger nuclease (ZFN)-mediated (Soldner et?al., 2011) or helper-dependent adenovirus (HDAdV)-mediated (Liu et?al., 2011b) gene modification. These technical developments will have far ranging effects on the rapidly growing field of regenerative medicine.     

Repeatable Construction Method for Engineered Zinc Finger Nuclease Based on Overlap Extension PCR and TA-Cloning

Zinc finger nuclease (ZFN) is a useful tool for endogenous site-directed genome modification. The development of an easier, less expensive and repeatedly usable construction method for various sequences of ZFNs should contribute to the further widespread use of this technology. Here, we establish a novel construction method for ZFNs. Zinc finger (ZF) fragments were synthesized by PCR using short primers coding DNA recognition helices of the ZF domain. DNA-binding domains composed of 4 to 6 ZFs were synthesized by overlap extension PCR of these PCR products, and the DNA-binding domains were joined with a nuclease vector by TA cloning. The short primers coding unique DNA recognition helices can be used repeatedly for other ZFN constructions. By using this novel OLTA (OverLap extension PCR and TA-cloning) method, arbitrary ZFN vectors were synthesized within 3 days, from the designing to the sequencing of the vector. Four different ZFN sets synthesized by OLTA showed nuclease activities at endogenous target loci. Genetically modified mice were successfully generated using ZFN vectors constructed by OLTA. This method, which enables the construction of intended ZFNs repeatedly and inexpensively in a short period of time, should contribute to the advancement of ZFN technology.     

利用ZFN删除转基因猪细胞中的多拷贝EGFP基因

锌指核酸酶（zinc finger nuclease, ZFN）是由特异性识别DNA的锌指结构域和Fok I切割结构域组成,能够在基因组特定位点上切割DNA,引起DNA双链断裂（double-strand break, DSB）. 通过DSB修复机制,可以使基因修饰的效率比传统方法提高102～104倍．目前,利用ZFN对动物内源基因进行敲除的研究较多,但对转基因动物中外源多拷贝基因进行敲除的报道较少．本研究首先利用荧光定量PCR法对本实验室培育的两头转基因猪中增强型绿色荧光蛋白（enhanced green fluorescent protein, EGFP）基因的拷贝数进行鉴定,发现其拷贝数分别为11.95和17.36拷贝;然后将靶向EGFP的一对ZFN转染进拷贝数为1736的EGFP转基因猪的成纤维细胞中,并通过流式和CEL-1酶切方法检测敲除效率. 结果表明,转染400 ng、800 ng和1 200 ng ZFN的切割效率分别为0.97%、1.39%和1.76%,可见随着转染ZFN剂量的增加,ZFN的切割效率逐渐提高．但是,不发绿色荧光的细胞比例却没有明显提高,因此认为,ZFN敲除转基因动物中多拷贝基因的效率还是比较低．     

Potential application of FoldX force field based protein modeling in zinc finger nucleases design

Engineered sequence-specific zinc finger nucleases (ZFNs) make the highly efficient modification of eukaryotic genomes possible. However, most current strategies for developing zinc finger nucleases with customized sequence specificities require the construction of numerous tandem arrays of zinc finger proteins (ZFPs), and subsequent largescale in vitro validation of their DNA binding affinities and specificities via bacterial selection. The labor and expertise required in this complex process limits the broad adoption of ZFN technology. An effective computational assisted design strategy will lower the complexity of the production of a pair of functional ZFNs. Here we used the FoldX force field to build 3D models of 420 ZFP-DNA complexes based on zinc finger arrays developed by the Zinc Finger Consortium using OPEN (oligomerized pool engineering). Using nonlinear and linear regression analysis, we found that the calculated protein-DNA binding energy in a modeled ZFP-DNA complex strongly correlates to the failure rate of the zinc finger array to show significant ZFN activity in human cells. In our models, less than 5% of the three-finger arrays with calculated protein-DNA binding energies lower than −13.132 kcal mol⁻¹ fail to form active ZFNs in human cells. By contrast, for arrays with calculated protein-DNA binding energies higher than −5 kcal mol⁻¹, as many as 40% lacked ZFN activity in human cells. Therefore, we suggest that the FoldX force field can be useful in reducing the failure rate and increasing efficiency in the design of ZFNs.     

Attenuation of Zinc Finger Nuclease Toxicity by Small-Molecule Regulation of Protein Levels

Zinc finger nucleases (ZFNs) have been used successfully to create genome-specific double-strand breaks and thereby stimulate gene targeting by several thousand fold. ZFNs are chimeric proteins composed of a specific DNA-binding domain linked to a non-specific DNA-cleavage domain. By changing key residues in the recognition helix of the specific DNA-binding domain, one can alter the ZFN binding specificity and thereby change the sequence to which a ZFN pair is being targeted. For these and other reasons, ZFNs are being pursued as reagents for genome modification, including use in gene therapy. In order for ZFNs to reach their full potential, it is important to attenuate the cytotoxic effects currently associated with many ZFNs. Here, we evaluate two potential strategies for reducing toxicity by regulating protein levels. Both strategies involve creating ZFNs with shortened half-lives and then regulating protein level with small molecules. First, we destabilize ZFNs by linking a ubiquitin moiety to the N-terminus and regulate ZFN levels using a proteasome inhibitor. Second, we destabilize ZFNs by linking a modified destabilizing FKBP12 domain to the N-terminus and regulate ZFN levels by using a small molecule that blocks the destabilization effect of the N-terminal domain. We show that by regulating protein levels, we can maintain high rates of ZFN-mediated gene targeting while reducing ZFN toxicity.     

利用人工锌指蛋白核酸酶进行植物基因定点突变和置换

基因定点突变技术在基因组原位改变基因特定序列,避免常规转基因过程中位置效应和插入失活。定点突变生物体不含转基因或标记基因,降低风险性。高等植物基因定点突变研究初见端倪,将可能为基因原位功能研究、作物遗传改良和分子设计提供有效策略。利用锌指蛋白核酸酶（Zinc Finger Nucleases, ZFN）引入DNA定点断裂（Double-Strand Breaks, DSBs）可以高效介导基因定点突变,使得ZFN在基因定点突变中倍受关注。文章综述了植物基因定点突变的一般策略,重点介绍了锌指蛋白的结构、原理、应用,特别是ZFN介导的植物基因定点突变与置换研究进展,并对ZFN介导的植物基因定点突变与置换应用前景进行了讨论。