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。     

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．     

Autonomous zinc-finger nuclease pairs for targeted chromosomal deletion

Zinc-finger nucleases (ZFNs) have been successfully used for rational genome engineering in a variety of cell types and organisms. ZFNs consist of a non-specific FokI endonuclease domain and a specific zinc-finger DNA-binding domain. Because the catalytic domain must dimerize to become active, two ZFN subunits are typically assembled at the cleavage site. The generation of obligate heterodimeric ZFNs was shown to significantly reduce ZFN-associated cytotoxicity in single-site genome editing strategies. To further expand the application range of ZFNs, we employed a combination of in silico protein modeling, in vitro cleavage assays, and in vivo recombination assays to identify autonomous ZFN pairs that lack cross-reactivity between each other. In the context of ZFNs designed to recognize two adjacent sites in the human HOXB13 locus, we demonstrate that two autonomous ZFN pairs can be directed simultaneously to two different sites to induce a chromosomal deletion in ∼10% of alleles. Notably, the autonomous ZFN pair induced a targeted chromosomal deletion with the same efficacy as previously published obligate heterodimeric ZFNs but with significantly less toxicity. These results demonstrate that autonomous ZFNs will prove useful in targeted genome engineering approaches wherever an application requires the expression of two distinct ZFN pairs.     

Unidirectional cloning by cleaving heterogeneous sites with a single sandwiched zinc finger nuclease

We previously developed a novel type of zinc finger nucleases (ZFNs), sandwiched ZFNs that can discriminate DNA substrates from cleavage products and thus cleave DNA much more efficiently than conventional ZFNs as well as perform with multiple turnovers like restriction endonucleases. In the present study, we used the sandwiched ZFN to unidirectionally clone exogenous genes into target vectors by cleaving heterogeneous sites that contained heterogeneous spacer DNAs between two zinc-finger protein binding sites with a single sandwiched ZFN. We demonstrated that the sandwiched ZFN cleaved a 40-fold excess of both insert and vector plasmids within 1 h and confirmed by sequencing that the resulting recombinants harbored the inserted DNA fragment in the desired orientation. Because sandwiched ZFNs can recognize and cleave a variety of long (?26-bp) target DNAs, they may not only expand the utility of ZFNs for construction of recombinant plasmids, but also serve as useful meganucleases for synthesis of artificial genomes.     

Sandwiched zinc-finger nucleases demonstrating higher homologous recombination rates than conventional zinc-finger nucleases in mammalian cells

We previously reported that our sandwiched zinc-finger nucleases (ZFNs), in which a DNA cleavage domain is inserted between two artificial zinc-finger proteins, cleave their target DNA much more efficiently than conventional ZFNs in vitro. In the present study, we compared DNA cleaving efficiencies of a sandwiched ZFN with those of its corresponding conventional ZFN in mammalian cells. Using a plasmid-based single-strand annealing reporter assay in HEK293 cells, we confirmed that the sandwiched ZFN induced homologous recombination more efficiently than the conventional ZFN; reporter activation by the sandwiched ZFN was more than eight times that of the conventional one. Western blot analysis showed that the sandwiched ZFN was expressed less frequently than the conventional ZFN, indicating that the greater DNA-cleaving activity of the sandwiched ZFN was not due to higher expression of the sandwiched ZFN. Furthermore, an MTT assay demonstrated that the sandwiched ZFN did not have any significant cytotoxicity under the DNA-cleavage conditions. Thus, because our sandwiched ZFN cleaved more efficiently than its corresponding conventional ZFN in HEK293 cells as well as in vitro, sandwiched ZFNs are expected to serve as an effective molecular tool for genome editing in living cells.     

Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases

Zinc‐finger nucleases (ZFNs) are engineered nucleases that induce DNA double‐strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non‐homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non‐homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP ‐ZFN mRNAs into the embryos of an EGFP ‐transgenic line and observed the mutation frequency in the target site of EGFP . We also examined the effects of the EGFP ‐ZFNs at off‐target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on‐target site with less effect on the off‐target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis.     

A toolbox and procedural notes for characterizing novel zinc finger nucleases for genome editing in plant cells

The induction of double-strand breaks (DSBs) in plant genomes can lead to increased homologous recombination or site-specific mutagenesis at the repair site. This phenomenon has the potential for use in gene targeting applications in plant cells upon the induction of site-specific genomic DSBs using zinc finger nucleases (ZFNs). Zinc finger nucleases are artificial restriction enzymes, custom-designed to cleave a specific DNA sequence. The tools and methods for ZFN assembly and validation could potentially boost their application for plant gene targeting. Here we report on the design of biochemical and in planta methods for the analysis of newly designed ZFNs. Cloning begins with de novo assembly of the DNA-binding regions of new ZFNs from overlapping oligonucleotides containing modified helices responsible for DNA-triplet recognition, and the fusion of the DNA-binding domain with a Fok I endonuclease domain in a dedicated plant expression cassette. Following the transfer of fully assembled ZFNs into Escherichia coli expression vectors, bacterial lysates were found to be most suitable for in vitro digestion analysis of palindromic target sequences. A set of three in planta activity assays was also developed to confirm the nucleic acid digestion activity of ZFNs in plant cells. The assays are based on the reconstruction of GUS expression following transient or stable delivery of a mutated uidA and ZFN-expressing cassettes into target plants cells. Our tools and assays offer cloning flexibility and simple assembly of tested ZFNs and their corresponding target sites into Agrobacterium tumefaciens binary plasmids, allowing efficient implementation of ZFN-validation assays in planta .     

Zinc finger protein-dependent and -independent contributions to the in vivo off-target activity of zinc finger nucleases

Zinc finger nucleases (ZFNs) facilitate tailor-made genomic modifications in vivo through the creation of targeted double-stranded breaks. They have been employed to modify the genomes of plants and animals, and cell-based therapies utilizing ZFNs are undergoing clinical trials. However, many ZFNs display dose-dependent toxicity presumably due to the generation of undesired double-stranded breaks at off-target sites. To evaluate the parameters influencing the functional specificity of ZFNs, we compared the in vivo activity of ZFN variants targeting the zebrafish kdrl locus, which display both high on-target activity and dose-dependent toxicity. We evaluated their functional specificity by assessing lesion frequency at 141 potential off-target sites using Illumina sequencing. Only a minority of these off-target sites accumulated lesions, where the thermodynamics of zinc finger–DNA recognition appear to be a defining feature of active sites. Surprisingly, we observed that both the specificity of the incorporated zinc fingers and the choice of the engineered nuclease domain could independently influence the fidelity of these ZFNs. The results of this study have implications for the assessment of likely off-target sites within a genome and point to both zinc finger-dependent and -independent characteristics that can be tailored to create ZFNs with greater precision.     

In vivo cleavage of transgene donors promotes nuclease‐mediated targeted integration

Targeted DNA integration is commonly used to eliminate position effects on transgene expression. Integration can be targeted to specific sites in the genome via both homology‐based and homology‐independent processes. Both pathways start the integration process with a site‐specific break in the chromosome, typically from a zinc‐finger nuclease (ZFN). We previously described an efficient homology‐independent targeted integration technique that captures short (<100 bp) pieces of DNA at chromosomal breaks created by ZFNs. We show here that inclusion of a nuclease target site on the donor plasmid followed by in vivo nuclease cleavage of both the donor and the chromosome results in efficient integration of large, transgene‐sized DNA molecules into the chromosomal double‐strand break. Successful targeted integration via in vivo donor linearization is demonstrated at five distinct loci in two mammalian cell types, highlighting the generality of the approach. Finally, we show that CHO cells, a cell type recalcitrant to homology‐based integration, are proficient at capture of in vivo‐linearized transgene donors. Moreover, we demonstrate knockout of the hamster FUT8 gene via the simultaneous ZFN‐ or TALE nuclease‐mediated integration of an antibody cassette. Our results enable efficient targeted transgene addition to cells and organisms that fare poorly with traditional homology‐driven approaches. Biotechnol. Bioeng. 2013; 110: 871–880. © 2012 Wiley Periodicals, Inc.     

In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites

Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described–one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.     

Targeted editing of goat genome with modular-assembly zinc finger nucleases based on activity prediction by computational molecular modeling

Zinc finger nuclease (ZFN) technology can mediate targeted genome modification to produce transgenic animals in a high-efficient and biological-safe way. Modular assembly is a rapid, convenient and open-source method for the synthesis of ZFNs. However, this biotechnology is hampered by multistep construction, low-efficiency editing and off-target cleavage. Here we synthesized and tested six pairs of three- or four-finger ZFNs to target one site in goat beta-lactoglobulin (BLG, a dominant allergen in goat milk) gene. Homology modeling was applied to build the structure model of ZFNs to predict their editing activities targeting at goat BLG gene. Goat fibroblast cells were transfected with plasmids that encoded ZFN pairs, and genomic DNA was isolated 72 h later for genome editing efficiency assay. The results of editing efficiency assay demonstrated that ZFNs with optimal interaction modes can edit goat BLG gene more efficiently, whereas ZFNs with unexpected interaction modes showed lower activities in editing BLG gene. We concluded that modular-assembly ZFNs can provide a rapid, public-available, and easy-to-practice platform for transgenic animal research and molecular modeling would help as a useful tool for ZFNs activity prediction.     

Binding of two zinc finger nuclease monomers to two specific sites is required for effective double-strand DNA cleavage

Custom-designed zinc finger nucleases (ZFNs) are becoming powerful tools in gene targeting-the process of replacing a gene within a genome by homologous recombination. Here, we have studied the DNA cleavage by one such ZFN, DeltaQNK-FN, in order to gain insight into how ZFNs cleave DNA and how two inverted sites promote double-strand cleavage. DNA cleavage by DeltaQNK-FN is greatly facilitated when two DeltaQNK-binding sites are close together in an inverted orientation. Substrate cleavage was not first order with respect to the concentration of DeltaQNK-FN, indicating that double-strand cleavage requires dimerization of the FokI cleavage domain. Rates of DNA cleavage decrease as the substrate concentrations increase, suggesting that the DeltaQNK-FN molecules are effectively "trapped" in a 1:1 complex on DNA when the DNA is in excess. The physical association of two ZFN monomers on DNA was monitored by using the biotin-pull-down assay, which showed that the formation of DeltaQNK-FN active complex required both binding of the two DeltaQNK-FN molecules to specific DNA sites and divalent metal ions.     

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.     

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.     

Targeted transgene integration in plant cells using designed zinc finger nucleases

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.     

锌指核酸酶技术在植物基因工程中的应用

锌指核酸酶(zinc finger nucleases,ZFNs)由3到4个锌指结构(zinc fingers,ZFs)和FokⅠ核酸内切酶的剪切结构域组成。锌指核酸酶(ZFNs)通过锌指结构(ZFs)与特异核酸位点结合,再利用FokⅠ的酶切作用切割DNA,引起特异位点DNA双链断裂(double strand break,DSB)。DNA双链断裂可以通过非同源末端连接(non-homologous end joining,NHEJ) 或同源重组(homologous recombination,HR)来修复。在修复过程中实现对基因组DNA的靶向修饰。介绍了锌指核酸酶结构、人工构建途径,作用机理和试验步骤,重点综述了锌指核酸酶技术在植物基因工程的应用。     

A novel arrangement of zinc finger nuclease system for in vivo targeted genome engineering: the tomato <Emphasis Type="Italic">LEC1</Emphasis>-<Emphasis Type="Italic">LIKE4</Emphasis> gene case

Key message

A selection-free, highly efficient targeted mutagenesis approach based on a novel ZFN monomer arrangement for genome engineering in tomato reveals plant trait modifications.

Abstract

How to achieve precise gene targeting in plants and especially in crops remains a long-sought goal for elucidating gene function and advancing molecular breeding. To address this issue, zinc finger nuclease (ZFN)-based technology was developed for the Solanum lycopersicum seed system. A ZFN architecture design with an intronic sequence between the two DNA recognition sites was evaluated for its efficiency in targeted gene mutagenesis. Custom engineered ZFNs for the developmental regulator LEAFY-COTYLEDON1-LIKE4 (L1L4) coding for the β subunit of nuclear factor Y, when transiently expressed in tomato seeds, cleaved the target site and stimulated imperfect repair driven by nonhomologous end-joining, thus, introducing mutations into the endogenous target site. The successful in planta application of the ZFN platform resulted in L1L4 mutations which conferred heterochronic phenotypes during development. Our results revealed that sequence changes upstream of the DNA binding domain of L1L4 can lead to phenotypic diversity including fruit organ. These results underscore the utility of engineered ZFN approach in targeted mutagenesis of tomato plant which may accelerate translational research and tomato breeding.