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 .     

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。     

Effects of DNA binding of the zinc finger and linkers for domain fusion on the catalytic activity of sequence-specific chimeric recombinases determined by a facile fluorescent system

Artificial zinc finger proteins (ZFPs) consist of Cys(2)-His(2)-type modules composed of ～30 amino acids with a ββα structure that coordinates a zinc ion. ZFPs that recognize specific DNA target sequences can substitute for the binding domains of enzymes that act on DNA to create designer enzymes with programmable sequence specificity. The most studied of these engineered enzymes are zinc finger nucleases (ZFNs). ZFNs have been widely used to model organisms and are currently in human clinical trials with an aim of therapeutic gene editing. Difficulties with ZFNs arise from unpredictable mutations caused by nonhomologous end joining and off-target DNA cleavage and mutagenesis. A more recent strategy that aims to address the shortcomings of ZFNs involves zinc finger recombinases (ZFRs). A thorough understanding of ZFRs and methods for their modification promises powerful new tools for gene manipulation in model organisms as well as in gene therapy. In an effort to design efficient and specific ZFRs, the effects of the DNA binding affinity of the zinc finger domains and the linker sequence between ZFPs and recombinase catalytic domains have been assessed. A plasmid system containing ZFR target sites was constructed for evaluation of catalytic activities of ZFRs with variable linker lengths and numbers of zinc finger modules. Recombination efficiencies were evaluated by restriction enzyme analysis of isolated plasmids after reaction in Escherichia coli and changes in EGFP fluorescence in mammalian cells. The results provide information relevant to the design of ZFRs that will be useful for sequence-specific genome modification.     

人工锌指核酸酶的设计与表达纯化

设计表达了四个锌指核酸酶,用于切断人基因组中的rRNA基因家族的内部转录间隔序列,造成双链断裂,以此提高针对多位点基因打靶的效率,为后续基因打靶应用于基因治疗研究奠定基础。首先,在人rRNA基因家族ITS1序列中找到两个合适的9 bp长的序列(中间间隔6 bp)为锌指蛋白识别位点,根据识别位点序列每个位点分别设计两个三锌指蛋白。通过设计引物进行重叠延伸PCR得到全长编码锌指蛋白的DNA,分别克隆到表达载体pET-28a（+）,构建重组质粒pET28a-ZFP,转化大肠杆菌RossettaTM（DE3）,实现带组氨酸标签的锌指融合蛋白的表达与纯化。同时,将限制性内切酶Fok I的切割结构域分别与四个锌指蛋白序列采用PCR拼接后克隆到表达载体pET-28a（+）,构建重组质粒pET28a-ZFN,转化到大肠杆菌RossettaTM（DE3）,实现带组氨酸标签的锌指核酸酶融合蛋白的表达并纯化。     

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.     

Progress of targeted genome modification approaches in higher plants

Transgene integration in plants is based on illegitimate recombination between non-homologous sequences. The low control of integration site and number of (trans/cis)gene copies might have negative consequences on the expression of transferred genes and their insertion within endogenous coding sequences. The first experiments conducted to use precise homologous recombination for gene integration commenced soon after the first demonstration that transgenic plants could be produced. Modern transgene targeting categories used in plant biology are: (a) homologous recombination-dependent gene targeting; (b) recombinase-mediated site-specific gene integration; (c) oligonucleotide-directed mutagenesis; (d) nuclease-mediated site-specific genome modifications. New tools enable precise gene replacement or stacking with exogenous sequences and targeted mutagenesis of endogeneous sequences. The possibility to engineer chimeric designer nucleases, which are able to target virtually any genomic site, and use them for inducing double-strand breaks in host DNA create new opportunities for both applied plant breeding and functional genomics. CRISPR is the most recent technology available for precise genome editing. Its rapid adoption in biological research is based on its inherent simplicity and efficacy. Its utilization, however, depends on available sequence information, especially for genome-wide analysis. We will review the approaches used for genome modification, specifically those for affecting gene integration and modification in higher plants. For each approach, the advantages and limitations will be noted. We also will speculate on how their actual commercial development and implementation in plant breeding will be affected by governmental regulations.     

Chimeric restriction enzymes: what is next?

Chimeric restriction enzymes are a novel class of engineered nucleases in which the non-specific DNA cleavage domain of Fokl (a type IIS restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely the helix-turn-helix motif, the zinc finger motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-F(N) was shown to find and cleave its target in vivo. This was tested by microinjection of DNA substrates and the enzyme into frog oocytes (Carroll et al., 1999). The injected enzyme made site-specific double-strand breaks in the targets even after assembly of the DNA into chromatin. In addition, this cleavage activated the target molecules for efficient homologous recombination. Since the recognition specificity of zinc fingers can be manipulated experimentally, chimeric nucleases could be engineered so as to target a specific site within a genome. The availability of such engineered chimeric restriction enzymes should make it feasible to do genome engineering, also commonly referred to as gene therapy.     

CRISPR/Cas9:基因编辑的新时代

基因编辑技术是通过核酸内切酶对基因组DNA进行定向改造的技术,可以实现对特定DNA碱基的缺失、替换等,常用的四种基因编辑工具分别是:巨型核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶以及CRISPR/Cas9系统.其中CRISPR/Cas9系统作为一种新型的基因组编辑技术具有组成简单、特异性好、切割效率高的优点.该文对...     

Gene targeting using zinc finger nucleases

The ability to achieve site-specific manipulation of the mammalian genome has widespread implications for basic and applied research. Gene targeting is a process in which a DNA molecule introduced into a cell replaces the corresponding chromosomal segment by homologous recombination, and thus presents a precise way to manipulate the genome. In the past, the application of gene targeting to mammalian cells has been limited by its low efficiency. Zinc finger nucleases (ZFNs) show promise in improving the efficiency of gene targeting by introducing DNA double-strand breaks in target genes, which then stimulate the cell's endogenous homologous recombination machinery. Recent results have shown that ZFNs can be used to create targeting frequencies of up to 20% in a human disease-causing gene. Future work will be needed to translate these in vitro findings to in vivo applications and to determine whether zinc finger nucleases create undesired genomic instability.     

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.     

Use of designer nucleases for targeted gene and genome editing in plants

The ability to efficiently inactivate or replace genes in model organisms allowed a rapid expansion of our understanding of many of the genetic, biochemical, molecular and cellular mechanisms that support life. With the advent of new techniques for manipulating genes and genomes that are applicable not only to single‐celled organisms, but also to more complex organisms such as animals and plants, the speed with which scientists and biotechnologists can expand fundamental knowledge and apply that knowledge to improvements in medicine, industry and agriculture is set to expand in an exponential fashion. At the heart of these advancements will be the use of gene editing tools such as zinc finger nucleases, modified meganucleases, hybrid DNA/RNA oligonucleotides, TAL effector nucleases and modified CRISPR/Cas9. Each of these tools has the ability to precisely target one specific DNA sequence within a genome and (except for DNA/RNA oligonucleotides) to create a double‐stranded DNA break. DNA repair to such breaks sometimes leads to gene knockouts or gene replacement by homologous recombination if exogenously supplied homologous DNA fragments are made available. Genome rearrangements are also possible to engineer. Creation and use of such genome rearrangements, gene knockouts and gene replacements by the plant science community is gaining significant momentum. To document some of this progress and to explore the technology's longer term potential, this review highlights present and future uses of designer nucleases to greatly expedite research with model plant systems and to engineer genes and genomes in major and minor crop species for enhanced food production.     

Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells

Custom-designed zinc finger nucleases (ZFNs), proteins designed to cut at specific DNA sequences, are becoming powerful tools in gene targeting—the process of replacing a gene within a genome by homologous recombination (HR). ZFNs that combine the non-specific cleavage domain (N) of FokI endonuclease with zinc finger proteins (ZFPs) offer a general way to deliver a site-specific double-strand break (DSB) to the genome. The development of ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically and permanently modify plant and mammalian genomes including the human genome via homology-directed repair of a targeted genomic DSB. The creation of designer ZFNs that cleave DNA at a pre-determined site depends on the reliable creation of ZFPs that can specifically recognize the chosen target site within a genome. The (Cys₂His₂) ZFPs offer the best framework for developing custom ZFN molecules with new sequence-specificities. Here, we explore the different approaches for generating the desired custom ZFNs with high sequence-specificity and affinity. We also discuss the potential of ZFN-mediated gene targeting for ‘directed mutagenesis’ and targeted ‘gene editing’ of the plant and mammalian genome as well as the potential of ZFN-based strategies as a form of gene therapy for human therapeutics in the future.     

Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains

This study concerns chimeric restriction enzymes that are hybrids between a zinc finger DNA-binding domain and the non-specific DNA-cleavage domain from the natural restriction enzyme FokI. Because of the flexibility of DNA recognition by zinc fingers, these enzymes are potential tools for cleaving DNA at arbitrarily selected sequences. Efficient double-strand cleavage by the chimeric nucleases requires two binding sites in close proximity. When cuts were mapped on the DNA strands, it was found that they occur in pairs separated by ~4 bp with a 5′ overhang, as for native FokI. Furthermore, amino acid changes in the dimer interface of the cleavage domain abolished activity. These results reflect a requirement for dimerization of the cleavage domain. The dependence of cleavage efficiency on the distance between two inverted binding sites was determined and both upper and lower limits were defined. Two different zinc finger combinations binding to non-identical sites also supported specific cleavage. Molecular modeling was employed to gain insight into the precise location of the cut sites. These results define requirements for effective targets of chimeric nucleases and will guide the design of novel specificities for directed DNA cleavage in vitro and in vivo.     

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。     

Zinc finger recombinases with adaptable DNA sequence specificity

Site-specific recombinases have become essential tools in genetics and molecular biology for the precise excision or integration of DNA sequences. However, their utility is currently limited to circumstances where the sites recognized by the recombinase enzyme have been introduced into the DNA being manipulated, or natural 'pseudosites' are already present. Many new applications would become feasible if recombinase activity could be targeted to chosen sequences in natural genomic DNA. Here we demonstrate efficient site-specific recombination at several sequences taken from a 1.9 kilobasepair locus of biotechnological interest (in the bovine β-casein gene), mediated by zinc finger recombinases (ZFRs), chimaeric enzymes with linked zinc finger (DNA recognition) and recombinase (catalytic) domains. In the "Z-sites" tested here, 22 bp casein gene sequences are flanked by 9 bp motifs recognized by zinc finger domains. Asymmetric Z-sites were recombined by the concomitant action of two ZFRs with different zinc finger DNA-binding specificities, and could be recombined with a heterologous site in the presence of a third recombinase. Our results show that engineered ZFRs may be designed to promote site-specific recombination at many natural DNA sequences.     

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.     

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。