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.     

锌指核酸酶技术的应用

锌指核酸酶(zinc finger nuclease,ZFN)技术是近年来发展起来的一种对基因组DNA实现靶向修饰的新技术。ZFN通过作用于基因组DNA上特异的靶位点产生DNA双链切口(double strand break,DSB),然后经过非同源末端连接(non-homologous end joining,NHEJ)或同源重组(homologous recombination,HR)途径实现对基因组DNA的靶向敲除或者替换。该技术近些年来已经被广泛应用于基因靶向修饰的研究。本文在简要介绍ZFN技术的基础上,重点综述了目前该技术在基因靶向修饰中的应用研究进展,并同时对该技术目前所需解决的一些问题以及未来的研究方向进行了分析。     

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.     

Expression, purification and characterization of cloning-grade zinc finger nuclease

The limited number of naturally occurring rare-cutting restriction enzymes and the slow and tedious engineering of existing restriction enzymes for novel specificities have prompted the design of new strategies for the development of restriction enzymes with specificities for long DNA sequences. One possibility is using zinc finger nucleases (ZFNs)—synthetic restriction enzymes that are custom-designed to target and cleave long DNA sequences and which have been recently shown useful for DNA cloning. Here we report on the purification and biochemical analysis of ZFN-10, a custom-made ZFN. We show that Ni-affinity and gel-filtration purification methods are sufficient to produce a cloning-grade enzyme. We show that ZFN-10 can function as an accurate and reliable ZFN using the same reagents and protocols used for naturally occurring and commercially available recombinant restriction enzymes. We also show that ZFN-10 tolerates a set of target-site substitutions which can be predicted from the specificities of recognition helices incorporated into the structure of its DNA-binding domain. The relative simplicity of ZFN-10 design, expression, purification and analysis suggests that novel ZFNs can potentially be designed and applied for various recombinant DNA applications.     

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.     

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。     

Structure based design of protein linkers for zinc finger nuclease

Zinc finger nucleases are a promising tool to edit DNA in many biological applications, in particular for gene knockout. Despite many efforts the number of genes that can be effectively targeted with ZFNs remains severely limited, as available constructs cannot address arbitrary gene sequences. Here, we develop a novel concept to significantly enhance the number of DNA sequences that can be targeted by ZFN. Using an efficient computational model, we provide an extensive library of possible linker molecules between individual zinc finger motifs in the construct that can skip up to 10 base pairs between adjacent zinc finger recognition sites in the DNA sequence, which increases the number of genes that can be efficiently targeted by more than an order of magnitude.     

Novel zinc finger nuclease created by combining the Cys2His2- and His4-type zinc finger domains

To improve the DNA hydrolytic activity of the zinc finger nuclease, we have created a new artificial zinc finger nuclease (ZWH4) by connecting two distinct zinc finger domains possessing different types of Zn(II) binding sites (Cys₂His₂- and His₄-types). The overall fold of ZWH4 is similar to that of the wild-type Sp1 zinc finger (Sp1(zf123)) as revealed by circular dichroism spectroscopy. The gel mobility shift assay demonstrated that ZWH4 binds to the GC box DNA, although the DNA-binding affinity is lower than that of Sp1(zf123). Evidently, ZWH4 hydrolyzes the covalently closed circular plasmid DNA (form I) containing the GC box (pBSGC) to the linear duplex DNA (form III) in the presence of a higher concentration (50 times) of the protein than DNA for a 24-h reaction. Of special interest is the fact that the novel mixed zinc finger protein containing the Cys₂His₂- and His₄-type domains was first created. The present results provide the useful information for the redesign strategy of an artificial nuclease based on the zinc finger motif.     

Adding fingers to an engineered zinc finger nuclease can reduce activity

Zinc finger nucleases (ZFNs) have been used to direct precise modifications of the genetic information in living cells at high efficiency. An important consideration in the design of ZFNs is the number of zinc fingers that are required for efficient and specific cleavage. We examined dimeric ZFNs composed of [1]+[1], [2]+[2], [3]+[3], [4]+[4], [5]+[5], and [6]+[6] zinc fingers, targeting 6, 12, 18, 24, 30, and 36 bp, respectively. We found that [1]+[1] and [2]+[2] fingers supported neither in vitro cleavage nor single-strand annealing in a cell-based recombination assay. An optimal ZFN activity was observed for [3]+[3] and [4]+[4] fingers. Surprisingly, [5]+[5] and [6]+[6] fingers exhibited significantly reduced activity. While the extra fingers were not found to dramatically increase toxicity, directly inhibit recombination, or perturb the ZFN target site, we demonstrate the ability of subsets of three fingers in six-finger arrays to bind independently to regions of the target site, possibly explaining the decrease in activity. These results have important implications for the design of new ZFNs, as they show that in some cases an excess of fingers may actually negatively affect the performance of engineered multifinger proteins. Maximal ZFN activity will require an optimization of both DNA binding affinity and specificity.     

Site-specific DNA cleavage by artificial zinc finger-type nuclease with cerium-binding peptide

The addition of a new function to native proteins is one of the most attractive protein-based designs. In this study, we have converted a C(2)H(2)-type zinc finger as a DNA-binding motif into a novel zinc finger-type nuclease by connecting two distinct zinc finger proteins (Sp1 and GLI) with a functional linker possessing DNA cleavage activity. As a DNA cleavage domain, we chose an analogue of the metal-binding loop (12 amino acid residues), peptide P1, which has been reported to exhibit a strong binding affinity for a lanthanide ion and DNA cleavage ability in the presence of Ce(IV). Our newly designed nucleases, Sp1(P1)GLI and Sp1(P1G)GLI, can strongly bind to a lanthanide ion and show a unique DNA cleavage pattern, in which certain positions between the two DNA-binding sites are specifically cleaved. The present result provides useful information for expanding the design strategy for artificial nucleases.     

PineappleDB: An online pineapple bioinformatics resource

Background  

A world first pineapple EST sequencing program has been undertaken to investigate genes expressed during non-climacteric fruit ripening and the nematode-plant interaction during root infection. Very little is known of how non-climacteric fruit ripening is controlled or of the molecular basis of the nematode-plant interaction. PineappleDB was developed to provide the research community with access to a curated bioinformatics resource housing the fruit, root and nematode infected gall expressed sequences.     

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.     

Targeted mutagenesis in the silkworm Bombyx mori using zinc finger nuclease mRNA injection

Targeted mutagenesis is one of the key methods for functional gene analysis. A simplified variant of gene targeting uses direct microinjection of custom-designed Zinc Finger Nuclease (ZFN) mRNAs into Drosophila embryos. To evaluate the applicability of this method to gene targeting in another insect, we mutagenized the Bombyx mori epidermal color marker gene BmBLOS2, which controls the formation of uric acid granules in the larval epidermis. Our results revealed that ZFN mRNA injection is effective to induce somatic, as well as germline, mutations in a targeted gene by non-homologous end joining (NHEJ). The ZFN-induced NHEJ mutations lack end-filling and blunt ligation products, and include mainly 7 bp or longer deletions, as well as single nucleotide insertions. These observations suggest that the B. mori double-strand break repair system relies on microhomologies rather than on a canonical ligase IV-dependent mechanism. The frequency of germline mutants in G₁ was sufficient to be used for gene targeting relying on a screen based solely on molecular methods.     

Human Splicing Finder: an online bioinformatics tool to predict splicing signals

Thousands of mutations are identified yearly. Although many directly affect protein expression, an increasing proportion of mutations is now believed to influence mRNA splicing. They mostly affect existing splice sites, but synonymous, non-synonymous or nonsense mutations can also create or disrupt splice sites or auxiliary cis-splicing sequences. To facilitate the analysis of the different mutations, we designed Human Splicing Finder (HSF), a tool to predict the effects of mutations on splicing signals or to identify splicing motifs in any human sequence. It contains all available matrices for auxiliary sequence prediction as well as new ones for binding sites of the 9G8 and Tra2-β Serine-Arginine proteins and the hnRNP A1 ribonucleoprotein. We also developed new Position Weight Matrices to assess the strength of 5′ and 3′ splice sites and branch points. We evaluated HSF efficiency using a set of 83 intronic and 35 exonic mutations known to result in splicing defects. We showed that the mutation effect was correctly predicted in almost all cases. HSF could thus represent a valuable resource for research, diagnostic and therapeutic (e.g. therapeutic exon skipping) purposes as well as for global studies, such as the GEN2PHEN European Project or the Human Variome Project.     

Restricted spacer tolerance of a zinc finger nuclease with a six amino acid linker

Zinc finger nucleases can be engineered to create highly efficient and precise changes to the genetic information within living cells. We report the investigation of an important parameter that defines the type of target site the nuclease can cleave. The active nuclease is a dimer, requiring that the DNA target site contain two zinc finger binding sites separated by a short spacer. Using a plasmid-based recombination assay in HEK 293T cells, we show that a 6 amino acid linker between the zinc finger DNA-binding domain and the FokI cleavage domain restricts nuclease activity to sites containing a 6 bp spacer. These observations concur with other recent studies, suggesting this information will be useful in the design of new potent and accurate zinc finger nucleases.     

Targeted gene exchange in plant cells mediated by a zinc finger nuclease double cut

Genome modification by homology‐directed repair (HDR) is an attractive tool for the controlled genetic manipulation of plants. Here, we report the HDR‐mediated gene exchange of expression cassettes in tobacco BY‐2 cells using a designed zinc finger nuclease (ZFN). The target contained a 7‐kb fragment flanked by two ZFN cutting sites. That fragment was replaced with a 4‐kb donor cassette, which integrates gene markers for selection (kanamycin resistance) and for scoring targeting (red fluorescent protein, RFP). Candidates resulting from cassette exchange were identified by molecular analysis of calli generated by transformation via direct DNA delivery. The precision of HDR‐mediated donor integration was evaluated by Southern blot analysis, sequencing of the integration locus and analysis of RFP fluorescence by flow cytometry. Screening of 1326 kanamycin‐resistant calli yielded 18 HDR events, 16 of which had a perfect cassette exchange at the insert junction and 13 of which produced functional RFP. Our results demonstrate that ZFN‐based HDR can be used for high frequency, precise, targeted exchange of fragments of sizes that are commercially relevant in plants.