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.     

Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects

Engineered zinc finger nucleases (ZFNs) induce DNA double-strand breaks at specific recognition sequences and can promote efficient introduction of desired insertions, deletions or substitutions at or near the cut site via homology-directed repair (HDR) with a double- and/or single-stranded donor DNA template. However, mutagenic events caused by error-prone non-homologous end-joining (NHEJ)-mediated repair are introduced with equal or higher frequency at the nuclease cleavage site. Furthermore, unintended mutations can also result from NHEJ-mediated repair of off-target nuclease cleavage sites. Here, we describe a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ.     

Directed Evolution of an Enhanced and Highly Efficient FokI Cleavage Domain for Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) are powerful tools for gene therapy and genetic engineering. The high specificity and affinity of these chimeric enzymes are based on custom-designed zinc finger proteins (ZFPs). To improve the performance of existing ZFN technology, we developed an in vivo evolution-based approach to improve the efficacy of the FokI cleavage domain (FCD). After multiple rounds of cycling mutagenesis and DNA shuffling, a more efficient nuclease variant (Sharkey) was generated. In vivo analyses indicated that Sharkey is > 15-fold more active than wild-type FCD on a diverse panel of cleavage sites. Further, a mammalian cell-based assay showed a three to sixfold improvement in targeted mutagenesis for ZFNs containing derivatives of the Sharkey cleavage domain. We also identified mutations that impart sequence specificity to the FCD that might be utilized in future studies to further refine ZFNs through cooperative specificity. In addition, Sharkey was observed to enhance the cleavage profiles of previously published and newly selected heterodimer ZFN architectures. This enhanced and highly efficient cleavage domain will aid in a variety of ZFN applications in medicine and biology.     

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.     

AAV-Mediated Delivery of Zinc Finger Nucleases Targeting Hepatitis B Virus Inhibits Active Replication

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy.     

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.     

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.     

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.     

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.     

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.     

Expression of 3-hydroxy-3-methylglutaryl-CoA reductase, p-hydroxybenzoate-m-geranyltransferase and genes of phenylpropanoid pathway exhibits positive correlation with shikonins content in arnebia [Arnebia euchroma (Royle) Johnston]

Background

Formation of site specific genomic double strand breaks (DSBs), induced by the expression of a pair of engineered zinc-finger nucleases (ZFNs), dramatically increases the rates of homologous recombination (HR) between a specific genomic target and a donor plasmid. However, for the safe use of ZFN induced HR in practical applications, possible adverse effects of the technology such as cytotoxicity and genotoxicity need to be well understood. In this work, off-target activity of a pair of ZFNs has been examined by measuring the ratio between HR and illegitimate genomic integration in cells that are growing exponentially, and in cells that have been arrested in the G2/M phase.

Results

A reporter cell line that contained consensus ZFN binding sites in an enhanced green fluorescent protein (EGFP) reporter gene was used to measure ratios between HR and non-homologous integration of a plasmid template. Both in human cells (HEK 293) containing the consensus ZFN binding sites and in cells lacking the ZFN binding sites, a 3.5 fold increase in the level of illegitimate integration was observed upon ZFN expression. Since the reporter gene containing the consensus ZFN target sites was found to be intact in cells where illegitimate integration had occurred, increased rates of illegitimate integration most likely resulted from the formation of off-target genomic DSBs. Additionally, in a fraction of the ZFN treated cells the co-occurrence of both specific HR and illegitimate integration was observed. As a mean to minimize unspecific effects, cell cycle manipulation of the target cells by induction of a transient G2/M cell cycle arrest was shown to stimulate the activity of HR while having little effect on the levels of illegitimate integration, thus resulting in a nearly eight fold increase in the ratio between the two processes.

Conclusions

The demonstration that ZFN expression, in addition to stimulating specific gene targeting by HR, leads to increased rates of illegitimate integration emphasizes the importance of careful characterization of ZFN treated cells. In order to reduce off-target events, reversible cell cycle arrest of the target cells in the G2/M phase is an efficient way for increasing the ratio between specific HR and illegitimate integration.     

Nuclear gene targeting in Chlamydomonas using engineered zinc‐finger nucleases

The unicellular green alga Chlamydomonas reinhardtii is a versatile model for fundamental and biotechnological research. A wide range of tools for genetic manipulation have been developed for this alga, but specific modification of nuclear genes is still not routinely possible. Here, we present a nuclear gene targeting strategy for Chlamydomonas that is based on the application of zinc‐finger nucleases (ZFNs). Our approach includes (i) design of gene‐specific ZFNs using available online tools, (ii) evaluation of the designed ZFNs in a Chlamydomonas in situ model system, (iii) optimization of ZFN activity by modification of the nuclease domain, and (iv) application of the most suitable enzymes for mutagenesis of an endogenous gene. Initially, we designed a set of ZFNs to target the COP3 gene that encodes the light‐activated ion channel channelrhodopsin‐1. To evaluate the designed ZFNs, we constructed a model strain by inserting a non‐functional aminoglycoside 3′‐phosphotransferase VIII (aphVIII) selection marker interspaced with a short COP3 target sequence into the nuclear genome. Upon co‐transformation of this recipient strain with the engineered ZFNs and an aphVIII DNA template, we were able to restore marker activity and select paromomycin‐resistant (Pm‐R) clones with expressing nucleases. Of these Pm‐R clones, 1% also contained a modified COP3 locus. In cases where cells were co‐transformed with a modified COP3 template, the COP3 locus was specifically modified by homologous recombination between COP3 and the supplied template DNA. We anticipate that this ZFN technology will be useful for studying the functions of individual genes in Chlamydomonas.     

Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection

Engineered zinc-finger nucleases (ZFNs) are promising tools for genome manipulation, and determining off-target cleavage sites of these enzymes is of great interest. We developed an in vitro selection method that interrogates 10(11) DNA sequences for cleavage by active, dimeric ZFNs. The method revealed hundreds of thousands of DNA sequences, some present in the human genome, that can be cleaved in vitro by two ZFNs: CCR5-224 and VF2468, which target the endogenous human CCR5 and VEGFA genes, respectively. Analysis of identified sites in one cultured human cell line revealed CCR5-224-induced changes at nine off-target loci, though this remains to be tested in other relevant cell types. Similarly, we observed 31 off-target sites cleaved by VF2468 in cultured human cells. Our findings establish an energy compensation model of ZFN specificity in which excess binding energy contributes to off-target ZFN cleavage and suggest strategies for the improvement of future ZFN design.     

Non-stem cancer cell kinetics modulate solid tumor progression

Background

Herpes simplex type II (HSV-2) is a member of the family herpesviridae. Human infection with this double stranded linear DNA virus causes genital ulcerative disease and existing treatment options only serve to resolve the symptomatology (ulcers) associated with active HSV-2 infection but do not eliminate latent virus. As a result, infection with HSV-2 follows a life-long relapsing (active versus latent) course. On the basis of a primitive bacterium anti-phage DNA defense, the restriction modification (R-M) system, we previously identified the Escherichia coli restriction enzyme (REase) EcoRII as a novel peptide to excise or irreversibly disrupt latent HSV-2 DNA from infected cells. However, sequences of the site specificity palindrome of EcoRII 5'-CCWGG-3' (W = A or T) are equally present within the human genome and are a potential source of host-genome toxicity. This feature has limited previous HSV-2 EcoRII based therapeutic models to microbicides only, and highlights the need to engineer artificial REases (zinc finger nucleases-ZFNs) with specificity to HSV-2 genomic-DNA only. Herein, the therapeutic-potential of zinc finger arrays (ZFAs) and ZFNs is identified and modeled, with unique specificity to the HSV-2 genome.

Methods and results

Using the whole genome of HSV-2 strain HG52 (Dolan A et al.,), and with the ZFN-consortium's CoDA-ZiFiT software pre-set at default, more than 28,000 ZFAs with specificity to HSV-2 DNA were identified. Using computational assembly (through in-silico linkage to the Flavobacterium okeanokoites endonuclease Fok I of the type IIS class), 684 ZFNs with specificity to the HSV-2 genome, were constructed. Graphic-analysis of the HSV-2 genome-cleavage pattern using the afore-identified ZFNs revealed that the highest cleavage-incidence occurred within the 30,950 base-pairs (~between the genomic context coordinates 0.80 and 1.00) at the 3' end of the HSV-2 genome. At approximately 3,095 bp before and after the 5' and 3' ends of the HSV-2 genome (genomic context coordinates 0.02 and 0.98, respectively) were specificity sites of ZFNs suited for the complete excision of over 60% of HSV-2 genomic material from within infected human cells, through the process of non-homologous end joining (NHEJ). Furthermore, a model concerning a recombinant (ICP10-PK mutant) replication competent HSV-2 viral vector for delivering and transducing a diploid copy (or pair) of the HSV-2-genome-specific ZFN genotype within neuronal tissue, is presented.

Conclusion

ZFNs with specificity to HSV-2 genomic DNA that are precursors of novel host-genome expressed HSV-2 gene-therapeutics or vaccines were identified.     

Different binding properties and function of CXXC zinc finger domains in Dnmt1 and Tet1

Several mammalian proteins involved in chromatin and DNA modification contain CXXC zinc finger domains. We compared the structure and function of the CXXC domains in the DNA methyltransferase Dnmt1 and the methylcytosine dioxygenase Tet1. Sequence alignment showed that both CXXC domains have a very similar framework but differ in the central tip region. Based on the known structure of a similar MLL1 domain we developed homology models and designed expression constructs for the isolated CXXC domains of Dnmt1 and Tet1 accordingly. We show that the CXXC domain of Tet1 has no DNA binding activity and is dispensable for catalytic activity in vivo. In contrast, the CXXC domain of Dnmt1 selectively binds DNA substrates containing unmethylated CpG sites. Surprisingly, a Dnmt1 mutant construct lacking the CXXC domain formed covalent complexes with cytosine bases both in vitro and in vivo and rescued DNA methylation patterns in dnmt1^−/− embryonic stem cells (ESCs) just as efficiently as wild type Dnmt1. Interestingly, neither wild type nor ΔCXXC Dnmt1 re-methylated imprinted CpG sites of the H19a promoter in dnmt1^−/− ESCs, arguing against a role of the CXXC domain in restraining Dnmt1 methyltransferase activity on unmethylated CpG sites.     

A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture

Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of target site recognition. Cleavage specificity is typically evaluated by low throughput assays (T7 endonuclease I assay, target amplification followed by high-throughput sequencing), which are limited to a subset of potential off-target sites. Here, we used ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites for two guide RNAs. RNA-guided Cas9 binding was highly specific to the target site while off-target binding occurred at much lower intensities. Cas9-bound regions were highly enriched in NGG sites, a sequence required for target site recognition by Streptococcus pyogenes Cas9. To determine the relationship between Cas9 binding and endonuclease activity, we applied targeted sequence capture, which allowed us to survey 1200 genomic loci simultaneously including potential off-target sites identified by ChIP-seq and by computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our results confirm the high-specificity of CRISPR endonucleases and demonstrate that sequence capture can be used as a high-throughput genome-wide approach to identify off-target activity.