Artificial Zinc Finger Proteins Research Articles

Zinc binding of a Cys2His2-type zinc finger protein is enhanced by the interaction with DNA

Zinc finger proteins specifically recognize DNA sequences and, therefore, play a crucial role in living organisms. In this study the Zn(II)-, and DNA-binding of 1MEY#, an artificial zinc finger protein consisting of three finger units was characterized by multiple methods. Fluorimetric, circular dichroism and isothermal calorimetric titrations were applied to determine the accurate stability constant of a zinc finger protein. Assuming that all three zinc finger subunits behave identically, the obtained thermodynamic data for the Zn(II) binding were ΔHbinding site = − (23.5 − 28.0) kcal/mol (depending on the applied protonation state of the cysteines) and logβ’pH 7.4 = 12.2 ± 0.1, being similar to those of the CP1 consensus zinc finger peptide. The specific DNA binding of the protein can be characterized by logβ’pH 7.4 = 8.20 ± 0.08, which is comparable to the affinity of the natural zinc finger proteins (Sp1, WT1, TFIIIA) toward DNA. This value is ~ 1.9 logβ’ unit higher than those determined for semi- or nonspecific DNA binding. Competitive circular dichroism and electrophoretic mobility shift measurements revealed that the conditional stability constant characteristic for Zn(II) binding of 1MEY# protein increased by 3.4 orders of magnitude in the presence of its target DNA sequence.Graphical abstract

Interactions of an Artificial Zinc Finger Protein with Cd(II) and Hg(II): Competition and Metal and DNA Binding

Cys2His2 zinc finger proteins are important for living organisms, as they—among other functions—specifically recognise DNA when Zn(II) is coordinated to the proteins, stabilising their ββα secondary structure. Therefore, competition with other metal ions may alter their original function. Toxic metal ions such as Cd(II) or Hg(II) might be especially dangerous because of their similar chemical properties to Zn(II). Most competition studies carried out so far have involved small zinc finger peptides. Therefore, we have investigated the interactions of toxic metal ions with a zinc finger proteins consisting of three finger units and the consequences on the DNA binding properties of the protein. Binding of one Cd(II) per finger subunit of the protein was shown by circular dichroism spectroscopy, fluorimetry and electrospray ionisation mass spectrometry. Cd(II) stabilised a similar secondary structure to that of the Zn(II)-bound protein but with a slightly lower affinity. In contrast, Hg(II) could displace Zn(II) quantitatively (logβ′ ≥ 16.7), demolishing the secondary structure, and further Hg(II) binding was also observed. Based on electrophoretic gel mobility shift assays, the Cd(II)-bound zinc finger protein could recognise the specific DNA target sequence similarly to the Zn(II)-loaded form but with a ~0.6 log units lower stability constant, while Hg(II) could destroy DNA binding completely.

Ectopic targeting of CG DNA methylation in Arabidopsis with the bacterial SssI methyltransferase

The ability to target epigenetic marks like DNA methylation to specific loci is important in both basic research and in crop plant engineering. However, heritability of targeted DNA methylation, how it impacts gene expression, and which epigenetic features are required for proper establishment are mostly unknown. Here, we show that targeting the CG-specific methyltransferase M.SssI with an artificial zinc finger protein can establish heritable CG methylation and silencing of a targeted locus in Arabidopsis. In addition, we observe highly heritable widespread ectopic CG methylation mainly over euchromatic regions. This hypermethylation shows little effect on transcription while it triggers a mild but significant reduction in the accumulation of H2A.Z and H3K27me3. Moreover, ectopic methylation occurs preferentially at less open chromatin that lacks positive histone marks. These results outline general principles of the heritability and interaction of CG methylation with other epigenomic features that should help guide future efforts to engineer epigenomes.

Global Reprogramming of Gene Transcription in Trichoderma reesei by Overexpressing an Artificial Transcription Factor for Improved Cellulase Production and Identification of Ypr1 as an Associated Regulator.

Synthetic biology studies on filamentous fungi are providing unprecedented opportunities for optimizing this important category of microbial cell factory. Artificial transcription factor can be designed and used to offer novel modes of regulation on gene transcription network. Trichoderma reesei is commonly used for cellulase production. In our previous studies, a plasmid library harboring genes encoding artificial zinc finger proteins (AZFPs) was constructed for engineering T. reesei, and the mutant strains with improved cellulase production were selected. However, the underlying mechanism by which AZFP function remain unclear. In this study, a T. reesei Rut-C30 mutant strain T. reesei U5 bearing an AZFP named as AZFP-U5 was focused, which secretes high level protein and shows significantly improved cellulase and xylanase production comparing with its parental strain. In addition, enhanced sugar release was achieved from lignocellulosic biomass using the crude cellulase from T. reesei U5. Comparative transcriptome analysis was further performed, which showed reprogramming of global gene transcription and elevated transcription of genes encoding glycoside hydrolases by overexpressing AZFP-U5. Furthermore, 15 candidate regulatory genes which showed remarkable higher transcription levels by AZFP-U5 insertion were overexpressed in T. reesei Rut-C30 to examine their effects on cellulase biosynthesis. Among these genes, TrC30_93861 (ypr1) and TrC30_74374 showed stimulating effects on filter paper activity (FPase), but deletion of these two genes did not affect cellulase activity. In addition, increased yellow pigment production in T. reesei Rut-C30 by overexpression of gene ypr1 was observed, and changes of cellulase gene transcription were revealed in the ypr1 deletion mutant, suggesting possible interaction between pigment production and cellulase gene transcription. The results in this study revealed novel aspects in regulation of cellulase gene expression by the artificial regulators. In addition, the candidate genes and processes identified in the transcriptome data can be further explored for synthetic biology design and metabolic engineering of T. reesei to enhance cellulase production.

Engineering the Effector Domain of the Artificial Transcription Factor to Improve Cellulase Production by Trichoderma reesei

Filamentous fungal strains of Trichoderma reesei have been widely used for cellulase production, and great effort has been devoted to enhancing their cellulase titers for the economic biorefinery of lignocellulosic biomass. In our previous studies, artificial zinc finger proteins (AZFPs) with the Gal4 effector domain were used to enhance cellulase biosynthesis in T. reesei, and it is of great interest to modify the AZFPs to further improve cellulase production. In this study, the endogenous activation domain from the transcription activator Xyr1 was used to replace the activation domain of Gal4 of the AZFP to explore impact on cellulase production. The cellulase producer T. reesei TU-6 was used as a host strain, and the engineered strains containing the Xyr1 and the Gal4 activation domains were named as T. reesei QS2 and T. reesei QS1, respectively. Compared to T. reesei QS1, activities of filter paper and endoglucanases in crude cellulase produced by T. reesei QS2 increased 24.6 and 50.4%, respectively. Real-time qPCR analysis also revealed significant up-regulation of major genes encoding cellulase in T. reesei QS2. Furthermore, the biomass hydrolytic performance of the cellulase was evaluated, and 83.8 and 97.9% more glucose was released during the hydrolysis of pretreated corn stover using crude enzyme produced by T. reesei QS2, when compared to the hydrolysis with cellulase produced by T. reesei QS1 and the parent strain T. reesei TU-6. As a result, we proved that the effector domain in the AZFPs can be optimized to construct more effective artificial transcription factors for engineering T. reesei to improve its cellulase production.

Identification of a novel repressor encoded by the putative gene ctf1 for cellulase biosynthesis in Trichoderma reesei through artificial zinc finger engineering.

Strains from Trichoderma reesei have been used for cellulase production with a long history. It has been well known that cellulase biosynthesis by the fungal species is controlled through regulators, and elucidation of their regulation network is of great importance for engineering T. reesei with robust cellulase production. However, progress in this regard is still very limited. In this study, T. reesei RUT-C30 was transformed with an artificial zinc finger protein (AZFP) library, and the mutant T. reesei M2 with improved cellulase production was screened. Compared to its parent strain, the filter paper activity and endo-β-glucanase activity in cellulases produced by T. reesei M2 increased 67.2% and 35.3%, respectively. Analysis by quantitative reverse transcription polymerase chain reaction indicated significant downregulation of the putative gene ctf1 in T. reesei M2, and its deletion mutants were thus developed for further studies. An increase of 36.9% in cellulase production was observed in the deletion mutants, but when ctf1 was constitutively overexpressed in T. reesei RUT-C30 under the control of the strong pdc1 promoter, cellulase production was substantially compromised. Comparative transcriptomic analysis revealed that the deletion of ctf1 upregulated transcription of gene encoding the regulator VIB1, but downregulated transcription of gene encoding another regulator RCE1, which consequently upregulated genes encoding the transcription factors XYR1 and ACE3 for the activation of genes encoding cellulolytic enzymes. As a result, ctf1 was characterized as a gene encoding a repressor for cellulase production in T. reesei RUT-C30, which is significant for further elucidating molecular mechanism underlying cellulase biosynthesis by the fungal species for rational design to develop robust strains for cellulase production. And in the meantime, AZFP transformation was validated to be an effective strategy for identifying functions of putative genes in the genome of T. reesei.

Artificial zinc finger protein mediated cellulase production in Trichoderma reesei Rut-C30

Trichoderma reesei Rut-C30 is widely used in industrial cellulase production, and development of cellulase hyper-producer is of great importance for economic lignocellulosic biorefinery. In this study, T. reesei Rut-C30 was engineered with an artificial zinc finger proteins (AZFPs) library. Two mutants T. reesei M1 and M2 with improved cellulase production were obtained. Compared to the parent strain, the filter paper activity (FPase) of T. reesei M1 and M2 increased 100% and 53%, respectively. In addition, the total amount of extracellular protein from the M1 mutant increased 69%, whereas the endo-β-glucanase (CMCase) activity of the M2 mutant is 64% higher compared to the parental strain. Furthermore, RT-qPCR analysis showed that the major cellulase genes exhibited significantly increased expression in both mutants, but different patterns were observed in the two mutants. On the other hand, the cellulase transcriptional repressor ace1 was down-regulated in both mutants, but the transcription level of the activator xyr1 was only up-regulated in the strain M1. These results demonstrated that different AZFPs exert diverse regulatory mechanisms on cellulase production in T. reesei. Analysis of the target genes of AZFPs from T. reesei M1 and M2 will not only benefit further exploration of the regulatory mechanisms of cellulase biosynthesis in T. reesei, but also enable development of cellulase hyper-producing strains by metabolic engineering.

Site-Specific Integration by Recruitment of a Complex of ΦC31 Integrase and Donor DNA to a Target Site by Using a Tandem, Artificial Zinc-Finger Protein.

To solve the problem of uncontrolled therapeutic gene integration, which is a critical drawback of retroviral vectors for gene therapy, the integration sites of exogenous genes should be precisely controlled not to perturb endogenous gene expression. To accomplish this, we explored the possibility of site-specific integration using two six-finger artificial zinc-finger proteins (AZPs) tandemly conjugated via a flexible peptide linker (designated "Tandem AZP"). A Tandem AZP in which two AZPs recognize specific 19 bp targets in a donor and acceptor DNA was expected to site-specifically recruit the donor DNA to the acceptor DNA. Thereafter, an exogenously added integrase was expected to integrate the donor DNA into a specific site in the acceptor DNA (as it might be in the human genome). We demonstrated in vitro that in the presence of Tandem AZP, ΦC31 integrase selectively integrated a donor plasmid into a target acceptor plasmid not only at 30 °C (the optimum temperature of the integrase) but also at 37 °C (for future application in humans). We expect that with further improvement of our current system, a combination of Tandem AZP with integrase/recombinase will enable site-specific integration in mammalian cells and provide safer gene therapy technology.

Robust erythroid differentiation system for rhesus hematopoietic progenitor cells allowing preclinical screening of genetic treatment strategies for the hemoglobinopathies

γ-globin expression can be induced by various gene modification strategies, which could be beneficial for hemoglobin (Hb) disorders. To translate promising ideas into clinics, large animal models have proven valuable to evaluate safety and efficacy of the approaches; however, in vitro erythroid differentiation methods have not been established to determine whether they can be modeled in nonhuman primates. We optimized erythroid differentiation culture to produce high-level adult Hb from rhesus hematopoietic progenitor cells by using low (LC) or high cytokine concentration (HC) protocols with or without feeder cells. In addition, we established rhesus globin protein analysis using reverse-phase high performance liquid chromatography and mass spectrometry. Robust adult Hb production at protein levels was observed in the LC protocol when feeder cells were used, whereas the HC protocol resulted in higher baseline fetal Hb levels (P < 0.01). We then compared lentiviral transduction of rhesus cells between serum-containing LC media and serum-free StemSpan-based differentiation media, revealing 100-fold more efficient transduction in serum-free differentiation media (P < 0.01). Finally, rhesus CD34+ cells were transduced with lentiviral vectors encoding artificial zinc finger proteins (ZF-Ldb1), which can reactivate γ-globin expression via tethering the transcriptional co-regulator Ldb1 to γ-globin promoters, and were differentiated in the optimized erythroid differentiation method. This resulted in marked increases of γ-globin levels compared with control groups (P < 0.01). In conclusion, we developed an efficient rhesus erythroid differentiation protocol from hematopoietic progenitor cells with low fetal and high adult Hb production. Further studies are warranted to optimize gene modification and transplantation of rhesus hematopoietic progenitor cells.

A peptide derived from enzymatic digestion of globulins from amaranth shows strong affinity binding to the replication origin of Tomato yellow leaf curl virus reducing viral replication in Nicotiana benthamiana

Tomato yellow leaf curl virus (TYLCV; genus Begomovirus; family Geminiviridae) infects mainly plants of the family Solanaceae, and the infection induces curling and chlorosis of leaves, dwarfing of the whole plant, and reduced fruit production. Alternatives for direct control of TYLCV and other geminiviruses have been reported, for example, the use of esterified whey proteins, peptide aptamer libraries or artificial zinc finger proteins. The two latter alternatives affect directly the replication of TYLCV as well as of other geminiviruses because the replication structures and sequences are highly conserved within this virus family. Because peptides and proteins offer a potential solution for virus replication control, in this study we show the isolation, biochemical characterization and antiviral activity of a peptide derived from globulins of amaranth seeds (Amaranthus hypochondriacus) that binds to the replication origin sequence (OriRep) of TYLCV and affects viral replication with a consequent reduction of disease symptoms in Nicotiana benthamiana. Aromatic peptides obtained from papain digests of extracted globulins and albumins of amaranth were tested by intrinsic fluorescent titration and localized surface resonance plasmon to analyze their binding affinity to OriRep of TYLCV. The peptide AmPep1 (molecular weight 2.076 KDa) showed the highest affinity value (Kd = 1.8 nM) for OriRep. This peptide shares a high amino acid similarity with a part of an amaranth 11S globulin, and the strong affinity of AmPep1 could be explained by the presence of tryptophan and lysine facilitating interaction with the secondary structure of OriRep. In order to evaluate the effect of the peptide on in vitro DNA synthesis, rolling circle amplification (RCA) was performed using as template DNA from plants infected with TYLCV or another begomovirus, pepper huasteco yellow vein virus (PHYVV), and adding AmPep1 peptide at different concentrations. The results showed a decrease in DNA synthesis of both viruses at increasing concentrations of AmPep1. To further confirm the antiviral activity of the peptide in vivo, AmPep1 was infiltrated into leaves of N. benthamiana plants previously infected with TYLCV. Plants treated with AmPep1 showed a significant decrease in virus titer compared with untreated N. benthamiana plants as well as reduced symptom progression due to the effect of AmPep1 curtailing TYLCV replication in the plant. The peptide also showed antiviral activity in plants infected with PHYVV. This is the first report, in which a peptide is directly used for DNA virus control in plants, supplied as exogenous application and without generation of transgenic lines.

Improved ethanol production at high temperature by consolidated bioprocessing using Saccharomyces cerevisiae strain engineered with artificial zinc finger protein

In this work, the consolidated bioprocessing (CBP) yeast Saccharomyces cerevisiae MNII/cocδBEC3 was transformed by an artificial zinc finger protein (AZFP) library to improve its thermal tolerance, and the strain MNII-AZFP with superior growth at 42°C was selected. Improved degradation of acid swollen cellulose by 45.9% led to an increase in ethanol production, when compared to the control strain. Moreover, the fermentation of Jerusalem artichoke stalk (JAS) by MNII-AZFP was shortened by 12h at 42°C with a concomitant improvement in ethanol production. Comparative transcriptomics analysis suggested that the AZFP in the mutant exerted beneficial effect by modulating the expression of multiple functional genes. These results provide a feasible strategy for efficient ethanol production from JAS and other cellulosic biomass through CBP based-fermentation at elevated temperatures.

Induction and regulation of cellulase expression in filamentous fungi: a review

Production of bioenergy and bio-based chemicals by using fermentable sugars released from low-cost renewable lignocellulosic biomass has received great attention. Efficient cellulolytic enzymes are crucial for lignocellulose bioconversion, but high cellulase production cost is limiting the bioconversion efficiency of cellulosic biomass and industrial applications of lignocellulose biorefinery. Studies on induction and regulation of cellulase in filamentous fungi will help to further develop superior fungal strains for efficient cellulase production and reduce cellulase production cost. With the advances in high-throughput sequencing and gene manipulation technology using fungal strains, an in-depth understanding of cellulase induction and regulation mechanisms of enzyme expression has been achieved. We reviewed recent progresses in the induction and regulation of cellulase expression in several model filamentous fungi, emphasizing sugar transporters, transcription factors and chromatin remodeling. Future prospects in application of artificial zinc finger proteins for cellulase induction and regulation in filamentous fungi were discussed.

Enhanced cellulase production from Trichoderma reesei Rut-C30 by engineering with an artificial zinc finger protein library.

Trichoderma reesei Rut-C30 is a well-known cellulase producer, and improvement of its cellulase production is of great interest. An artificial zinc finger protein (AZFP) library is constructed for expression in T. reesei Rut-C30, and a mutant strain T. reesei U3 is selected based on its enhanced cellulase production. The U3 mutant shows a 55% rise in filter paper activity and 8.1-fold increased β-glucosidase activity, when compared to the native strain T. reesei Rut-C30. It is demonstrated that enhanced β-glucosidase activity was due to elevated transcription level of β-glucosidase gene in the U3 mutant. Moreover, significant elevation in transcription levels of several putative Azfp-U3 target genes is detected in the U3 mutant, including genes encoding hypothetical transcription factors and a putative glycoside hydrolase. Furthermore, U3 cellulase shows 115% higher glucose yield from pretreated corn stover, when compared to the cellulase of T. reesei Rut-C30. These results demonstrate that AZFP can be used to improve cellulase production in T. reesei Rut-C30. Our current work offers the establishment of an alternative strategy to develop fungal cell factories for improved production of high value industrial products.

Directing an artificial zinc finger protein to new targets by fusion to a non-DNA-binding domain.

Transcription factors are often regarded as having two separable components: a DNA-binding domain (DBD) and a functional domain (FD), with the DBD thought to determine target gene recognition. While this holds true for DNA binding in vitro, it appears that in vivo FDs can also influence genomic targeting. We fused the FD from the well-characterized transcription factor Krüppel-like Factor 3 (KLF3) to an artificial zinc finger (AZF) protein originally designed to target the Vascular Endothelial Growth Factor-A (VEGF-A) gene promoter. We compared genome-wide occupancy of the KLF3FD-AZF fusion to that observed with AZF. AZF bound to the VEGF-A promoter as predicted, but was also found to occupy approximately 25 000 other sites, a large number of which contained the expected AZF recognition sequence, GCTGGGGGC. Interestingly, addition of the KLF3 FD re-distributes the fusion protein to new sites, with total DNA occupancy detected at around 50 000 sites. A portion of these sites correspond to known KLF3-bound regions, while others contained sequences similar but not identical to the expected AZF recognition sequence. These results show that FDs can influence and may be useful in directing AZF DNA-binding proteins to specific targets and provide insights into how natural transcription factors operate.

Application and potential of genome engineering by artificial enzymes

Artificial zinc finger proteins (ZFPs) consist of Cys2-His2-type modules composed of approximately 30 amino acids that adopt a ββα structure and coordinate a zinc ion. ZFPs recognizing specific DNA target sequences can substitute for the binding domains of various DNA-modifying enzymes to create designer nucleases, recombinases, and methylases with programmable sequence specificity. Enzymatic genome editing and modification can be applied to many fields of basic research and medicine. The recent development of new platforms using transcription activator-like effector (TALE) proteins or the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) system has expanded the range of possibilities for genome-editing technologies. These technologies empower investigators with the ability to efficiently knockout or regulate the functions of genes of interest. In this review, we discuss historical advancements in artificial ZFP applications and important issues that may influence the future of genome editing and engineering technologies. The development of artificial ZFPs has greatly increased the feasibility of manipulating endogenous gene functions through transcriptional control and gene modification. Advances in the ZFP, TALE, and CRISPR/Cas platforms have paved the way for the next generation of genome engineering approaches. Perspectives for the future of genome engineering are also discussed, including applications of targeting specific genomic alleles and studies in synthetic biology.

Improvement of acetic acid tolerance of Saccharomyces cerevisiae using a zinc-finger-based artificial transcription factor and identification of novel genes involved in acetic acid tolerance.

Acetic acid is present in cellulosic hydrolysate as a potent inhibitor, and the superior acetic acid tolerance of Saccharomyces cerevisiae ensures good cell viability and efficient ethanol production when cellulosic raw materials are used as substrates. In this study, a mutant strain of S. cerevisiae ATCC4126 (Sc4126-M01) with improved acetic acid tolerance was obtained through screening strains transformed with an artificial zinc finger protein transcription factor (ZFP-TF) library. Further analysis indicated that improved acetic acid tolerance was associated with improved catalase (CAT) activity. The ZFP coding sequence associated with the improved phenotype was identified, and real-time RT-PCR analysis revealed that three of the possible genes involved in the enhanced acetic acidtolerance regulated by this ZFP-TF, namely YFL040W, QDR3, and IKS1, showed decreased transcription levels in Sc4126-M01 in the presence of acetic acid, compared to those in the control strain. Sc4126-M01 mutants having QDR3 and IKS1 deletion (ΔQDR3 and ΔIKS1) exhibited higher acetic acid tolerance than the wild-type strain under acetic acid treatment. Glucose consumption rate and ethanol productivity in the presence of 5g/L acetic acid were improved in the ΔQDR3 mutant compared to the wild-type strain. Our studies demonstrated that the synthetic ZFP-TF library can be used to improve acetic acid tolerance of S. cerevisiae and that the employment of an artificial transcription factor can facilitate the exploration of novel functional genes involved in stress tolerance of S. cerevisiae.

Inhibition of DNA Replication of Human Papillomavirus by Using Zinc Finger–Single-Chain FokI Dimer Hybrid

Previously, we reported that an artificial zinc-finger protein (AZP)-staphylococcal nuclease (SNase) hybrid (designated AZP-SNase) inhibited DNA replication of human papillomavirus type 18 (HPV-18) in mammalian cells by binding to and cleaving a specific HPV-18 ori plasmid. Although the AZP-SNase did not show any side effects under the experimental conditions, the SNase is potentially able to cleave RNA as well as DNA. In the present study, to make AZP hybrid nucleases that cleave only viral DNA, we switched the SNase moiety in the AZP-SNase to the single-chain FokI dimer (scFokI) that we had developed previously. We demonstrated that transfection with a plasmid expressing the resulting hybrid nuclease (designated AZP-scFokI) inhibited HPV-18 DNA replication in transient replication assays using mammalian cells more efficiently than AZP-SNase. Then, by linker-mediated PCR analysis, we confirmed that AZP-scFokI cleaved an HPV-18 ori plasmid around its binding site in mammalian cells. Finally, a modified MTT assay revealed that AZP-scFokI did not show any significant cytotoxicity. Thus, the newly developed AZP-scFokI hybrid is expected to serve as a novel antiviral reagent for the neutralization of human DNA viruses with less fewer potential side effects.

Inhibiting replication of begomoviruses using artificial zinc finger nucleases that target viral-conserved nucleotide motif

Geminiviridae consists of a large group of single-stranded DNA viruses that cause tremendous losses worldwide. Frequent mixed infection and high rates of recombination and mutation allow them to adapt rapidly to new hosts and overcome hosts' resistances. Therefore, an effective strategy able to confer plants with resistance against multiple begomoviruses is needed. In the present study, artificial zinc finger proteins were designed based on a conserved sequence motif of begomoviruses. DNA-binding affinities and specificities of these artificial zinc fingers were evaluated using electrophoretic mobility shift assay. Artificial zinc finger nuclease (AZFNs) were then constructed based on the ones with the highest DNA-binding affinities. In vitro digest and transient expression assay showed that these AZFNs can efficiently cleave the target sequence and inhibit the replication of different begomoviruses. These results suggest that artificial zinc finger protein technology may be used to achieve resistance against multiple begomoviruses.

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.

C-terminal in Sp1-like artificial zinc-finger proteins plays crucial roles in determining their DNA binding affinity

BackgroundIt is well known that the C-terminal zinc-finger-3 in transcription factor Sp1 contributes more than the N-terminal zinc-finger-1 in determining Sp1’s DNA binding capacity. Sp1-like artificial poly-zinc-finger proteins (ZFPs) are powerful biotechnological tools for gene-specific recognization and manipulation. It is important to understand whether the C-terminal fingers in the Sp1-like artificial ZFPs remain crucial for their DNA binding ability. Recently, a set of p16 promoter-specific seven-ZFPs (7ZFPs) has been constructed to reactivate the expression of methylation-silenced p16. These 7ZFPs contain one N-terminal three-zinc-finger domain of Sp1 (3ZF), two Sp1-like two-zinc-finger domains derived from the Sp1 finger-2 and finger-3 (2ZF) in the middle and C-terminal regions.ResultsIn the present study, sets of variants for several representative 7ZFPs with the p16-binding affinity were further constructed. This was accomplished through finger replacements and key amino acid mutations in the N-terminal fingers, C-terminal fingers, and linker peptide, respectively. Their p16-binding activity was analysed using gel mobility shift assays. Results showed that the motif replacement or a key amino acid mutation (S > R) at position +2 of the α-helix in the C-terminal 2ZF domain completely abolished their p16-binding affinity. Deletion of three amino acids in a consensus linker (TGEKP > TG) between finger-7 and the 6 × Histidine-tag in the C-terminal also dramatically abolished their binding affinity. In contrast, the replacement of the finger-3 in the N-terminal 3ZF domain did not affect their binding affinity, but decreased their binding stability.ConclusionsAltogether, the present study show that the C-terminal region may play crucial roles in determining the DNA binding affinity of Sp1-like artificial ZFPs.