Epigenome editing for targeted DNA (de)methylation: a new perspective in modulating gene expression.

Traditionally, it has been believed that inheritance is driven as phenotypic variations resulting from changes in DNA sequence. However, this paradigm has been challenged and redefined in the contemporary era of epigenetics. The changes in DNA methylation, histone modification, non-coding RNA biogenesis, and chromatin remodeling play crucial roles in genomic functions and regulation of gene expression. More importantly, some of these changes are inherited to the next generations as a part of epigenetic memory and play significant roles in gene expression. The sum total of all changes in DNA bases, histone proteins, and ncRNA biogenesis constitutes the epigenome. Continuous progress in deciphering epigenetic regulations and the existence of heritable epigenetic/epiallelic variations associated with trait of interest enables to deploy epigenome editing tools to modulate gene expression. DNA methylation marks can be utilized in epigenome editing for the manipulation of gene expression. Initially, genome/epigenome editing technologies relied on zinc-finger protein or transcriptional activator-like effector protein. However, the discovery of clustered regulatory interspaced short palindromic repeats CRISPR)/deadCRISPR-associated protein 9 (dCas9) enabled epigenome editing to be more specific/efficient for targeted DNA (de)methylation. One of the major concerns has been the off-target effects, wherein epigenome editing may unintentionally modify gene/regulatory element which may cause unintended change/harmful effects. Moreover, epigenome editing of germline cell raises several ethical/safety issues. This review focuses on the recent developments in epigenome editing tools/techniques, technological limitations, and future perspectives of this emerging technology in therapeutics for human diseases as well as plant improvement to achieve sustainable developmental goals.

Progress in gene editing tools, implications and success in plants: a review.

Genetic modifications are made through diverse mutagenesis techniques for crop improvement programs. Among these mutagenesis tools, the traditional methods involve chemical and radiation-induced mutagenesis, resulting in off-target and unintended mutations in the genome. However, recent advances have introduced site-directed nucleases (SDNs) for gene editing, significantly reducing off-target changes in the genome compared to induced mutagenesis and naturally occurring mutations in breeding populations. SDNs have revolutionized genetic engineering, enabling precise gene editing in recent decades. One widely used method, homology-directed repair (HDR), has been effective for accurate base substitution and gene alterations in some plant species. However, its application has been limited due to the inefficiency of HDR in plant cells and the prevalence of the error-prone repair pathway known as non-homologous end joining (NHEJ). The discovery of CRISPR-Cas has been a game-changer in this field. This system induces mutations by creating double-strand breaks (DSBs) in the genome and repairing them through associated repair pathways like NHEJ. As a result, the CRISPR-Cas system has been extensively used to transform plants for gene function analysis and to enhance desirable traits. Researchers have made significant progress in genetic engineering in recent years, particularly in understanding the CRISPR-Cas mechanism. This has led to various CRISPR-Cas variants, including CRISPR-Cas13, CRISPR interference, CRISPR activation, base editors, primes editors, and CRASPASE, a new CRISPR-Cas system for genetic engineering that cleaves proteins. Moreover, gene editing technologies like the prime editor and base editor approaches offer excellent opportunities for plant genome engineering. These cutting-edge tools have opened up new avenues for rapidly manipulating plant genomes. This review article provides a comprehensive overview of the current state of plant genetic engineering, focusing on recently developed tools for gene alteration and their potential applications in plant research.

Production of therapeutic levels of human FIX-R338L by engineered B cells using GMP-compatible medium.

B cells can differentiate into plasmablast and plasma cells, capable of producing antibodies for decades. Gene editing using zinc-finger nucleases (ZFN) enables the engineering of B cells capable of secreting sustained and high levels of therapeutic proteins. In this study, we established an advanced in vitro good manufacturing practice-compatible culturing system characterized by robust and consistent expansion rate, high viability, and efficient B cell differentiation. Using this process, an optimized B cell editing protocol was developed by combining ZFN/adeno-associated virus 6 technology to achieve site-specific insertion of the human factor IX R338L Padua into the silent TRAC locus. In vitro analysis revealed high levels of secreted human immunoglobulins and human factor IX-Padua. Following intravenous infusion in a mouse model, human plasma cells were detected in spleen and bone marrow, indicating successful and potentially long-term engraftment in vivo. Moreover, high levels of human immunoglobin and therapeutic levels of human factor IX-Padua were detected in mouse plasma, correlating with 15% of normal human factor IX activity. These data suggest that the proposed process promotes the production of functional and differentiated engineered B cells. In conclusion, this study represents an important step toward the development of a manufacturing platform for potential B cell-derived therapeutic products.

Unclasping potentials of genomics and gene editing in chickpea to fight climate change and global hunger threat.

Genomics and genome editing promise enormous opportunities for crop improvement and elementary research. Precise modification in the specific targeted location of a genome has profited over the unplanned insertional events which are generally accomplished employing unadventurous means of genetic modifications. The advent of new genome editing procedures viz; zinc finger nucleases (ZFNs), homing endonucleases, transcription activator like effector nucleases (TALENs), Base Editors (BEs), and Primer Editors (PEs) enable molecular scientists to modulate gene expressions or create novel genes with high precision and efficiency. However, all these techniques are exorbitant and tedious since their prerequisites are difficult processes that necessitate protein engineering. Contrary to first generation genome modifying methods, CRISPR/Cas9 is simple to construct, and clones can hypothetically target several locations in the genome with different guide RNAs. Following the model of the application in crop with the help of the CRISPR/Cas9 module, various customized Cas9 cassettes have been cast off to advance mark discrimination and diminish random cuts. The present study discusses the progression in genome editing apparatuses, and their applications in chickpea crop development, scientific limitations, and future perspectives for biofortifying cytokinin dehydrogenase, nitrate reductase, superoxide dismutase to induce drought resistance, heat tolerance and higher yield in chickpea to encounter global climate change, hunger and nutritional threats.

Construction and Evaluation of Zinc Finger Nucleases.

Zinc finger nucleases (ZFNs) are programmable nucleases that have contributed significantly to past genome-editing research. They are now utilized much less owing to the advent of transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein system (CRISPR-Cas). These new methods allow for easier generation of reagents that target genomic sequences of interest and are less labor-intensive than ZFNs at targeting desired sequences. However, fundamental ZFN patents have expired, enabling a wide range of their distribution for clinical and industrial applications. This article introduces a ZFN construction protocol that uses bacterial one-hybrid (B1H) selection and single-strand annealing (SSA) assay.

Manipulation of Murine Mitochondrial DNA Heteroplasmy with mtZFNs.

Mouse models of mitochondrial DNA mutations hold promise in the development and optimization of mitochondrial gene therapy technology and for gathering pre-clinical data prior to human trials. Their suitability for this purpose stems from the high similarity of human and murine mitochondrial genomes and the increasing availability of rationally designed AAV vectors capable of selectively transducing murine tissues. Our laboratory routinely optimizes mitochondrially targeted zinc finger nucleases (mtZFNs), the compactness of which makes them highly suitable for downstream AAV-based in vivo mitochondrial gene therapy. This chapter discusses the necessary precautions for the robust and precise genotyping of the murine mitochondrial genome as well as the optimization of mtZFNs intended for subsequent use in vivo.

[Application of genome editing technology in gene therapy].

Genome editing has been attracting increasing attention as a new treatment for several refractory diseases since the CRISPR-Cas discovery has facilitated easy modification of target chromosomal DNA. The concept of treating refractory diseases by genome editing has been achieved in various animal models, and genome editing has been applied to human clinical trials for ß-thalassemia, sickle cell disease, mucopolysaccharidosis, transthyretin amyloidosis, HIV infection, and CAR-T therapy. The genome editing technology targets the germline in industrial applications in animals and plants and is directed at the chromosomal DNA of the somatic cells in human therapeutic applications. Genome editing therapy for germline cells is currently forbidden due to ethical and safety concerns. Concerns regarding genome editing technology include safety (off-target effects) as well as technical aspects (low homologous recombination). Various technological innovations for genome editing are expected to expand its clinical application to various diseases in the future.

First-in-human in vivo genome editing via AAV-zinc-finger nucleases for mucopolysaccharidosis I/II and hemophilia B.

Zinc-finger nuclease (ZFN)-based in vivo genome editing is a novel treatment that can potentially provide lifelong protein replacement with single intravenous administration. Three first-in-human open-label ascending single-dose phase 1/2 studies were performed in parallel (starting November 2017) primarily to assess safety and tolerability of ZFN in vivo editing therapy in mucopolysaccharidosis I (MPS I) (n = 3), MPS II (n = 9), and hemophilia B (n = 1). Treatment was well tolerated with no serious treatment-related adverse events. At the 1e13 vg/kg dose, evidence of genome editing was detected through albumin-transgene fusion transcripts in liver for MPS II (n = 2) and MPS I (n = 1) subjects. The MPS I subject also had a transient increase in leukocyte iduronidase activity to the lower normal range. At the 5e13 vg/kg dose, one MPS II subject had a transient increase in plasma iduronate-2-sulfatase approaching normal levels and one MPS I subject approached mid-normal levels of leukocyte iduronidase activity with no evidence of genome editing. The hemophilia B subject was not able to decrease use of factor IX concentrate; genome editing could not be assessed. Overall, ZFN in vivo editing therapy had a favorable safety profile with evidence of targeted genome editing in liver, but no long-term enzyme expression in blood.

Modulating Mitochondrial DNA Heteroplasmy with Mitochondrially Targeted Endonucleases.

Mitochondria, mainly known as energy factories of eukaryotic cells, also exert several additional signaling and metabolic functions and are today recognized as major cellular biosynthetic and signaling hubs. Mitochondria possess their own genome (mitochondrial DNA-mtDNA), that encodes proteins essential for oxidative phosphorylation, and mutations in it are an important contributor to human disease. The mtDNA mutations often exist in heteroplasmic conditions, with both healthy and mutant versions of the mtDNA residing in patients' cells and the level of mutant mtDNA may vary between different tissues and organs and affect the clinical outcome of the disease. Thus, shifting the ratio between healthy and mutant mtDNA in patients' cells provides an intriguing therapeutic option for mtDNA diseases. In this review we describe current strategies for modulating mitochondrial heteroplasmy levels with engineered endonucleases including mitochondrially targeted TALENs and Zinc finger nucleases (ZFNs) and discuss their therapeutic potential. These gene therapy tools could in the future provide therapeutic help both for patients with mitochondrial disease as well as in preventing the transfer of pathogenic mtDNA mutations from a mother to her offspring.

Transplantation of human cells into Interleukin-2 receptor gamma gene knockout pigs under several conditions.

Introduction: Previously, we performed gene knockout (KO) of interleukin-2 receptor gamma (IL2RG) in porcine fetal fibroblasts using zinc finger nuclease-encoding mRNAs, subsequently generating IL2RG KO pigs using these cells through somatic cell nuclear transfer. The IL2RG KO pigs lacked a thymus and were deficient in T lymphocytes and natural killer cells, similar to human X-linked severe combined immunodeficiency (SCID) patients. The present study aimed to evaluate whether pigs can support the growth of xenografted human cells and have the potential to be an effective animal model. Methods: The IL2RG XKOY pigs used in this study were obtained by mating IL2RG XKOX females with wild-type boars. This permitted the routine production of IL2RG KO pigs via natural breeding without complicated somatic cell cloning procedures; therefore, a sufficient number of pigs could be prepared. We transplanted human HeLa S3 cells expressing the tandem dimer tomato into the ears and pancreas of IL2RG KO pigs. Additionally, a newly developed method for the aseptic rearing of SCID pigs was used in case of necessity. Results: Tumors from the transplanted cells quickly developed in all pigs and were verified by histology and immunohistochemistry. We also transplanted these cells into the pancreas of designated pathogen-free pigs housed in novel biocontainment facilities, and large tumors were confirmed. Conclusions: IL2RG KO pigs have the potential to become useful animal models in a variety of translational biology fields.

Intracellular RNase activity dampens zinc finger nuclease-mediated gene editing in hematopoietic stem and progenitor cells.

Over the past decade, numerous gene-editing platforms which alter host DNA in a highly specific and targeted fashion have been described. Two notable examples are zinc finger nucleases (ZFNs), the first gene-editing platform to be tested in clinical trials, and more recently, CRISPR/Cas9. Although CRISPR/Cas9 approaches have become arguably the most popular platform in the field, the therapeutic advantages and disadvantages of each strategy are only beginning to emerge. We have established a nonhuman primate (NHP) model that serves as a strong predictor of successful gene therapy and gene-editing approaches in humans; our recent work shows that ZFN-edited hematopoietic stem and progenitor cells (HSPCs) engraft at lower levels than CRISPR/Cas9-edited cells. Here, we investigate the mechanisms underlying this difference. We show that optimized culture conditions, including defined serum-free media, augment engraftment of gene-edited NHP HSPCs in a mouse xenograft model. Furthermore, we identify intracellular RNases as major barriers for mRNA-encoded nucleases relative to preformed enzymatically active CRISPR/Cas9 ribonucleoprotein (RNP) complexes. We conclude that CRISPR/Cas9 RNP gene editing is more stable and efficient than ZFN mRNA-based delivery and identify co-delivered RNase inhibitors as a strategy to enhance the expression of gene-editing proteins from mRNA intermediates.

Applications of CPPs in Genome Editing of Plants.

Cell penetrating peptides (CPPs) are short peptides that are able to translocate themselves and their cargo into cells. The progressive and continuous application of CPPs in various fields of basic and applied research shows that they are efficient delivery vectors for an assortment of biomolecules, including nucleic acids and proteins. This feature makes CPPs an excellent tool for modification of plant genomes through transgenesis and genome editing. In this review, we present the progress during the last three decades in application of CPPs for delivery of DNA, RNA, and proteins into plant cells and tissues. Moreover, we highlight the exploiting of CPPs as advantageous and beneficial tool for plant genome editing via delivery of nuclease proteins, and provide a practical example of genome alternation through CPP-delivered nucleases. Finally, the current exploitation of peptides in organelle-specific DNA delivery and modification of organellar genomes is discussed.

Targeted integration of EpCAM-specific CAR in human induced pluripotent stem cells and their differentiation into NK cells.

BACKGROUND: Redirection of natural killer (NK) cells with chimeric antigen receptors (CAR) is attractive in developing off-the-shelf CAR therapeutics for cancer treatment. However, the site-specific integration of a CAR gene into NK cells remains challenging. METHODS: In the present study, we genetically modified human induced pluripotent stem cells (iPSCs) with a zinc finger nuclease (ZFN) technology to introduce a cDNA encoding an anti-EpCAM CAR into the adeno-associated virus integration site 1, a "safe harbour" for transgene insertion into human genome, and next differentiated the modified iPSCs into CAR-expressing iNK cells. RESULTS: We detected the targeted integration in 4 out of 5 selected iPSC clones, 3 of which were biallelically modified. Southern blotting analysis revealed no random integration events. iNK cells were successfully derived from the modified iPSCs with a 47-day protocol, which were morphologically similar to peripheral blood NK cells, displayed NK phenotype (CD56+CD3-), and expressed NK receptors. The CAR expression of the iPSC-derived NK cells was confirmed with RT-PCR and flow cytometry analysis. In vitro cytotoxicity assay further confirmed their lytic activity against NK cell-resistant, EpCAM-positive cancer cells, but not to EpCAM-positive normal cells, demonstrating the retained tolerability of the CAR-iNK cells towards normal cells. CONCLUSION: Looking ahead, the modified iPSCs generated in the current study hold a great potential as a practically unlimited source to generate anti-EpCAM CAR iNK cells.

Mutagenic repair of a ZFN-induced double-strand break in yeast: Effects of cleavage site sequence and spacer size.

Double-strand breaks are repaired by error-free homologous recombination or by relatively error-prone pathways that directly join broken ends. Both types of repair have been extensively studied in Saccharomyces cerevisiae using enzymes HO or I-SceI, which create breaks with 4-nt 3' overhangs. In the current study, a galactose-regulated zinc-finger nuclease (ZFN) designed to cleave the Drosophila rosy locus was used to generate breaks with 4-nt 5' overhangs at out-of-frame cleavage sites inserted into the yeast LYS2 gene. Mutagenic repair was examined following selection of prototrophs on lysine-deficient medium containing galactose or surviving colonies on galactose-containing rich medium. Following cleavage of the original rosy spacer (ACGAAT), most Lys+ colonies contained 1- or 4-bp insertions at the cleavage site while most survivors had either a 2-bp insertion or a large deletion. Small insertions reflected nonhomologous end joining (NHEJ) and large deletions were the product of microhomology-mediated end joining (MMEJ). Changing the original ACGAAT spacer to either AGCAAT, ACGCGT or CTATTA altered the molecular features of NHEJ events as well as their frequency relative to MMEJ. Altering the optimal 6-bp spacer size between the zinc-finger protein binding sites to 5 bp or 7 bp eliminated the effect of continuous ZFN expression on survival, but Lys+ prototrophs were still generated. Analysis of Lys+ revertants after cleavage of the 5-bp spacer indicated that both the position and spacing of ZFN-generated nicks were variable. Results provide insight into effects of overhang sequence on mutagenic outcomes and demonstrate ZFN cleavage of 5- or 7-bp spacers in vivo.

Improvement of the efficiency and quality in developing a new CHO host cell line.

Chinese hamster ovary (CHO) cells are a ubiquitous tool for industrial therapeutic recombinant protein production. However, consistently generating high-producing clones remains a major challenge during the cell line development process. The glutamine synthetase (GS) and dihydrofolate reductase (DHFR) selection systems are commonly used CHO expression platforms based on controlling the balance of expression between the transgenic and endogenous GS or DHFR genes. Since the expression of the endogenous selection gene in CHO hosts can interfere with selection, generating a corresponding null CHO cell line is required to improve selection stringency, productivity, and stability. However, the efficiency of generating bi-allelic genetic knockouts using conventional protocols is very low (<5%). This significantly affects clone screening efficiency and reduces the chance of identifying robust knockout host cell lines. In this study, we use the GS expression system as an example to improve the genome editing process with zinc finger nucleases (ZFNs), resulting in improved GS-knockout efficiency of up to 46.8%. Furthermore, we demonstrate a process capable of enriching knockout CHO hosts with robust bioprocess traits. This integrated host development process yields a larger number of GS-knockout hosts with desired growth and recombinant protein expression characteristics.

Current status of the application of gene editing in pigs.

Genetically modified animals, especially rodents, are widely used in biomedical research. However, non-rodent models are required for efficient translational medicine and preclinical studies. Owing to the similarity in the physiological traits of pigs and humans, genetically modified pigs may be a valuable resource for biomedical research. Somatic cell nuclear transfer (SCNT) using genetically modified somatic cells has been the primary method for the generation of genetically modified pigs. However, site-specific gene modification in porcine cells is inefficient and requires laborious and time-consuming processes. Recent improvements in gene-editing systems, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (CRISPR/Cas) system, represent major advances. The efficient introduction of site-specific modifications into cells via gene editors dramatically reduces the effort and time required to generate genetically modified pigs. Furthermore, gene editors enable direct gene modification during embryogenesis, bypassing the SCNT procedure. The application of gene editors has progressively expanded, and a range of strategies is now available for porcine gene engineering. This review provides an overview of approaches for the generation of genetically modified pigs using gene editors, and highlights the current trends, as well as the limitations, of gene editing in pigs.

ZFN-mediated in vivo gene editing in hepatocytes leads to supraphysiologic α-Gal A activity and effective substrate reduction in Fabry mice.

Fabry disease, a lysosomal storage disorder resulting from the deficient activity of α-galactosidase A (α-Gal A), is characterized by cardiac, renal, and/or cerebrovascular disease due to progressive accumulation of the enzyme's substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). We report here the preclinical evaluation of liver-targeted in vivo genome editing using zinc-finger nuclease (ZFN) technology to insert the human α-galactosidase A (hGLA) cDNA into the albumin "safe harbor" locus of Fabry mice, thereby generating an albumin-α-Gal A fusion protein. The mature α-Gal A protein is secreted into the circulation for subsequent mannose-6-phosphate receptor-mediated tissue uptake. Donor vector optimization studies showed that replacing the hGLA cDNA signal peptide sequence with that of human iduronate 2-sulfatase (IDS) achieved higher transgene expression. Intravenous adeno-associated virus (AAV) 2/8-mediated co-delivery of the IDS-hGLA donor and ZFNs targeting the albumin locus resulted in continuous, supraphysiological plasma and tissue α-Gal A activities, which essentially normalized Gb3 and Lyso-Gb3 levels in key tissues of pathology. Notably, this was achieved with <10% of hepatocytes being edited to express hGLA, occurring mostly via non-homologous end joining (NHEJ) rather than homology-directed repair (HDR). These studies indicate that ZFN-mediated in vivo genome editing has the potential to be an effective one-time therapy for Fabry disease.

Can genetic engineering-based methods for gene function identification be eclipsed by genome editing in plants? A comparison of methodologies.

Finding and explaining the functions of genes in plants have promising applications in crop improvement and bioprospecting and hence, it is important to compare various techniques available for gene function identification in plants. Today, the most popular technology among researchers to identify the functions of genes is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-based genome editing method. But by no means can we say that CRISPR/Cas9 is the go-to method for all purposes. It comes with its own baggage. Researchers will agree and have lived through at least seven more technologies deployed to find the functions of genes, which come under three umbrellas: 1. genetic engineering, 2. transient expression, and 3. chemical/physical mutagenesis. Each of the methods evolved when the previous one ran into an insurmountable problem. In this review, we compare the eight technologies against one another on 14 parameters. This review lays bare the pros and cons, and similarities and dissimilarities of various methods. Every method comes with its advantages and disadvantages. For example, the CRISPR/Cas9-based genome editing is an excellent method for modifying gene sequences, creating allelic versions of genes, thereby aiding the understanding of gene function. But it comes with the baggage of unwanted or off-target mutations. Then, we have methods based on random or targeted knockout of the gene, knockdown, and overexpression of the gene. Targeted disruption of genes is required for complete knockout of gene function, which may not be accomplished by editing. We have also discussed the strategies to overcome the shortcomings of the targeted gene-knockout and the CRISPR/Cas9-based methods. This review serves as a comprehensive guide towards the understanding and comparison of various technologies available for gene function identification in plants and hence, it will find application for crop improvement and bioprospecting related research.

Acquisition of chromosome 1q duplication in parental and genome-edited human-induced pluripotent stem cell-derived neural stem cells results in their higher proliferation rate in vitro and in vivo.

OBJECTIVES: Genetic engineering of human-induced pluripotent stem cell-derived neural stem cells (hiPSC-NSC) may increase the risk of genomic aberrations. Therefore, we asked whether genetic modification of hiPSC-NSCs exacerbates chromosomal abnormalities that may occur during passaging and whether they may cause any functional perturbations in NSCs in vitro and in vivo. MATERIALS AND METHODS: The transgenic cassette was inserted into the AAVS1 locus, and the genetic integrity of zinc-finger nuclease (ZFN)-modified hiPSC-NSCs was assessed by the SNP-based karyotyping. The hiPSC-NSC proliferation was assessed in vitro by the EdU incorporation assay and in vivo by staining of brain slices with Ki-67 antibody at 2 and 8 weeks after transplantation of ZFN-NSCs with and without chromosomal aberration into the striatum of immunodeficient rats. RESULTS: During early passages, no chromosomal abnormalities were detected in unmodified or ZFN-modified hiPSC-NSCs. However, at higher passages both cell populations acquired duplication of the entire long arm of chromosome 1, dup(1)q. ZNF-NSCs carrying dup(1)q exhibited higher proliferation rate than karyotypically intact cells, which was partly mediated by increased expression of AKT3 located on Chr1q. Compared to karyotypically normal ZNF-NSCs, cells with dup(1)q also exhibited increased proliferation in vivo 2 weeks, but not 2 months, after transplantation. CONCLUSIONS: These results demonstrate that, independently of ZFN-editing, hiPSC-NSCs have a propensity for acquiring dup(1)q and this aberration results in increased proliferation which might compromise downstream hiPSC-NSC applications.

Targeting of cholera toxin A (ctxA) gene by zinc finger nuclease: pitfalls of using gene editing tools in prokaryotes.

BACKGROUND AND PURPOSE: The study was launched to use zinc finger nuclease (ZFN) technology to disrupt the cholera toxin gene (ctxA) for inhibiting CT toxin production in Vibrio cholera (V. cholera). EXPERIMENTAL APPROACH: An engineered ZFN was designed to target the catalytic site of the ctxA gene. The coding sequence of ZFN was cloned to pKD46, pTZ57R T/A vector, and E2-crimson plasmid and transformed to Escherichia coli (E. coli) Top10 and V. cholera. The efficiency of ZFN was evaluated by colony counting. FINDINGS/RESULTS: No expression was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting in transformed E. coli. The ctxA gene sequencing did not show any mutation. Polymerase chain reaction on pKD46-ZFN plasmid had negative results. Transformation of E. coli Top10 with T/A vectors containing whole ZFN sequence led to 7 colonies all of which contained bacteria with self-ligated vector. Transformation with left array ZFN led to 24 colonies of which 6 contained bacteria with self-ligated vector and 18 of them contained bacteria with vector/left array. Transformation of V. cholera with E2-crimson vectors containing whole ZFN did not produce any colonies. Transformation with left array vectors led to 17 colonies containing bacteria with vector/left array. Left array protein band was captured using western blot assay. CONCLUSIONS AND IMPLICATIONS: ZFN might have off target on bacterial genome causing lethal double-strand DNA break due to lack of non-homologous end joining (NHEJ) mechanism. It is recommended to develop ZFNs against bacterial genes, engineered packaging host with NHEJ repair system is essential.