Transcription Activator-like Nucleases Research Articles

Therapeutic Application of Genome Editing Technologies in Viral Diseases.

Viral infections can be fatal and consequently, they are a serious threat to human health. Therefore, the development of vaccines and appropriate antiviral therapeutic agents is essential. Depending on the virus, it can cause an acute or a chronic infection. The characteristics of viruses can act as inhibiting factors for the development of appropriate treatment methods. Genome editing technology, including the use of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) proteins, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), is a technology that can directly target and modify genomic sequences in almost all eukaryotic cells. The development of this technology has greatly expanded its applicability in life science research and gene therapy development. Research on the use of this technology to develop therapeutics for viral diseases is being conducted for various purposes, such as eliminating latent infections or providing resistance to new infections. In this review, we will look at the current status of the development of viral therapeutic agents using genome editing technology and discuss how this technology can be used as a new treatment approach for viral diseases.

A Comprehensive Review of Indel Detection Methods for Identification of Zebrafish Knockout Mutants Generated by Genome-Editing Nucleases.

The use of zebrafish in functional genomics and disease modeling has become popular due to the ease of targeted mutagenesis with genome editing nucleases, i.e., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9). These nucleases, specifically CRISPR/Cas9, are routinely used to generate gene knockout mutants by causing a double stranded break at the desired site in the target gene and selecting for frameshift insertions or deletions (indels) caused by the errors during the repair process. Thus, a variety of methods have been developed to identify fish with indels during the process of mutant generation and phenotypic analysis. These methods range from PCR and gel-based low-throughput methods to high-throughput methods requiring specific reagents and/or equipment. Here, we provide a comprehensive review of currently used indel detection methods in zebrafish. By discussing the molecular basis for each method as well as their pros and cons, we hope that this review will serve as a comprehensive resource for zebrafish researchers, allowing them to choose the most appropriate method depending upon their budget, access to required equipment and the throughput needs of the projects.

Btg2 mutation induces renal injury and impairs blood pressure control in female rats.

Hypertension (HTN) is a complex disease influenced by heritable genetic elements and environmental interactions. Dietary salt is among the most influential modifiable factors contributing to increased blood pressure (BP). It is well established that men and women develop BP impairment in different patterns and a recent emphasis has been placed on identifying mechanisms leading to the differences observed between the sexes in HTN development. The current work reported here builds on an extensive genetic mapping experiment that sought to identify genetic determinants of salt-sensitive (SS) HTN using the Dahl SS rat. BTG antiproliferation factor 2 (Btg2) was previously identified by our group as a candidate gene contributing to SS HTN in female rats. In the current study, Btg2 was mutated using transcription activator-like effector nuclease (TALEN)-targeted gene disruption on the SSBN congenic rat background. The Btg2 mutated rats exhibited impaired BP and proteinuria responses to a high-salt diet compared with wild-type rats. Differences in body weight, mutant pup viability, skeletal morphology, and adult nephron density suggest a potential role for Btg2 in developmental signaling pathways. Subsequent cell cycle gene expression assessment provides several additional signaling pathways that Btg2 may function through during salt handling in the kidney. The expression analysis also identified several potential upstream targets that can be explored to further isolate therapeutic approaches for SS HTN.

CRISPR/Cas9 technology and its application in horticultural crops

Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated 9 (CRISPR/Cas9) system has recently become one popular technology due to its efficiency, precision, and simplicity compared with other genome editing tools such as Zinc Finger Nucleases (ZFNs) and Transcription Activator Like Effector Nucleases (TALENs). Horticultural crops provide energy and health-keeping nutrients to humankind. Genome-editing technology has become widely adopted in horticultural breeding with the increasing demand for high yield and better-quality horticultural crops. Here, we describe the CRISPR/Cas9 system construction, its optimization, including sgRNA promoter, sgRNA design, Cas9 protein promoter, SpCas9 variants and orthologs, and vector delivery methods. We also summarized the application of this technology in horticultural plants for stress responses enhancement, fruit quality improvement, and cultivation traits modification. This detailed review was compiled to help establish comprehensive understanding of the CRISPR/Cas9 systems and provide a reference for further developing this technology to manipulate horticultural plant traits effectively.

Establishment of CRFK cells for vaccine production by inactivating endogenous retrovirus with TALEN technology

Endogenous retroviruses (ERVs) are retroviral sequences present in the host genomes. Although most ERVs are inactivated, some are produced as replication-competent viruses from host cells. We previously reported that several live-attenuated vaccines for companion animals prepared using the Crandell-Rees feline kidney (CRFK) cell line were contaminated with a replication-competent feline ERV termed RD-114 virus. We also found that the infectious RD-114 virus can be generated by recombination between multiple RD-114 virus-related proviruses (RDRSs) in CRFK cells. In this study, we knocked out RDRS env genes using the genome-editing tool TAL Effector Nuclease (TALEN) to reduce the risk of contamination by infectious ERVs in vaccine products. As a result, we succeeded in establishing RDRS knockout CRFK cells (RDKO_CRFK cells) that do not produce infectious RD-114 virus. The growth kinetics of feline herpesvirus type 1, calicivirus, and panleukopenia virus in RDKO_CRFK cells differed from those in parental cells, but all of them showed high titers exceeding 107 TCID50/mL. Infectious RD-114 virus was undetectable in the viral stocks propagated in RDKO_CRFK cells. This study suggested that RDRS env gene-knockout CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with no risk of contamination with infectious ERV.

An Insight into Modern Targeted Genome-Editing Technologies with a Special Focus on CRISPR/Cas9 and its Applications.

Genome-editing technology has enabled scientists to make changes in model organisms’ DNA at the genomic level to get biotechnologically important products from them. Most commonly employed technologies for this purpose are transcription activator like effector nucleases (TALENs), homing-endonucleases or meganucleases, zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9). Among these tools, CRISPR/Cas9 is most preferred because it's easy to use, has a small mutation rate, has great effectiveness, low cost of development, and decreased rate of advancement. CRISPR/Cas9 has a lot of applications in plants, animals, humans, and microbes. It also has applications in many fields such as horticulture, cancer, food biotechnology, and targeted human genome treatments. CRISPR technology has shown great potential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic to provide early and easy detection methods, possible treatment, and vaccine development. In the present review, genome-editing tools with their basic assembly and features have been discussed. Exceptional notice has been paid to CRISPR technology on basis of its structure and significant applications in humans, plants, animals, and microbes such as bacteria, viruses, and fungi. The review has also shed a little light on current CRISPR challenges and future perspectives.Graphical abstract

Gene Editing of the Catfish Gonadotropin-Releasing Hormone Gene and Hormone Therapy to Control the Reproduction in Channel Catfish, Ictalurus punctatus.

Simple SummaryGonadotropin-releasing hormone (GnRH) plays a pivotal role in fish reproduction. In the present study, transcription activator-like effector nuclease (TALEN) plasmids targeting the catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). After low fertility was observed, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants (individuals with human-induced sequence changes at the cfGnRH locus). Gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.Transcription activator-like effector nuclease (TALEN) plasmids targeting the channel catfish gonadotropin-releasing hormone (cfGnRH) gene were delivered into fertilized eggs with double electroporation to sterilize channel catfish (Ictalurus punctatus). Targeted cfGnRH fish were sequenced and base deletion, substitution, and insertion were detected. The gene mutagenesis was achieved in 52.9% of P1 fish. P1 mutants (individuals with human-induced sequence changes at the cfGnRH locus) had lower spawning rates (20.0–50.0%) when there was no hormone therapy compared to the control pairs (66.7%) as well as having lower average egg hatch rates (2.0% versus 32.3–74.3%) except for one cfGnRH mutated female that had a 66.0% hatch rate. After low fertility was observed in 2016, application of luteinizing hormone-releasing hormone analog (LHRHa) hormone therapy resulted in good spawning and hatch rates for mutants in 2017, which were not significantly different from the controls (p > 0.05). No exogenous DNA fragments were detected in the genome of mutant P1 fish, indicating no integration of the plasmids. No obvious effects on other economically important traits were observed after the knockout of the reproductive gene in the P1 fish. Growth rates, survival, and appearance between mutant and control individuals were not different. While complete knock-out of reproductive output was not achieved, as these were mosaic P1 brood stock, gene editing of channel catfish for the reproductive confinement of gene-engineered, domestic, and invasive fish to prevent gene flow into the natural environment appears promising.

Expanding the targeting scope of FokI‐dCas nuclease systems with SpRY and Mb2Cas12a

CRISPR-Cas9 and Cas12a are widely used sequence-specific nucleases (SSNs) for genome editing. The nuclease domains of Cas proteins can induce DNA double strand breaks upon RNA guided DNA targeting. Zinc finger nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) have been popular SSNs prior to CRISPR. Both ZFNs and TALENs are based on reconstitution of two monomers with each consisting of a DNA binding domain and a FokI nuclease domain. Inspired by the configuration of ZFNs and TALENs, dimeric FokI-dCas9 systems were previously demonstrated in human cells. Such configuration, based on a pair of guide RNAs (gRNAs), offers great improvement on targeting specificity. To expand the targeting scope of dimeric FokI-dCas systems, the PAM (protospacer adjacent motif)-less SpRY Cas9 variant and the PAM-relaxed Mb2Cas12a system were explored. Rice cells showed that FokI-dSpRY had more robust editing efficiency than a paired SpRY nickase system. Furthermore, a dimeric FokI-dMb2Cas12a system wasdeveloped that displayed comparable editing activity to Mb2Cas12a nuclease in rice cells. Finally, a single-chain FokI-FokI-dMb2Cas12a system wasdeveloped that cuts DNA outside its targeting sequence, which could be useful for many versatile applications. Together, this work greatly expanded the FokI based CRISPR-Cas systems for genome editing.

Current and Possible Future Therapeutic Options for Huntington's Disease.

Huntington’s disease (HD) is an autosomal neurodegenerative disease that is characterized by an excessive number of CAG trinucleotide repeats within the huntingtin gene (HTT). HD patients can present with a variety of symptoms including chorea, behavioural and psychiatric abnormalities and cognitive decline. Each patient has a unique combination of symptoms, and although these can be managed using a range of medications and non-drug treatments there is currently no cure for the disease. Current therapies prescribed for HD can be categorized by the symptom they treat. These categories include chorea medication, antipsychotic medication, antidepressants, mood stabilizing medication as well as non-drug therapies. Fortunately, there are also many new HD therapeutics currently undergoing clinical trials that target the disease at its origin; lowering the levels of mutant huntingtin protein (mHTT). Currently, much attention is being directed to antisense oligonucleotide (ASO) therapies, which bind to pre-RNA or mRNA and can alter protein expression via RNA degradation, blocking translation or splice modulation. Other potential therapies in clinical development include RNA interference (RNAi) therapies, RNA targeting small molecule therapies, stem cell therapies, antibody therapies, non-RNA targeting small molecule therapies and neuroinflammation targeted therapies. Potential therapies in pre-clinical development include Zinc-Finger Protein (ZFP) therapies, transcription activator-like effector nuclease (TALEN) therapies and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated system (Cas) therapies. This comprehensive review aims to discuss the efficacy of current HD treatments and explore the clinical trial progress of emerging potential HD therapeutics.

Applications of Genome Editing Tools in Stem Cells Towards Regenerative Medicine: An Update

Precise and site-specific genome editing through application of emerging and modern gene engineering techniques, namely zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR/ Cas9) have swiftly progressed the application and use of the stem cell technology in the sphere of in-vitro disease modelling and regenerative medicine. Genome editing tools facilitate the manipulation of genes in various types of cells with target-specific nucleases. These tools aid in elucidating the genetics and etiology behind different diseases and have immense promise as novel therapeutics for correcting the genetic mutations, making alterations, and curing diseases permanently, which are not responding and resistant to traditional therapies. These genome engineering tools have evolved in the field of biomedical research and have also been shown to have a significant improvement in clinical trials. However, their widespread use in the research revealed potential safety issues, which need to be addressed before implementing such techniques for clinical purposes. Significant and valiant attempts need to be made in order to surpass those hurdles. The current review outlines the advancements of several genome engineering tools and describes suitable strategies for their application towards regenerative medicine.

Generation of genetic modified pigs devoid of GGTA1 and expressing the human leukocyte antigen-G5

Pigs are considered as ideal donors for xenotransplantation because they have many physiological and anatomical characteristics similar to human beings. However, antibody-mediated immunity, which includes both natural and induced antibody responses, is a major challenge for the success of pig-to-primate xenotransplantation. Various genetic modification methods help to tailor pigs to be appropriate donors for xenotransplantation. In this study, we applied transcription activator-like effector nuclease (TALEN) to knock out the porcine α-1, 3-galactosyltransferase gene GGTA1, which encodes Gal epitopes that induce hyperacute immune rejection in pig-to-human xenotransplantation. Meanwhile, human leukocyte antigen-G5 gene HLA-G5, which acts as an immunosuppressive factor, was co-transfected with TALEN into porcine fetal fibroblasts. The cell colonies of GGTA1 biallelic knockout with positive transgene for HLA-G5 were chosen as nuclear donors to generate genetic modified piglets through a single round of somatic cell nuclear transfer. As a result, we successfully obtained 20 modified piglets that were positive for GGTA1 knockout (GTKO) and half of them expressed the HLA-G5 protein. Gal epitopes on the cell membrane of GTKO/HLA-G5 piglets were completely absent. Western blotting and immunofluorescence showed that HLA-G5 was expressed in the modified piglets. Functionally, the fibroblasts from the GTKO/HLA-G5 piglets showed enhanced resistance to complement-mediated lysis ability compared with those from GTKO-only or wild-type pigs. These results indicate that the GTKO/HLA-G5 pigs could be a valuable donor model to facilitate laboratory studies and clinics for xenotransplantation.

Zygotic nanos3 Mutant Medaka (Oryzias latipes) Displays Gradual Loss of Germ Cells and Precocious Spermatogenesis During Gonadal Development.

Nanos is one of the components of germ plasm and is evolutionarily conserved from flies to mammals. In medaka (Oryzias latipes), maternally provided nanos3 is essential for maintenance of primordial germ cells (PGCs). Here, we generated nanos3 loss-of-function mutants by using transcription activator-like effector nuclease (TALEN) and examined the function of zygotic nanos3 in medaka. Zygotic nanos3 homozygous (-/-) mutants derived from nanos3 heterozygous mothers formed germ cells. However, after hatching, the number of germ cells decreased considerably, resulting in infertility of both nanos3-/- females and males. Surprisingly, both nanos3-/- XX and XY mutants underwent precocious spermatogenesis during early gonadal development, as seen in loss-of-function mutants of foxl3, the germline sex-determination gene, in medaka. Therefore, in addition to the maintenance of germ cells, these results suggest that zygotic nanos3 affects the proper regulation of germline sex in XX medaka.

Modified Gene Editing Systems: Diverse Bioengineering Tools and Crop Improvement.

Gene-editing systems have emerged as bioengineering tools in recent years. Classical gene-editing systems include zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9), and these tools allow specific sequences to be targeted and edited. Various modified gene-editing systems have been established based on classical gene-editing systems. Base editors (BEs) can accurately carry out base substitution on target sequences, while prime editors (PEs) can replace or insert sequences. CRISPR systems targeting mitochondrial genomes and RNA have also been explored and established. Multiple gene-editing techniques based on CRISPR/Cas9 have been established and applied to genome engineering. Modified gene-editing systems also make transgene-free plants more readily available. In this review, we discuss the modifications made to gene-editing systems in recent years and summarize the capabilities, deficiencies, and applications of these modified gene-editing systems. Finally, we discuss the future developmental direction and challenges of modified gene-editing systems.

Effect of cryopreservation on A172 and U251 glioma cells infected with lentiviral vectors designed for CRISPR/Cas9-mediated aquaporin-8 knock-out.

Among the three existing targeted gene editing technologies, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats-CRISPR-associated 9 (CRISPR-Cas9), the latter is widely used owing to its simplicity, efficiency, and low cost. Here, we routinely infected A172 and U251 cells with lentiviral vectors, in which aquaporin-8 (AQP8) was knocked out using CRISPR/Cas9. Our results indicated that cryopreservation did not significantly alter the viral infection efficiency, but influenced AQP8 expression in the infected cells at both protein and mRNA levels compared with the non-cryopreserved samples. Further, AQP8 expression at protein and mRNA levels in recovered cryopreserved infected cells did not significantly differ from those in the blank and negative controls, indicating that the lentivirus was still infectious at low temperatures. However, it failed to release the AQP8-targeting guide RNA in the infected cells, or the guide RNA was released, but underwent changes that caused it to malfunction in the cells with CRISPR/Cas9-mediated AQP8 knock-out. Our findings possibly provide some insights into the reliability of lentiviruses as CRISPR/Cas9 vectors.

Targeted introduction of heritable point mutations into the plant mitochondrial genome

The development of technologies for the genetic manipulation of mitochondrial genomes remains a major challenge. Here we report a method for the targeted introduction of mutations into plant mitochondrial DNA (mtDNA) that we refer to as transcription activator-like effector nuclease (TALEN) gene-drive mutagenesis (GDM), or TALEN-GDM. The method combines TALEN-induced site-specific cleavage of the mtDNA with selection for mutations that confer resistance to the TALEN cut. Applying TALEN-GDM to the tobacco mitochondrial nad9 gene, we isolated a large set of mutants carrying single amino acid substitutions in the Nad9 protein. The mutants could be purified to homochondriomy and stably inherited their edited mtDNA in the expected maternal fashion. TALEN-GDM induces both transitions and transversions, and can access most nucleotide positions within the TALEN binding site. Our work provides an efficient method for targeted mitochondrial genome editing that produces genetically stable, homochondriomic and fertile plants with specific point mutations in their mtDNA.

Recent Advances in the Production of Genome-Edited Rats.

The rat is an important animal model for understanding gene function and developing human disease models. Knocking out a gene function in rats was difficult until recently, when a series of genome editing (GE) technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the type II bacterial clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated Cas9 (CRISPR/Cas9) systems were successfully applied for gene modification (as exemplified by gene-specific knockout and knock-in) in the endogenous target genes of various organisms including rats. Owing to its simple application for gene modification and its ease of use, the CRISPR/Cas9 system is now commonly used worldwide. The most important aspect of this process is the selection of the method used to deliver GE components to rat embryos. In earlier stages, the microinjection (MI) of GE components into the cytoplasm and/or nuclei of a zygote was frequently employed. However, this method is associated with the use of an expensive manipulator system, the skills required to operate it, and the egg transfer (ET) of MI-treated embryos to recipient females for further development. In vitro electroporation (EP) of zygotes is next recognized as a simple and rapid method to introduce GE components to produce GE animals. Furthermore, in vitro transduction of rat embryos with adeno-associated viruses is potentially effective for obtaining GE rats. However, these two approaches also require ET. The use of gene-engineered embryonic stem cells or spermatogonial stem cells appears to be of interest to obtain GE rats; however, the procedure itself is difficult and laborious. Genome-editing via oviductal nucleic acids delivery (GONAD) (or improved GONAD (i-GONAD)) is a novel method allowing for the in situ production of GE zygotes existing within the oviductal lumen. This can be performed by the simple intraoviductal injection of GE components and subsequent in vivo EP toward the injected oviducts and does not require ET. In this review, we describe the development of various approaches for producing GE rats together with an assessment of their technical advantages and limitations, and present new GE-related technologies and current achievements using those rats in relation to human diseases.

Intrauterine Fetal Gene Therapy: Is That the Future and Is That Future Now?

Researchers are looking into techniques to intervene sooner and earlier in the disease process thanks to advances in disease genetics, etiologies, and prenatal diagnosis. We conducted a literature search in PubMed-indexed journals to provide an overview of the evolution of gene therapy, rationale for prenatal gene therapy, uses and risks of gene therapy, and ethical issues following the usage of gene therapy. Recent animal research has revealed that transmitting genetic material to a growing fetus through viral and non-viral vectors is conceivable besides proving how gene-editing technology is achieved by various mechanisms that utilize zinc finger nucleases, TAL effector nucleases, and clustered short palindromic repeats-Cas9 complex. This review offers an overview of the current knowledge in the field of prenatal gene therapy, as well as potential future research avenues. In addition, it weighs the risks of prenatal gene therapy, such as oncogenesis, genetic mutation transfer from mother to child, and fetal disruption, against the expected benefits, such as preventing the development of severe early-onset illness symptoms, targeting previously inaccessible organs, and establishing tolerance to the therapeutic transgenic protein, all of which lead to permanent somatic gene correction.This review discusses the scientific, ethical, legal, and sociological implications of these groundbreaking genetic disease prevention techniques, as well as the parameters that must be satisfied for a future clinical application to be considered.

Article Review: Editing genome of Toxoplasma gondii by CRISPR/Cas9 system

It is well known that finding out the genetic information of any organism helps in understanding how performs its metabolism, and knowing the extent of the effect that gene modification affects the performance of these functions. Many techniques have been used to editing genome of parasites such as Zinc fingers and Transcription activator-like effector nucleases (TALENs), But the development of clustered regularly interspaced short palindromic repeats/ CRISPR-associated (CRISPR Cas9) system created the quantum leap in this area, as this technique relies on identifying the portion to be modified using a guide from Guide RNAs (gRNA) that identifies this part very accurately. Not only that, but the enzyme present with it called CAS9 works as a molecular scissor to cut DNA at this part and allow the required modification.Since 2014, the date on which the first article about use CRISPR/Cas9 in the editing of Toxoplasma gondii genome was published, after that continued, developed new methods and protocols that facilitate the researchers work, we will attempt in this paper to review some of these major achievements.

Gene Editing: a Powerful Tool for Cancer Immunotherapy

Recently, Gene editing has emerged as one of the recent promising tools for gene therapy. This technique is remarkable in biotechnology and medicine since it enables genomic editing in vivo with high accuracy. The most important gene-editing enzymes are zinc finger nucleases (ZFNs), homing meganucleases, transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9). The immune system plays an important role in the removal of abnormal and cancerous cells and their role in the defense against foreign pathogens. Immune cells routinely remove abnormal cells by screening cell function, gene mutations, and cancerous cell formation. The CRISPER technology was widely applied to introduce a therapeutic regimen in cancer treatment based on tumor genome editing. Additionally, it was used in cancer immunotherapy. For example, CRISPR technology has introduced an alternative to the conventional clinical drug, Herceptin, targeting HER2 in breast carcinoma. Moreover, it was used in CAR-T cell generation and immune cell checkpoint inhibition. Researchers are seeking to fight many hard diseases by using CRISPR technology. However, many challenges still exist. Some of these challenges include the requirement of PAM sequence, the possibility of on target deletion or addition, off-target effects, Cas9-DSB complex formation, the lack of perfect delivery methods, and low HDR output. In this review, we outline the application of CRISPR technology in cancer immunotherapy and the challenges that hinder the implementation of this technology.

Gene manipulation in the Mucorales fungus Rhizopus oryzae using TALENs with exonuclease overexpression.

The Mucorales fungal genus Rhizopus is used for the industrial production of organic acids, enzymes and fermented foods. The metabolic engineering efficiency of Rhizopus could be improved using gene manipulation; however, exogenous DNA rarely integrates into the host genome. Consequently, a genetic tool for Mucorales fungi needs to be developed. Recently, programmable nucleases that generate DNA double-strand breaks (DSBs) at specific genomic loci have been used for genome editing in various organisms. In this study, we examined gene disruption in Rhizopus oryzae using transcription activator-like effector nucleases (TALENs), with and without exonuclease overexpression. TALENs with an overexpressing exonuclease induced DSBs, followed by target site deletions. Although DSBs are repaired mainly by nonhomologous end joining in most organisms, our results suggested that in R. oryzae microhomology-mediated end joining was the major DSB repair system. Our gene manipulation method using TALENs coupled with exonuclease overexpression contributes to basic scientific knowledge and the metabolic engineering of Rhizopus.