Abstract 108: TALEN-mediated Gene-Editing in Zebrafish for Studying Titin Biology

Background: Titin, the largest protein found in nature, has been found to be crucial for heart and muscle development and mutations on titin caused human diseases. Especially, truncated mutations on TITIN were found to be a major causative gene (more than 25%) for familiar dilated cardiomyopathy (DCM) but the mechanism is lacking. Due to the gigantic size and number of exons, genetic study of titin has been challenging. To facilitate genetic analysis of titin biology, we leveraged the Transcription Activator-Like Effects Nuclease (TALEN) technology in zebrafish to edit the titin gene directly. Results: We generated TALEN pairs targeting different domains of zebrafish titin, including Z-disc domain, N2B domain, and A domains. Because zebrafish contain two titin homologues, ttna and ttnb , TALEN pairs targeting the corresponding loci for both homologues were made. The mutations are efficiently introduced on the titin using TALENs. In addition to these truncated mutants, TALEN technology can also create internal deletions. The titin mutant fish were identified and further phenotypic analysis has been carried out. Conclusions: Combined with TALEN technology, zebrafish can provide a convenient vertebrate model for genetic studies of titin biology. Moreover, this gene-editing technology will facilitate the use of the zebrafish model as a powerful research tool for modeling human diseases and investigating their pathogenesis.

Download Full-text

Abstract 344: Finding the Achilles’ heel of Muscle Giant---TALEN-mediated Gene-editing in Zebrafish Titin

Circulation Research ◽

10.1161/res.117.suppl_1.344 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Yu-Huan Shih ◽

Xiaolei Xu

Keyword(s):

Alternative Splicing ◽

Gene Editing ◽

Muscle Development ◽

Exon Skipping ◽

Transcription Activator ◽

Detailed Characterization ◽

A Domains ◽

Underlying Mechanisms ◽

Sarcomere Assembly

Background: TITIN (TTN) has more than 300 exons and encodes a gigantic protein that is crucial for heart and muscle development. Mutations in TTN caused a variety of human diseases including cardiomyopathy and muscular dystrophy. Recently, dilated cardiomyopathy-associated mutations on TTN have been found more frequently in exons encoding A-band domains but less in exons encoding the N-terminal Z-disc domains, suggesting that mutations in different exons of TTN cause distinct consequences. To elucidate the underlying mechanisms, we leveraged the Transcription Activator-Like Effects Nuclease (TALEN) technology in zebrafish to introduce truncating mutations in different exons of ttn, and then study their effects on heart and somites. Results: We generated truncational mutations in different exons of zebrafish titins encoding Z-disc, N2B, Novex-3, and A domains, respectively. Because zebrafish contains two titin homologues, ttna and ttnb, we introduced mutations in both genes at the corresponding loci. While both Z-disc and A band mutations on ttna disrupted sarcomere assembly in heart and somites, Z-disc or A band mutations on ttnb only affect somites without affecting the heart. Interestingly, a Z-disc mutation on ttna resulted in milder phenotypes than an A-band mutation, while a Z-disc mutation on ttnb generated severer phenotypes than an A-band mutation. No phenotype was observed in the homozygous fish in either ttna-novex-3 or ttnb-N2B mutant fish. Conclusions: A spectrum of truncational mutations in ttna and ttnb has been generated in zebrafish using the TALEN technology. Mutations in different exons result in different phenotypes. Detailed characterization of these mutants and double mutants will be presented, which shall elicit distinct contribution of alternative splicing and exon skipping as two candidate mechanisms during pathogenesis of Titinopathies.

Download Full-text

mtor Haploinsufficiency Ameliorates Renal Cysts and Cilia Abnormality in Adult Zebrafish tmem67 Mutants

Journal of the American Society of Nephrology ◽

10.1681/asn.2020070991 ◽

2021 ◽

pp. ASN.2020070991

Author(s):

Ping Zhu ◽

Qi Qiu ◽

Peter C. Harris ◽

Xiaolei Xu ◽

Xueying Lin

Keyword(s):

Renal Cysts ◽

Cyst Formation ◽

Adult Zebrafish ◽

Transcription Activator ◽

Primary Defect ◽

Causative Gene ◽

Mtor Inhibition ◽

Adaptive Action ◽

Single Nephron ◽

Vertebrate Model

BackgroundAlthough zebrafish embryos have been used to study ciliogenesis and model polycystic kidney disease (PKD), adult zebrafish remain unexplored.MethodsTranscription activator-like effector nucleases (TALEN) technology was used to generate mutant for tmem67, the homolog of the mammalian causative gene for Meckel syndrome type 3 (MKS3). Classic 2D and optical-clearing 3D imaging of an isolated adult zebrafish kidney were used to examine cystic and ciliary phenotypes. A hypomorphic mtor strain or rapamycin was used to inhibit mTOR activity.ResultsAdult tmem67 zebrafish developed progressive mesonephric cysts that share conserved features of mammalian cystogenesis, including a switch of cyst origin with age and an increase in proliferation of cyst-lining epithelial cells. The mutants had shorter and fewer distal single cilia and greater numbers of multiciliated cells (MCCs). Absence of a single cilium preceded cystogenesis, and expansion of MCCs occurred after pronephric cyst formation and was inversely correlated with the severity of renal cysts in young adult zebrafish, suggesting a primary defect and an adaptive action, respectively. Finally, the mutants exhibited hyperactive mTOR signaling. mTOR inhibition ameliorated renal cysts in both the embryonic and adult zebrafish models; however, it only rescued ciliary abnormalities in the adult mutants.ConclusionsAdult zebrafish tmem67 mutants offer a new vertebrate model for renal cystic diseases, in which cilia morphology can be analyzed at a single-nephron resolution and mTOR inhibition proves to be a candidate therapeutic strategy.

Download Full-text

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-019-0089-y ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 85

Author(s):

Hongyi Li ◽

Yang Yang ◽

Weiqi Hong ◽

Mengyuan Huang ◽

Min Wu ◽

...

Keyword(s):

Animal Models ◽

Genome Editing ◽

Gene Editing ◽

Basic Research ◽

Human Diseases ◽

Eukaryotic Cells ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

Major Genome ◽

Almost All

AbstractBased on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)–Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.

Download Full-text

Exploiting DNA Endonucleases to Advance Mechanisms of DNA Repair

Biology ◽

10.3390/biology10060530 ◽

2021 ◽

Vol 10 (6) ◽

pp. 530

Author(s):

Marlo K. Thompson ◽

Robert W. Sobol ◽

Aishwarya Prakash

Keyword(s):

Dna Repair ◽

Mammalian Cells ◽

Genome Stability ◽

Gene Editing ◽

Adaptive Immune System ◽

Zinc Finger Nucleases ◽

Specific Gene ◽

Target Sequence ◽

Transcription Activator ◽

Low Cytotoxicity

The earliest methods of genome editing, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALENs), utilize customizable DNA-binding motifs to target the genome at specific loci. While these approaches provided sequence-specific gene-editing capacity, the laborious process of designing and synthesizing recombinant nucleases to recognize a specific target sequence, combined with limited target choices and poor editing efficiency, ultimately minimized the broad utility of these systems. The discovery of clustered regularly interspaced short palindromic repeat sequences (CRISPR) in Escherichia coli dates to 1987, yet it was another 20 years before CRISPR and the CRISPR-associated (Cas) proteins were identified as part of the microbial adaptive immune system, by targeting phage DNA, to fight bacteriophage reinfection. By 2013, CRISPR/Cas9 systems had been engineered to allow gene editing in mammalian cells. The ease of design, low cytotoxicity, and increased efficiency have made CRISPR/Cas9 and its related systems the designer nucleases of choice for many. In this review, we discuss the various CRISPR systems and their broad utility in genome manipulation. We will explore how CRISPR-controlled modifications have advanced our understanding of the mechanisms of genome stability, using the modulation of DNA repair genes as examples.

Download Full-text

Zebrafish Model in Ophthalmology to Study Disease Mechanism and Drug Discovery

Pharmaceuticals ◽

10.3390/ph14080716 ◽

2021 ◽

Vol 14 (8) ◽

pp. 716

Author(s):

Yiwen Hong ◽

Yan Luo

Keyword(s):

Drug Discovery ◽

Behavioral Assessment ◽

Age Related Macular Degeneration ◽

Disease Mechanism ◽

Zebrafish Model ◽

Age Related ◽

Eye Disorders ◽

Vertebrate Model ◽

Drug Treatments ◽

Novel Drug

Visual impairment and blindness are common and seriously affect people’s work and quality of life in the world. Therefore, the effective therapies for eye diseases are of high priority. Zebrafish (Danio rerio) is an alternative vertebrate model as a useful tool for the mechanism elucidation and drug discovery of various eye disorders, such as cataracts, glaucoma, diabetic retinopathy, age-related macular degeneration, photoreceptor degeneration, etc. The genetic and embryonic accessibility of zebrafish in combination with a behavioral assessment of visual function has made it a very popular model in ophthalmology. Zebrafish has also been widely used in ocular drug discovery, such as the screening of new anti-angiogenic compounds or neuroprotective drugs, and the oculotoxicity test. In this review, we summarized the applications of zebrafish as the models of eye disorders to study disease mechanism and investigate novel drug treatments.

Download Full-text

VGLL2-NCOA2 leverages developmental programs for pediatric sarcomagenesis

10.1101/2021.09.15.460544 ◽

2021 ◽

Author(s):

Sarah Watson ◽

Collette LaVigne ◽

Lin Xu ◽

Whitney Murchison ◽

Didier Surdez ◽

...

Keyword(s):

Skeletal Muscle ◽

Structural Changes ◽

Muscle Development ◽

Fusion Gene ◽

Transcriptional Level ◽

Mesenchymal Tumors ◽

Developmental Programs ◽

Zebrafish Model ◽

Ras Family ◽

Therapeutic Opportunity

Clinical sequencing efforts are uncovering numerous fusion genes in childhood solid tumors, yet few methods exist to delineate fusion-oncogenes from structural changes of unknown significance. One such novel fusion gene is VGLL2-NCOA2, which was described by us and others in patients with congenital sarcomas but has lacked functional validation. To determine if this fusion is an oncogene, and how it is driving disease, we developed a vertebrate zebrafish model and mouse allograft model of human VGLL2-NCOA2 driven sarcomagenesis. We found that VGLL2-NCOA2 is indeed an oncogene and is sufficient to generate mesenchymal tumors that recapitulate the human disease at the histological and transcriptional level. Zebrafish VGLL2-NCOA2 tumors display features of arrested skeletal muscle development, and a subset transcriptionally cluster with somitogenesis in developing embryos. By comparing tumor and embryonic gene expression signatures, we identified developmentally regulated targets that VGLL2-NCOA2 potentially leverages for tumorigenesis. These targets highlight the core biology of the disease and could represent therapeutic opportunities. Specifically, a RAS family GTPase, arf6/ARF6, involved in actin remodeling and rapid cycling of endocytic vesicles at the plasma membrane, is highly expressed during zebrafish somitogenesis and in VGLL2-NCOA2 tumors. In zebrafish tumors, arf6 protein is highly expressed and is absent from mature skeletal muscle. In VGLL2-NCOA2 mouse allograft models and patient tumors, ARF6 mRNA is overexpressed as compared to skeletal muscle or normal controls. More broadly, ARF6 is overexpressed in adult and pediatric sarcoma subtypes as compared to mature skeletal muscle. Overall, our cross-species comparative oncology approach provides evidence that VGLL2-NCOA2 is an oncogene which leverages developmental programs for tumorigenesis, and that one of these programs, the reactivation or persistence of arf6/ARF6, could represent a therapeutic opportunity.

Download Full-text

Gene editing as a promising approach for respiratory diseases

Journal of Medical Genetics ◽

10.1136/jmedgenet-2017-104960 ◽

2018 ◽

Vol 55 (3) ◽

pp. 143-149 ◽

Cited By ~ 2

Author(s):

Yichun Bai ◽

Yang Liu ◽

Zhenlei Su ◽

Yana Ma ◽

Chonghua Ren ◽

...

Keyword(s):

Respiratory Diseases ◽

Gene Editing ◽

Gene Mutations ◽

Well Being ◽

Short Review ◽

Zinc Finger Nucleases ◽

Transcription Activator ◽

Mortality And Morbidity ◽

Chronic Respiratory Diseases ◽

Difficulty Breathing

Respiratory diseases, which are leading causes of mortality and morbidity in the world, are dysfunctions of the nasopharynx, the trachea, the bronchus, the lung and the pleural cavity. Symptoms of chronic respiratory diseases, such as cough, sneezing and difficulty breathing, may seriously affect the productivity, sleep quality and physical and mental well-being of patients, and patients with acute respiratory diseases may have difficulty breathing, anoxia and even life-threatening respiratory failure. Respiratory diseases are generally heterogeneous, with multifaceted causes including smoking, ageing, air pollution, infection and gene mutations. Clinically, a single pulmonary disease can exhibit more than one phenotype or coexist with multiple organ disorders. To correct abnormal function or repair injured respiratory tissues, one of the most promising techniques is to correct mutated genes by gene editing, as some gene mutations have been clearly demonstrated to be associated with genetic or heterogeneous respiratory diseases. Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) systems are three innovative gene editing technologies developed recently. In this short review, we have summarised the structure and operating principles of the ZFNs, TALENs and CRISPR/Cas9 systems and their preclinical and clinical applications in respiratory diseases.

Download Full-text

Melanogenic Inhibition and Toxicity Assessment of Flavokawain A and B on B16/F10 Melanoma Cells and Zebrafish (Danio rerio)

Molecules ◽

10.3390/molecules25153403 ◽

2020 ◽

Vol 25 (15) ◽

pp. 3403

Author(s):

Nurshafika Mohd Sakeh ◽

Nurliyana Najwa Md Razip ◽

Farah Idayu Mohd Ma’in ◽

Mohammad Nazri Abdul Bahari ◽

Naimah Latif ◽

...

Keyword(s):

Danio Rerio ◽

Toxicity Assessment ◽

Tyrosinase Activity ◽

The Other ◽

Quantitative Real Time Pcr ◽

Melanin Content ◽

Melanin Production ◽

Zebrafish Model ◽

Vertebrate Model ◽

Toxicity Effects

Excessive production of melanin implicates hyperpigmentation disorders. Flavokawain A (FLA) and flavokawain B (FLB) have been reported with anti-melanogenic activity, but their melanogenic inhibition and toxicity effects on the vertebrate model of zebrafish are still unknown. In the present study, cytotoxic as well as melanogenic effects of FLA and FLB on cellular melanin content and tyrosinase activity were evaluated in α-MSH-induced B16/F10 cells. Master regulator of microphthalmia-associated transcription factor (Mitf) and the other downstream melanogenic-related genes were verified via quantitative real time PCR (qPCR). Toxicity assessment and melanogenesis inhibition on zebrafish model was further observed. FLA and FLB significantly reduced the specific cellular melanin content by 4.3-fold and 9.6-fold decrement, respectively in α-MSH-induced B16/F10 cells. Concomitantly, FLA significantly reduced the specific cellular tyrosinase activity by 7-fold whilst FLB by 9-fold. The decrement of melanin production and tyrosinase activity were correlated with the mRNA suppression of Mitf which in turn down-regulate Tyr, Trp-1 and Trp-2. FLA and FLB exhibited non-toxic effects on the zebrafish model at 25 and 6.25 µM, respectively. Further experiments on the zebrafish model demonstrated successful phenotype-based depigmenting activity of FLA and FLB under induced melanogenesis. To sum up, our findings provide an important first key step for both of the chalcone derivatives to be further studied and developed as potent depigmenting agents.

Download Full-text

Immunodeficient Rabbit Models: History, Current Status and Future Perspectives

Applied Sciences ◽

10.3390/app10207369 ◽

2020 ◽

Vol 10 (20) ◽

pp. 7369

Author(s):

Jun Song ◽

Brooke Pallas ◽

Dongshan Yang ◽

Jifeng Zhang ◽

Yash Agarwal ◽

...

Keyword(s):

Gene Editing ◽

Interleukin 2 ◽

Current Status ◽

Loss Of Function ◽

Future Perspectives ◽

Transcription Activator ◽

Immune Genes ◽

Forkhead Box ◽

Recombination Activating Gene ◽

Model Family

Production of immunodeficient (ID) models in non-murine animal species had been extremely challenging until the advent of gene-editing tools: first zinc finger nuclease (ZFN), then transcription activator-like effector nuclease (TALEN), and most recently clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR)/Cas9. We and others used those gene-editing tools to develop ID rabbits carrying a loss of function mutation in essential immune genes, such as forkhead box protein N1 (FOXN1), recombination activating gene 1/2 (RAG1/2), and interleukin 2 receptor subunit gamma (IL2RG). Like their mouse counterparts, ID rabbits have profound defects in their immune system and are prone to bacterial and pneumocystis infections without prophylactic antibiotics. In addition to their use as preclinical models for primary immunodeficient diseases, ID rabbits are expected to contribute significantly to regenerative medicine and cancer research, where they serve as recipients for allo- and xeno-grafts, with notable advantages over mouse models, including a longer lifespan and a much larger body size. Here we provide a concise review of the history and current status of the development of ID rabbits, as well as future perspectives of this new member in the animal model family.

Download Full-text

Progress and Challenges in the Improvement of Ornamental Plants by Genome Editing

Plants ◽

10.3390/plants9060687 ◽

2020 ◽

Vol 9 (6) ◽

pp. 687 ◽

Cited By ~ 3

Author(s):

Chang Ho Ahn ◽

Mummadireddy Ramya ◽

Hye Ryun An ◽

Pil Man Park ◽

Yae-Jin Kim ◽

...

Keyword(s):

Genome Editing ◽

Gene Editing ◽

Genetically Modified Organisms ◽

Ornamental Plants ◽

Safety Evaluation ◽

Evaluation Process ◽

Vase Life ◽

Transcription Activator ◽

A Genome ◽

Dna Specificity

Biotechnological approaches have been used to modify the floral color, size, and fragrance of ornamental plants, as well as to increase disease resistance and vase life. Together with the advancement of whole genome sequencing technologies, new plant breeding techniques have rapidly emerged in recent years. Compared to the early versions of gene editing tools, such as meganucleases (MNs), zinc fingers (ZFNs), and transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeat (CRISPR) is capable of altering a genome more efficiently and with higher accuracy. Most recently, new CRISPR systems, including base editors and prime editors, confer reduced off-target activity with improved DNA specificity and an expanded targeting scope. However, there are still controversial issues worldwide for the recognition of genome-edited plants, including whether genome-edited plants are genetically modified organisms and require a safety evaluation process. In the current review, we briefly summarize the current progress in gene editing systems and also introduce successful/representative cases of the CRISPR system application for the improvement of ornamental plants with desirable traits. Furthermore, potential challenges and future prospects in the use of genome-editing tools for ornamental plants are also discussed.

Download Full-text