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Baldini A, Battaglia F, Perrella G. The generation of novel epialleles in plants: the prospective behind re-shaping the epigenome. FRONTIERS IN PLANT SCIENCE 2025; 16:1544744. [PMID: 40190658 PMCID: PMC11968746 DOI: 10.3389/fpls.2025.1544744] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 12/13/2024] [Accepted: 02/07/2025] [Indexed: 04/09/2025]
Abstract
Chromatin organization is a relevant layer of control of gene expression during plant development. Chromatin states strictly depend on associated features such as DNA methylation, histone modifications and histone variants. Thus, epigenome editing has become of primary interest to alter gene expression without disrupting genomic sequences. Different tools have been developed to address this challenge, starting with modular Zinc Finger Proteins (ZFPs) and Transcription Activator Like Effectors (TALEs). However, the discovery of CRISPR/Cas9 system and the adaptability of technologies based on enzymatically dead Cas9 (dCas9) have paved the way towards a reliable and adaptable epigenome editing in a great variety of organisms. In this review, we will focus on the application of targeted epigenome editing technologies in plants, summarizing the most updated advances in this field. The promising results obtained by altering the expression state of targets involved in flowering time and abiotic stress resistance are crucial not only for elucidating the molecular interactions that underly chromatin dynamics, but also for future applications in breeding programs as an alternative route to genetic manipulation towards the achievement of higher quality crops particularly in terms of nutritional properties, yield and tolerance.
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Affiliation(s)
| | | | - Giorgio Perrella
- Department of Biosciences, Università degli Studi di Milano, Milan, Italy
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2
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Ahmadikhah A, Zarabizadeh H, Nayeri S, Abbasi MS. Advancements in genome editing tools for genetic studies and crop improvement. FRONTIERS IN PLANT SCIENCE 2025; 15:1370675. [PMID: 39963359 PMCID: PMC11830681 DOI: 10.3389/fpls.2024.1370675] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Accepted: 12/31/2024] [Indexed: 02/20/2025]
Abstract
The rapid increase in global population poses a significant challenge to food security, compounded by the adverse effects of climate change, which limit crop productivity through both biotic and abiotic stressors. Despite decades of progress in plant breeding and genetic engineering, the development of new crop varieties with desirable agronomic traits remains a time-consuming process. Traditional breeding methods often fall short of addressing the urgent need for improved crop varieties. Genome editing technologies, which enable precise modifications at specific genomic loci, have emerged as powerful tools for enhancing crop traits. These technologies, including RNA interference, Meganucleases, ZFNs, TALENs, and CRISPR/Cas systems, allow for the targeted insertion, deletion, or alteration of DNA fragments, facilitating improvements in traits such as herbicide and insect resistance, nutritional quality, and stress tolerance. Among these, CRISPR/Cas9 stands out for its simplicity, efficiency, and ability to reduce off-target effects, making it a valuable tool in both agricultural biotechnology and plant functional genomics. This review examines the functional mechanisms and applications of various genome editing technologies for crop improvement, highlighting their advantages and limitations. It also explores the ethical considerations associated with genome editing in agriculture and discusses the potential of these technologies to contribute to sustainable food production in the face of growing global challenges.
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Affiliation(s)
- Asadollah Ahmadikhah
- Department of Cellular and Molecular Biology, Faculty of Life Sciences and Biotechnology, Shahid Beheshti University, Tehran, Iran
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3
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Katayama S, Watanabe M, Kato Y, Nomura W, Yamamoto T. Engineering of Zinc Finger Nucleases Through Structural Modeling Improves Genome Editing Efficiency in Cells. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2024; 11:e2310255. [PMID: 38600709 PMCID: PMC11187957 DOI: 10.1002/advs.202310255] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/27/2023] [Revised: 02/28/2024] [Indexed: 04/12/2024]
Abstract
Genome Editing is widely used in biomedical research and medicine. Zinc finger nucleases (ZFNs) are smaller in size than transcription activator-like effector (TALE) nucleases (TALENs) and CRISPR-Cas9. Therefore, ZFN-encoding DNAs can be easily packaged into a viral vector with limited cargo space, such as adeno-associated virus (AAV) vectors, for in vivo and clinical applications. ZFNs have great potential for translational research and clinical use. However, constructing functional ZFNs and improving their genome editing efficiency is extremely difficult. Here, the efficient construction of functional ZFNs and the improvement of their genome editing efficiency using AlphaFold, Coot, and Rosetta are described. Plasmids encoding ZFNs consisting of six fingers using publicly available zinc-finger resources are assembled. Two functional ZFNs from the ten ZFNs tested are successfully obtained. Furthermore, the engineering of ZFNs using AlphaFold, Coot, or Rosetta increases the efficiency of genome editing by 5%, demonstrating the effectiveness of engineering ZFNs based on structural modeling.
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Affiliation(s)
- Shota Katayama
- Genome Editing Innovation CenterHiroshima UniversityHigashi‐Hiroshima739‐0046Japan
| | - Masahiro Watanabe
- Research Institute for Sustainable ChemistryNational Institute of Advanced Industrial Science and Technology (AIST)Higashi‐Hiroshima739‐0046Japan
| | - Yoshio Kato
- Biomedical Research InstituteNational Institute of Advanced Industrial Science and Technology (AIST)Ibaraki305‐8566Japan
| | - Wataru Nomura
- Graduate School of Biomedical and Health SciencesHiroshima UniversityHiroshima734‐8553Japan
| | - Takashi Yamamoto
- Genome Editing Innovation CenterHiroshima UniversityHigashi‐Hiroshima739‐0046Japan
- Division of Integrated Sciences for LifeGraduate School of Integrated Sciences for LifeHiroshima UniversityHigashi‐Hiroshima739‐8526Japan
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4
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Bhuyan SJ, Kumar M, Ramrao Devde P, Rai AC, Mishra AK, Singh PK, Siddique KHM. Progress in gene editing tools, implications and success in plants: a review. Front Genome Ed 2023; 5:1272678. [PMID: 38144710 PMCID: PMC10744593 DOI: 10.3389/fgeed.2023.1272678] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2023] [Accepted: 11/13/2023] [Indexed: 12/26/2023] Open
Abstract
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.
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Affiliation(s)
- Suman Jyoti Bhuyan
- Department of Biotechnology, Mizoram University (A Central University), Pachhunga University College Campus, Aizawl, Mizoram, India
| | - Manoj Kumar
- Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel
| | - Pandurang Ramrao Devde
- Department of Biotechnology, Mizoram University (A Central University), Pachhunga University College Campus, Aizawl, Mizoram, India
| | - Avinash Chandra Rai
- Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel
| | | | - Prashant Kumar Singh
- Department of Biotechnology, Mizoram University (A Central University), Pachhunga University College Campus, Aizawl, Mizoram, India
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Liu W, Zuo S, Shao Y, Bi K, Zhao J, Huang L, Xu Z, Lian J. Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli. Nucleic Acids Res 2023; 51:8293-8307. [PMID: 37471041 PMCID: PMC10450171 DOI: 10.1093/nar/gkad607] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2023] [Revised: 07/02/2023] [Accepted: 07/07/2023] [Indexed: 07/21/2023] Open
Abstract
While there are several genome editing techniques available, few are suitable for dynamic and simultaneous mutagenesis of arbitrary targeted sequences in prokaryotes. Here, to address these limitations, we present a versatile and multiplex retron-mediated genome editing system (REGES). First, through systematic optimization of REGES, we achieve efficiency of ∼100%, 85 ± 3%, 69 ± 14% and 25 ± 14% for single-, double-, triple- and quadruple-locus genome editing, respectively. In addition, we employ REGES to generate pooled and barcoded variant libraries with degenerate RBS sequences to fine-tune the expression level of endogenous and exogenous genes, such as transcriptional factors to improve ethanol tolerance and biotin biosynthesis. Finally, we demonstrate REGES-mediated continuous in vivo protein evolution, by combining retron, polymerase-mediated base editing and error-prone transcription. By these case studies, we demonstrate REGES as a powerful multiplex genome editing and continuous evolution tool with broad applications in synthetic biology and metabolic engineering.
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Affiliation(s)
- Wenqian Liu
- Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
- Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
| | - Siqi Zuo
- Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
- Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
| | - Youran Shao
- Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
- Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
| | - Ke Bi
- Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
- Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
| | - Jiarun Zhao
- Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
- Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
| | - Lei Huang
- Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
- Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
| | - Zhinan Xu
- Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
- Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
| | - Jiazhang Lian
- Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
- Institute of Bioengineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China
- ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang University, Hangzhou 311215, China
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Basu U, Riaz Ahmed S, Bhat BA, Anwar Z, Ali A, Ijaz A, Gulzar A, Bibi A, Tyagi A, Nebapure SM, Goud CA, Ahanger SA, Ali S, Mushtaq M. A CRISPR way for accelerating cereal crop improvement: Progress and challenges. Front Genet 2023; 13:866976. [PMID: 36685816 PMCID: PMC9852743 DOI: 10.3389/fgene.2022.866976] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2022] [Accepted: 11/21/2022] [Indexed: 01/09/2023] Open
Abstract
Humans rely heavily on cereal grains as a key source of nutrients, hence regular improvement of cereal crops is essential for ensuring food security. The current food crisis at the global level is due to the rising population and harsh climatic conditions which prompts scientists to develop smart resilient cereal crops to attain food security. Cereal crop improvement in the past generally depended on imprecise methods like random mutagenesis and conventional genetic recombination which results in high off targeting risks. In this context, we have witnessed the application of targeted mutagenesis using versatile CRISPR-Cas systems for cereal crop improvement in sustainable agriculture. Accelerated crop improvement using molecular breeding methods based on CRISPR-Cas genome editing (GE) is an unprecedented tool for plant biotechnology and agriculture. The last decade has shown the fidelity, accuracy, low levels of off-target effects, and the high efficacy of CRISPR technology to induce targeted mutagenesis for the improvement of cereal crops such as wheat, rice, maize, barley, and millets. Since the genomic databases of these cereal crops are available, several modifications using GE technologies have been performed to attain desirable results. This review provides a brief overview of GE technologies and includes an elaborate account of the mechanisms and applications of CRISPR-Cas editing systems to induce targeted mutagenesis in cereal crops for improving the desired traits. Further, we describe recent developments in CRISPR-Cas-based targeted mutagenesis through base editing and prime editing to develop resilient cereal crop plants, possibly providing new dimensions in the field of cereal crop genome editing.
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Affiliation(s)
- Umer Basu
- Division of Entomology, ICAR-Indian Agricultural Research Institute, New Delhi, India
| | - Syed Riaz Ahmed
- Nuclear Institute for Agriculture and Biology College, Pakistan Institute of Engineering and Applied Sciences (NIAB-C, PIEAS), Faisalabad, Pakistan
| | | | - Zunaira Anwar
- Nuclear Institute for Agriculture and Biology College, Pakistan Institute of Engineering and Applied Sciences (NIAB-C, PIEAS), Faisalabad, Pakistan
| | - Ahmad Ali
- National Key Laboratory of Crop Genetic Improvement, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, China
| | - Aqsa Ijaz
- Nuclear Institute for Agriculture and Biology College, Pakistan Institute of Engineering and Applied Sciences (NIAB-C, PIEAS), Faisalabad, Pakistan
| | - Addafar Gulzar
- Division of Plant Pathology, Faculty of Agriculture, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Wadura Sopore, India
| | - Amir Bibi
- Department of Plant Breeding and Genetics, Faculty of Agriculture Sciences, University of Agriculture Faisalabad, Faisalabad, Pakistan
| | - Anshika Tyagi
- Department of Biotechnology, Yeungnam University, Gyeongsan, South Korea
| | - Suresh M. Nebapure
- Division of Entomology, ICAR-Indian Agricultural Research Institute, New Delhi, India
| | - Chengeshpur Anjali Goud
- Institute of Biotechnology, Professor Jayashanker Telangana State Agriculture University, Hyderabad, India
| | - Shafat Ahmad Ahanger
- Division of Plant Pathology, Faculty of Agriculture, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Wadura Sopore, India,*Correspondence: Shafat Ahmad Ahanger, ; Sajad Ali, ; Muntazir Mushtaq,
| | - Sajad Ali
- Department of Biotechnology, Yeungnam University, Gyeongsan, South Korea,*Correspondence: Shafat Ahmad Ahanger, ; Sajad Ali, ; Muntazir Mushtaq,
| | - Muntazir Mushtaq
- ICAR-National Bureau of Plant Genetic Resources, Division of Germplasm Evaluation, Pusa Campus, New Delhi, India,*Correspondence: Shafat Ahmad Ahanger, ; Sajad Ali, ; Muntazir Mushtaq,
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7
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Nguyen Tran MT, Kc R, Hewitt AW. A Taxonomic and Phylogenetic Classification of Diverse Base Editors. CRISPR J 2022; 5:311-328. [PMID: 35244489 DOI: 10.1089/crispr.2021.0095] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Base editors mediate the targeted conversion of single nucleobases in a therapeutically relevant manner. Herein, we present a hypothetical taxonomic and phylogenetic framework for the classification of more than 200 different DNA base editors, and we categorize them based on their described properties. Following evaluation of their in situ activity windows, which were derived by cataloguing their activity in published literature, organization is done hierarchically, with specific base editor signatures being subcategorized according to their on-target activity or nonspecific, genome- or transcriptome-wide activity. Based on this categorization, we curate a phylogenetic framework, based on protein homology alignment, and describe a taxonomic structure that clusters base editor variants on their target chemistry, endonuclease component, identity of their deaminase component, and their described properties into discrete taxa. Thus, we establish a hypothetical taxonomic structure that can describe and organize current and potentially future base editing variants into clearly defined groups that are defined by their characteristics. Finally, we summarize our findings into a navigable database (ShinyApp in R) that allows users to select through our repository to nominate ideal base editor candidates as a starting point for further testing in their specific application.
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Affiliation(s)
- Minh Thuan Nguyen Tran
- Menzies Institute for Medical Research, School of Medicine, University of Tasmania, Tasmania, Australia; and The University of Melbourne, Melbourne, Australia
| | - Rajendra Kc
- Menzies Institute for Medical Research, School of Medicine, University of Tasmania, Tasmania, Australia; and The University of Melbourne, Melbourne, Australia
| | - Alex W Hewitt
- Menzies Institute for Medical Research, School of Medicine, University of Tasmania, Tasmania, Australia; and The University of Melbourne, Melbourne, Australia.,Centre for Eye Research Australia, The University of Melbourne, Melbourne, Australia
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8
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Zinc-Finger-Protein-Based Microfluidic Electrophoretic Mobility Reversal Assay for Quantitative Double-Stranded DNA Analysis. BIOCHIP JOURNAL 2021. [DOI: 10.1007/s13206-021-00038-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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9
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Pacheco MB, Camilo V, Henrique R, Jerónimo C. Epigenetic Editing in Prostate Cancer: Challenges and Opportunities. Epigenetics 2021; 17:564-588. [PMID: 34130596 DOI: 10.1080/15592294.2021.1939477] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022] Open
Abstract
Epigenome editing consists of fusing a predesigned DNA recognition unit to the catalytic domain of a chromatin modifying enzyme leading to the introduction or removal of an epigenetic mark at a specific locus. These platforms enabled the study of the mechanisms and roles of epigenetic changes in several research domains such as those addressing pathogenesis and progression of cancer. Despite the continued efforts required to overcome some limitations, which include specificity, off-target effects, efficacy, and longevity, these tools have been rapidly progressing and improving.Since prostate cancer is characterized by multiple genetic and epigenetic alterations that affect different signalling pathways, epigenetic editing constitutes a promising strategy to hamper cancer progression. Therefore, by modulating chromatin structure through epigenome editing, its conformation might be better understood and events that drive prostate carcinogenesis might be further unveiled.This review describes the different epigenome engineering tools, their mechanisms concerning gene's expression and regulation, highlighting the challenges and opportunities concerning prostate cancer research.
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Affiliation(s)
- Mariana Brütt Pacheco
- Cancer Biology and Epigenetics Group, Research Center (GEBC CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto) & Porto Comprehensive Cancer Center (P.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal
| | - Vânia Camilo
- Cancer Biology and Epigenetics Group, Research Center (GEBC CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto) & Porto Comprehensive Cancer Center (P.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal
| | - Rui Henrique
- Cancer Biology and Epigenetics Group, Research Center (GEBC CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto) & Porto Comprehensive Cancer Center (P.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal.,Department of Pathology, Portuguese Oncology Institute of Porto (IPOP), R. DR. António Bernardino De Almeida, Porto, Portugal.,Department of Pathology and Molecular Immunology, School of Medicine & Biomedical Sciences, University of Porto (ICBAS-UP), Rua Jorge Viterbo Ferreira 228, Porto, Portugal
| | - Carmen Jerónimo
- Cancer Biology and Epigenetics Group, Research Center (GEBC CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto) & Porto Comprehensive Cancer Center (P.CCC), R. Dr. António Bernardino de Almeida, Porto, Portugal.,Department of Pathology and Molecular Immunology, School of Medicine & Biomedical Sciences, University of Porto (ICBAS-UP), Rua Jorge Viterbo Ferreira 228, Porto, Portugal
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10
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Ha DT, Nguyen VT, Kim MS. Graphene Oxide-Based Simple and Rapid Detection of Antibiotic Resistance Gene via Quantum Dot-Labeled Zinc Finger Proteins. Anal Chem 2021; 93:8459-8466. [PMID: 34097379 DOI: 10.1021/acs.analchem.1c00560] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
With the increasing rise of antibiotic-resistant pathogens, a simple and rapid detection of antibiotic resistance gene (ARG) is crucial to mitigate the spreading of antibiotic resistance. DNA-binding zinc finger proteins (ZFPs) can be engineered to recognize specific double-stranded (ds) DNA sequences in ARG. Here, we designed a simple and rapid method to detect ARG in bacteria utilizing engineered ZFPs and 2D nanosheet graphene oxide (GO) as a sensing platform. Our approach relies on the on and off effect of fluorescence signal in the presence and absence of target ARG, respectively. By taking advantage of the unique quenching capability of GO due to its electronic property, quantum dot (QD)-labeled ZFPs are adsorbed onto the GO sheets, and their fluorescence signal is quenched by proximal GO sheets through fluorescence resonance energy transfer (FRET). In the presence of target DNA, ZFP binding to the target DNA induces dissociation from GO, thereby restoring the fluorescence signal. Our system detects target DNA through restoration of QD emission as the restored signal increases directly with target DNA concentrations. Engineered ZFPs were able to detect specific dsDNA of the tetracycline resistance gene tetM with high specificity after only 10 min incubation on our GO-based sensing system. Our sensing system employed one-step FRET-based ZFP and GO combined technology to enable rapid and quantitative detection of ARG, providing a limit of detection as low as 1 nM. This study demonstrated the application of GO in conjunction with engineered DNA-binding domains for the direct detection of dsDNA with great potential as a rapid and reliable screening and detecton method against the growing threat of antibiotic resistant bacteria.
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Affiliation(s)
- Dat Thinh Ha
- Department of Chemistry, Western Kentucky University, Bowling Green, Kentucky 42101, United States
| | - Van-Thuan Nguyen
- Department of Chemistry, Western Kentucky University, Bowling Green, Kentucky 42101, United States
| | - Moon-Soo Kim
- Department of Chemistry, Western Kentucky University, Bowling Green, Kentucky 42101, United States
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11
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Abstract
Conventional methods of DNA sequence insertion into plants, using Agrobacterium-mediated transformation or microprojectile bombardment, result in the integration of the DNA at random sites in the genome. These plants may exhibit altered agronomic traits as a consequence of disruption or silencing of genes that serve a critical function. Also, genes of interest inserted at random sites are often not expressed at the desired level. For these reasons, targeted DNA insertion at suitable genomic sites in plants is a desirable alternative. In this paper we review approaches of targeted DNA insertion in plant genomes, discuss current technical challenges, and describe promising applications of targeted DNA insertion for crop genetic improvement.
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12
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Current Status of Gene Therapy Research in Polyglutamine Spinocerebellar Ataxias. Int J Mol Sci 2021; 22:ijms22084249. [PMID: 33921915 PMCID: PMC8074016 DOI: 10.3390/ijms22084249] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2021] [Revised: 04/14/2021] [Accepted: 04/14/2021] [Indexed: 12/26/2022] Open
Abstract
Polyglutamine spinocerebellar ataxias (PolyQ SCAs) are a group of 6 rare autosomal dominant diseases, which arise from an abnormal CAG repeat expansion in the coding region of their causative gene. These neurodegenerative ataxic disorders are characterized by progressive cerebellar degeneration, which translates into progressive ataxia, the main clinical feature, often accompanied by oculomotor deficits and dysarthria. Currently, PolyQ SCAs treatment is limited only to symptomatic mitigation, and no therapy is available to stop or delay the disease progression, which culminates with death. Over the last years, many promising gene therapy approaches were investigated in preclinical studies and could lead to a future treatment to stop or delay the disease development. Here, we summed up the most promising of these therapies, categorizing them in gene augmentation therapy, gene silencing strategies, and gene edition approaches. While several of the reviewed strategies are promising, there is still a gap from the preclinical results obtained and their translation to clinical studies. However, there is an increase in the number of approved gene therapies, as well as a constant development in their safety and efficacy profiles. Thus, it is expected that in a near future some of the promising strategies reviewed here could be tested in a clinical setting and if successful provide hope for SCAs patients.
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13
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Bao XR, Pan Y, Lee CM, Davis TH, Bao G. Tools for experimental and computational analyses of off-target editing by programmable nucleases. Nat Protoc 2021; 16:10-26. [PMID: 33288953 PMCID: PMC8049448 DOI: 10.1038/s41596-020-00431-y] [Citation(s) in RCA: 56] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2020] [Accepted: 09/30/2020] [Indexed: 12/14/2022]
Abstract
Genome editing using programmable nucleases is revolutionizing life science and medicine. Off-target editing by these nucleases remains a considerable concern, especially in therapeutic applications. Here we review tools developed for identifying potential off-target editing sites and compare the ability of these tools to properly analyze off-target effects. Recent advances in both in silico and experimental tools for off-target analysis have generated remarkably concordant results for sites with high off-target editing activity. However, no single tool is able to accurately predict low-frequency off-target editing, presenting a bottleneck in therapeutic genome editing, because even a small number of cells with off-target editing can be detrimental. Therefore, we recommend that at least one in silico tool and one experimental tool should be used together to identify potential off-target sites, and amplicon-based next-generation sequencing (NGS) should be used as the gold standard assay for assessing the true off-target effects at these candidate sites. Future work to improve off-target analysis includes expanding the true off-target editing dataset to evaluate new experimental techniques and to train machine learning algorithms; performing analysis using the particular genome of the cells in question rather than the reference genome; and applying novel NGS techniques to improve the sensitivity of amplicon-based off-target editing quantification.
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Affiliation(s)
- X Robert Bao
- ILISATech, Houston, TX, USA
- Arsenal Biosciences, South San Francisco, CA, USA
| | - Yidan Pan
- Department of Bioengineering, Rice University, Houston, TX, USA
| | - Ciaran M Lee
- APC Microbiome Ireland, University College Cork, Cork, Ireland
| | - Timothy H Davis
- Department of Bioengineering, Rice University, Houston, TX, USA
| | - Gang Bao
- Department of Bioengineering, Rice University, Houston, TX, USA.
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14
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Ha DT, Ghosh S, Ahn CH, Segal DJ, Kim MS. Pathogen-specific DNA sensing with engineered zinc finger proteins immobilized on a polymer chip. Analyst 2019; 143:4009-4016. [PMID: 30043772 DOI: 10.1039/c8an00395e] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Abstract
A specific double-stranded DNA sensing system is of great interest for diagnostic and other biomedical applications. Zinc finger domains, which recognize double-stranded DNA, can be engineered to form custom DNA-binding proteins for the recognition of specific DNA sequences. As a proof of concept, a sequence-enabled reassembly of a TEM-1 β-lactamase system (SEER-LAC) was previously demonstrated to develop zinc finger protein (ZFP) arrays for the detection of a double-stranded bacterial DNA sequence. Here, we implemented the SEER-LAC system to demonstrate the direct detection of pathogen-specific DNA sequences present in E. coli O157:H7 on a lab-on-a-chip. ZFPs custom-designed to detect Shiga toxin in E. coli O157:H7 were immobilized on a cyclic olefin copolymer (COC) chip, which can function as a non-PCR based molecular diagnostic device. Pathogen-specific double-stranded DNA was directly detected by using engineered ZFPs immobilized on the COC chip with high specificity, providing a detection limit of 10 fmol of target DNA in a colorimetric assay. Therefore, in this study, we demonstrated the great potential of ZFP arrays on the COC chip for further development of a simple and novel lab-on-a-chip technology for the detection of pathogens.
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Affiliation(s)
- Dat Thinh Ha
- Department of Chemistry, Western Kentucky University, Bowling Green, Kentucky 42101, USA.
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15
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Porter SN, Levine RM, Pruett-Miller SM. A Practical Guide to Genome Editing Using Targeted Nuclease Technologies. Compr Physiol 2019; 9:665-714. [PMID: 30873595 DOI: 10.1002/cphy.c180022] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
Genome engineering using programmable nucleases is a rapidly evolving technique that enables precise genetic manipulations within complex genomes. Although this technology first surfaced with the creation of meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases, CRISPR-Cas9 has been the most widely adopted platform because of its ease of use. This comprehensive review presents a basic overview of genome engineering and discusses the major technological advances in the field. In addition to nucleases, we discuss CRISPR-derived base editors and epigenetic modifiers. We also delve into practical applications of these tools, including creating custom-edited cell and animal models as well as performing genetic screens. Finally, we discuss the potential for therapeutic applications and ethical considerations related to employing this technology in humans. © 2019 American Physiological Society. Compr Physiol 9:665-714, 2019.
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Affiliation(s)
- Shaina N Porter
- Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Rachel M Levine
- Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
| | - Shondra M Pruett-Miller
- Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA
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16
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Paschon DE, Lussier S, Wangzor T, Xia DF, Li PW, Hinkley SJ, Scarlott NA, Lam SC, Waite AJ, Truong LN, Gandhi N, Kadam BN, Patil DP, Shivak DA, Lee GK, Holmes MC, Zhang L, Miller JC, Rebar EJ. Diversifying the structure of zinc finger nucleases for high-precision genome editing. Nat Commun 2019; 10:1133. [PMID: 30850604 PMCID: PMC6408524 DOI: 10.1038/s41467-019-08867-x] [Citation(s) in RCA: 75] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2018] [Accepted: 01/28/2019] [Indexed: 12/27/2022] Open
Abstract
Genome editing for therapeutic applications often requires cleavage within a narrow sequence window. Here, to enable such high-precision targeting with zinc-finger nucleases (ZFNs), we have developed an expanded set of architectures that collectively increase the configurational options available for design by a factor of 64. These new architectures feature the functional attachment of the FokI cleavage domain to the amino terminus of one or both zinc-finger proteins (ZFPs) in the ZFN dimer, as well as the option to skip bases between the target triplets of otherwise adjacent fingers in each zinc-finger array. Using our new architectures, we demonstrate targeting of an arbitrarily chosen 28 bp genomic locus at a density that approaches 1.0 (i.e., efficient ZFNs available for targeting almost every base step). We show that these new architectures may be used for targeting three loci of therapeutic significance with a high degree of precision, efficiency, and specificity.
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Affiliation(s)
- David E Paschon
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Stephanie Lussier
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Tenzin Wangzor
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Danny F Xia
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Patrick W Li
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Sarah J Hinkley
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Nicholas A Scarlott
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Stephen C Lam
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Adam J Waite
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Lynn N Truong
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Nimisha Gandhi
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Bhakti N Kadam
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Deepak P Patil
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - David A Shivak
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Gary K Lee
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Michael C Holmes
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Lei Zhang
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Jeffrey C Miller
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA
| | - Edward J Rebar
- Sangamo Therapeutics, Inc., 501 Canal Boulevard, Suite A100, Richmond, California, 94804, USA.
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Nomura W. Development of Toolboxes for Precision Genome/Epigenome Editing and Imaging of Epigenetics. CHEM REC 2018; 18:1717-1726. [PMID: 30066981 DOI: 10.1002/tcr.201800036] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2018] [Accepted: 07/17/2018] [Indexed: 12/17/2022]
Abstract
Zinc finger (ZF) proteins are composed of repeated ββα modules and coordinate a zinc ion. ZF domains recognizing specific DNA target sequences can be substituted for the binding domains of various DNA-modifying enzymes to create designer nucleases, recombinases, and methyltransferases 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 CRISPR-Cas9 system has expanded the range of possibilities for genome-editing technologies. In addition, these DNA binding domains can also be utilized to build a toolbox for epigenetic controls by fusing them with protein- or DNA-modifying enzymes. Here, our research on epigenome editing including the development of artificial zinc finger recombinase (ZFR), split DNA methyltransferase, and fluorescence imaging of histone proteins by ZIP tag-probe system is introduced. Advances in the ZF, TALE, and CRISPR-Cas9 platforms have paved the way for the next generation of genome/epigenome engineering approaches.
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Affiliation(s)
- Wataru Nomura
- Institute of Biomaterials and Bioenginerring, Tokyo Medical and Dental University, 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo, 101-0062, Japan
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18
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Huang N, Huang Z, Gao M, Luo Z, Zhou F, Liu L, Xiao Q, Wang X, Feng W. Induction of apoptosis in imatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption of bcr-abl oncogene with zinc finger nucleases. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2018; 37:62. [PMID: 29554925 PMCID: PMC5859405 DOI: 10.1186/s13046-018-0732-4] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/28/2018] [Accepted: 03/12/2018] [Indexed: 12/26/2022]
Abstract
Background The bcr-abl fusion gene is the pathological origin of chronic myeloid leukemia (CML) and plays a critical role in the resistance of imatinib. Thus, bcr-abl disruption-based novel therapeutic strategy may warrant exploration. In our study, we were surprised to find that the characteristics of bcr-abl sequences met the design requirements of zinc finger nucleases (ZFNs). Methods We constructed the ZFNs targeting bcr-abl with high specificity through simple modular assembly approach. Western blotting was conducted to detect the expression of BCR-ABL and phosphorylation of its downstream STAT5, ERK and CRKL in CML cells. CCK8 assay, colony-forming assay and flow cytometry (FCM) were used to evaluate the effect of the ZFNs on the viablity and apoptosis of CML cells and CML CD34+ cells. Moreover, mice model was used to determine the ability of ZFNs in disrupting the leukemogenesis of bcr-abl in vivo. Results The ZFNs skillfully mediated 8-base NotI enzyme cutting site addition in bcr-abl gene of imatinib sensitive and resistant CML cells by homology-directed repair (HDR), which led to a stop codon and terminated the translation of BCR-ABL protein. As expected, the disruption of bcr-abl gene induced cell apoptosis and inhibited cell proliferation. Notably, we obtained similar result in CD34+ cells from CML patients. Moreover, the ZFNs significantly reduced the oncogenicity of CML cells in mice. Conclusion These results reveal that the bcr-abl gene disruption based on ZFNs may provide a treatment choice for imatinib resistant or intolerant CML patients. Electronic supplementary material The online version of this article (10.1186/s13046-018-0732-4) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Ningshu Huang
- Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China
| | - Zhenglan Huang
- Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China
| | - Miao Gao
- Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People's Republic of China
| | - Zhenhong Luo
- Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China
| | - Fangzhu Zhou
- Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China
| | - Lin Liu
- Department of Hematology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, People's Republic of China
| | - Qing Xiao
- Department of Hematology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, People's Republic of China
| | - Xin Wang
- Department of Hematology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, People's Republic of China
| | - Wenli Feng
- Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China.
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19
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Huang N, Huang Z, Gao M, Luo Z, Zhou F, Liu L, Xiao Q, Wang X, Feng W. Induction of apoptosis in imatinib sensitive and resistant chronic myeloid leukemia cells by efficient disruption of bcr-abl oncogene with zinc finger nucleases. JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH : CR 2018. [PMID: 29554925 DOI: 10.1186/s13046-018-0732-4.] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
Abstract
BACKGROUND The bcr-abl fusion gene is the pathological origin of chronic myeloid leukemia (CML) and plays a critical role in the resistance of imatinib. Thus, bcr-abl disruption-based novel therapeutic strategy may warrant exploration. In our study, we were surprised to find that the characteristics of bcr-abl sequences met the design requirements of zinc finger nucleases (ZFNs). METHODS We constructed the ZFNs targeting bcr-abl with high specificity through simple modular assembly approach. Western blotting was conducted to detect the expression of BCR-ABL and phosphorylation of its downstream STAT5, ERK and CRKL in CML cells. CCK8 assay, colony-forming assay and flow cytometry (FCM) were used to evaluate the effect of the ZFNs on the viablity and apoptosis of CML cells and CML CD34+ cells. Moreover, mice model was used to determine the ability of ZFNs in disrupting the leukemogenesis of bcr-abl in vivo. RESULTS The ZFNs skillfully mediated 8-base NotI enzyme cutting site addition in bcr-abl gene of imatinib sensitive and resistant CML cells by homology-directed repair (HDR), which led to a stop codon and terminated the translation of BCR-ABL protein. As expected, the disruption of bcr-abl gene induced cell apoptosis and inhibited cell proliferation. Notably, we obtained similar result in CD34+ cells from CML patients. Moreover, the ZFNs significantly reduced the oncogenicity of CML cells in mice. CONCLUSION These results reveal that the bcr-abl gene disruption based on ZFNs may provide a treatment choice for imatinib resistant or intolerant CML patients.
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Affiliation(s)
- Ningshu Huang
- Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China
| | - Zhenglan Huang
- Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China
| | - Miao Gao
- Department of Laboratory Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing, 400016, People's Republic of China
| | - Zhenhong Luo
- Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China
| | - Fangzhu Zhou
- Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China
| | - Lin Liu
- Department of Hematology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, People's Republic of China
| | - Qing Xiao
- Department of Hematology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, People's Republic of China
| | - Xin Wang
- Department of Hematology, The First Affiliated Hospital, Chongqing Medical University, Chongqing, 400016, People's Republic of China
| | - Wenli Feng
- Department of Clinical Hematology, Key Laboratory of Laboratory Medical Diagnostics Designated by the Ministry of Education, Chongqing Medical University, No.1, Yixueyuan Road, Chongqing, 400016, People's Republic of China.
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20
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Waryah CB, Moses C, Arooj M, Blancafort P. Zinc Fingers, TALEs, and CRISPR Systems: A Comparison of Tools for Epigenome Editing. Methods Mol Biol 2018. [PMID: 29524128 DOI: 10.1007/978-1-4939-7774-1_2] [Citation(s) in RCA: 52] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The completion of genome, epigenome, and transcriptome mapping in multiple cell types has created a demand for precision biomolecular tools that allow researchers to functionally manipulate DNA, reconfigure chromatin structure, and ultimately reshape gene expression patterns. Epigenetic editing tools provide the ability to interrogate the relationship between epigenetic modifications and gene expression. Importantly, this information can be exploited to reprogram cell fate for both basic research and therapeutic applications. Three different molecular platforms for epigenetic editing have been developed: zinc finger proteins (ZFs), transcription activator-like effectors (TALEs), and the system of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins. These platforms serve as custom DNA-binding domains (DBDs), which are fused to epigenetic modifying domains to manipulate epigenetic marks at specific sites in the genome. The addition and/or removal of epigenetic modifications reconfigures local chromatin structure, with the potential to provoke long-lasting changes in gene transcription. Here we summarize the molecular structure and mechanism of action of ZF, TALE, and CRISPR platforms and describe their applications for the locus-specific manipulation of the epigenome. The advantages and disadvantages of each platform will be discussed with regard to genomic specificity, potency in regulating gene expression, and reprogramming cell phenotypes, as well as ease of design, construction, and delivery. Finally, we outline potential applications for these tools in molecular biology and biomedicine and identify possible barriers to their future clinical implementation.
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Affiliation(s)
- Charlene Babra Waryah
- Cancer Epigenetics Group, The Harry Perkins Institute of Medical Research, Nedlands, Perth, WA, Australia
| | - Colette Moses
- Cancer Epigenetics Group, The Harry Perkins Institute of Medical Research, Nedlands, Perth, WA, Australia
- School of Human Sciences, The University of Western Australia, Perth, WA, Australia
| | - Mahira Arooj
- Cancer Epigenetics Group, The Harry Perkins Institute of Medical Research, Nedlands, Perth, WA, Australia
- School of Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth, WA, Australia
| | - Pilar Blancafort
- Cancer Epigenetics Group, The Harry Perkins Institute of Medical Research, Nedlands, Perth, WA, Australia.
- School of Human Sciences, The University of Western Australia, Perth, WA, Australia.
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21
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Sanagala R, Moola AK, Bollipo Diana RK. A review on advanced methods in plant gene targeting. J Genet Eng Biotechnol 2017; 15:317-321. [PMID: 30647669 PMCID: PMC6296621 DOI: 10.1016/j.jgeb.2017.07.004] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2016] [Revised: 06/29/2017] [Accepted: 07/03/2017] [Indexed: 12/26/2022]
Abstract
Plant genetic engineering is one of the most significant tools implemented in the modern molecular crop breeding techniques. The conventional approaches of plant genetic transformation include Agrobacterium tumefaciens, particle bombardment, DNA uptake into protoplast. The transgenic events derived by these methods carry the transgenes that are integrated at random sites in the plant genome. Novel techniques that mediate integration of foreign genes at specific pre-determined locations circumvent many problems associated with the existing methods of gene transfer. The recent years have witnessed the emergence of gene targeting techniques by employing zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindrome repeats (CRISPR). The present review focuses on the various approaches and their performance of plant gene targeting and suggests future directions in the important areas of plant molecular biology.
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Affiliation(s)
- Raghavendrarao Sanagala
- National Research Centre on Plant Biotechnology, Lal Bahadur Shastri Building, Pusa Campus, New Delhi 110012, India
- Department of Botany, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620024, India
| | - Anil Kumar Moola
- Department of Botany, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620024, India
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22
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Boyle M, Mann J. WITHDRAWN: Epigenetics in Chronic Liver Disease. J Hepatol 2017:S0168-8278(17)32255-9. [PMID: 28855099 DOI: 10.1016/j.jhep.2017.08.016] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/01/2017] [Revised: 08/17/2017] [Accepted: 08/18/2017] [Indexed: 12/04/2022]
Abstract
This article has been withdrawn at the request of the editors. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.
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Affiliation(s)
- Marie Boyle
- Institute of Cellular Medicine, Faculty of Medical Sciences, 4(th) Floor, William Leech Building, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK
| | - Jelena Mann
- Institute of Cellular Medicine, Faculty of Medical Sciences, 4(th) Floor, William Leech Building, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
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23
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Kim MS, Kini AG. Engineering and Application of Zinc Finger Proteins and TALEs for Biomedical Research. Mol Cells 2017; 40:533-541. [PMID: 28835021 PMCID: PMC5582299 DOI: 10.14348/molcells.2017.0139] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2017] [Revised: 08/10/2017] [Accepted: 08/11/2017] [Indexed: 12/13/2022] Open
Abstract
Engineered DNA-binding domains provide a powerful technology for numerous biomedical studies due to their ability to recognize specific DNA sequences. Zinc fingers (ZF) are one of the most common DNA-binding domains and have been extensively studied for a variety of applications, such as gene regulation, genome engineering and diagnostics. Another novel DNA-binding domain known as a transcriptional activator-like effector (TALE) has been more recently discovered, which has a previously undescribed DNA-binding mode. Due to their modular architecture and flexibility, TALEs have been rapidly developed into artificial gene targeting reagents. Here, we describe the methods used to design these DNA-binding proteins and their key applications in biomedical research.
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Affiliation(s)
- Moon-Soo Kim
- Department of Chemistry, Western Kentucky University, 1906 College Heights Blvd., Bowling Green, KY 42101,
USA
| | - Anu Ganesh Kini
- Department of Chemistry, Western Kentucky University, 1906 College Heights Blvd., Bowling Green, KY 42101,
USA
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24
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Malzahn A, Lowder L, Qi Y. Plant genome editing with TALEN and CRISPR. Cell Biosci 2017; 7:21. [PMID: 28451378 PMCID: PMC5404292 DOI: 10.1186/s13578-017-0148-4] [Citation(s) in RCA: 116] [Impact Index Per Article: 14.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2017] [Accepted: 04/19/2017] [Indexed: 11/10/2022] Open
Abstract
Genome editing promises giant leaps forward in advancing biotechnology, agriculture, and basic research. The process relies on the use of sequence specific nucleases (SSNs) to make DNA double stranded breaks at user defined genomic loci, which are subsequently repaired by two main DNA repair pathways: non-homologous end joining (NHEJ) and homology directed repair (HDR). NHEJ can result in frameshift mutations that often create genetic knockouts. These knockout lines are useful for functional and reverse genetic studies but also have applications in agriculture. HDR has a variety of applications as it can be used for gene replacement, gene stacking, and for creating various fusion proteins. In recent years, transcription activator-like effector nucleases and clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR associated protein 9 or CRISPR from Prevotella and Francisella 1 have emerged as the preferred SSNs for research purposes. Here, we review their applications in plant research, discuss current limitations, and predict future research directions in plant genome editing.
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Affiliation(s)
- Aimee Malzahn
- Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742 USA
| | - Levi Lowder
- Department of Biology, East Carolina University, Greenville, NC 27858 USA
| | - Yiping Qi
- Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD 20742 USA
- Institute for Bioscience and Biotechnology Research, University of Maryland, Rockville, MD 20850 USA
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25
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Genome Editing for the β-Hemoglobinopathies. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2017; 1013:203-217. [DOI: 10.1007/978-1-4939-7299-9_8] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/12/2023]
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26
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Leitão AL, Costa MC, Enguita FJ. Applications of genome editing by programmable nucleases to the metabolic engineering of secondary metabolites. J Biotechnol 2016; 241:50-60. [PMID: 27845165 DOI: 10.1016/j.jbiotec.2016.11.009] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2016] [Revised: 11/06/2016] [Accepted: 11/10/2016] [Indexed: 12/17/2022]
Abstract
Genome engineering is a branch of modern biotechnology composed of a cohort of protocols designed to construct and modify a genotype with the main objective of giving rise to a desired phenotype. Conceptually, genome engineering is based on the so called genome editing technologies, a group of genetic techniques that allow either to delete or to insert genetic information in a particular genomic locus. Ten years ago, genome editing tools were limited to virus-driven integration and homologous DNA recombination. However, nowadays the uprising of programmable nucleases is rapidly changing this paradigm. There are two main families of modern tools for genome editing depending on the molecule that controls the specificity of the system and drives the editor machinery to its place of action. Enzymes such as Zn-finger and TALEN nucleases are protein-driven genome editors; while CRISPR system is a nucleic acid-guided editing system. Genome editing techniques are still not widely applied for the design of new compounds with pharmacological activity, but they are starting to be considered as promising tools for rational genome manipulation in biotechnology applications. In this review we will discuss the potential applications of programmable nucleases for the metabolic engineering of secondary metabolites with biological activity.
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Affiliation(s)
- Ana Lúcia Leitão
- Departamento de Ciências e Tecnologia da Biomassa, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, Campus de Caparica, 2829-516 Caparica, Portugal; MEtRICs, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, Campus de Caparica, 2829-516 Caparica, Portugal.
| | - Marina C Costa
- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal
| | - Francisco J Enguita
- Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal.
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27
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Kim MS, Kim J. Multiplexed detection of pathogen-specific DNA using engineered zinc finger proteins without target amplification. ANALYTICAL METHODS : ADVANCING METHODS AND APPLICATIONS 2016; 8:6696-6700. [PMID: 28127406 PMCID: PMC5258119 DOI: 10.1039/c6ay02102f] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/06/2023]
Abstract
Multiplexed detection of pathogen-specific DNA sequences in a simple and reliable way is in great demand for clinical and biomedical applications. However, there is still a lack of available DNA detection methods that are simple and pathogen-selective for point-of-care (POC) testing. Here, we report a novel zinc finger protein (ZFP)-based chemiluminescent method for direct detection of pathogenic double-stranded DNA (dsDNA) in a multiplexed platform. ZFPs are custom-designed to identify unique pathogenic DNA sequences. ZFP-based chemiluminescent detection of dsDNA provides sufficient sensitivity (≤50 fmol) and high specificity without target DNA amplification. Our study addresses the potential of developing a simple and selective pathogen detection method in a multiplexed fashion needed for POC application.
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Affiliation(s)
- Moon-Soo Kim
- Department of Chemistry, Western Kentucky University, Bowling Green, KY 42101 USA
| | - Juhwa Kim
- Department of Chemistry, Western Kentucky University, Bowling Green, KY 42101 USA
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Lee J, Chung JH, Kim HM, Kim DW, Kim H. Designed nucleases for targeted genome editing. PLANT BIOTECHNOLOGY JOURNAL 2016; 14:448-62. [PMID: 26369767 PMCID: PMC11389202 DOI: 10.1111/pbi.12465] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/01/2015] [Revised: 08/07/2015] [Accepted: 08/07/2015] [Indexed: 05/20/2023]
Abstract
Targeted genome-editing technology using designed nucleases has been evolving rapidly, and its applications are widely expanding in research, medicine and biotechnology. Using this genome-modifying technology, researchers can precisely and efficiently insert, remove or change specific sequences in various cultured cells, micro-organisms, animals and plants. This genome editing is based on the generation of double-strand breaks (DSBs), repair of which modifies the genome through nonhomologous end-joining (NHEJ) or homology-directed repair (HDR). In addition, designed nickase-induced generation of single-strand breaks can also lead to precise genome editing through HDR, albeit at relatively lower efficiencies than that induced by nucleases. Three kinds of designed nucleases have been used for targeted DSB formation: zinc-finger nucleases, transcription activator-like effector nucleases, and RNA-guided engineered nucleases derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) system. A growing number of researchers are using genome-editing technologies, which have become more accessible and affordable since the discovery and adaptation of CRISPR-Cas9. Here, the repair mechanism and outcomes of DSBs are reviewed and the three types of designed nucleases are discussed with the hope that such understanding will facilitate applications to genome editing.
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Affiliation(s)
- Junwon Lee
- Department of Physiology and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
| | - Jae-Hee Chung
- Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea
- Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea
| | - Ho Min Kim
- Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Korea
| | - Dong-Wook Kim
- Department of Physiology and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
| | - Hyongbum Kim
- Department of Pharmacology and Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea
- Graduate Program of Nano Science and Technology, Yonsei University, Seoul, Korea
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29
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The Development and Use of Zinc-Finger Nucleases. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2016. [DOI: 10.1007/978-1-4939-3509-3_2] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
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30
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Lu JW, Ho YJ, Yang YJ, Liao HA, Ciou SC, Lin LI, Ou DL. Zebrafish as a disease model for studying human hepatocellular carcinoma. World J Gastroenterol 2015; 21:12042-12058. [PMID: 26576090 PMCID: PMC4641123 DOI: 10.3748/wjg.v21.i42.12042] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/16/2015] [Revised: 05/28/2015] [Accepted: 08/31/2015] [Indexed: 02/07/2023] Open
Abstract
Liver cancer is one of the world’s most common cancers and the second leading cause of cancer deaths. Hepatocellular carcinoma (HCC), a primary hepatic cancer, accounts for 90%-95% of liver cancer cases. The pathogenesis of HCC consists of a stepwise process of liver damage that extends over decades, due to hepatitis, fatty liver, fibrosis, and cirrhosis before developing fully into HCC. Multiple risk factors are highly correlated with HCC, including infection with the hepatitis B or C viruses, alcohol abuse, aflatoxin exposure, and metabolic diseases. Over the last decade, genetic alterations, which include the regulation of multiple oncogenes or tumor suppressor genes and the activation of tumorigenesis-related pathways, have also been identified as important factors in HCC. Recently, zebrafish have become an important living vertebrate model organism, especially for translational medical research. In studies focusing on the biology of cancer, carcinogen induced tumors in zebrafish were found to have many similarities to human tumors. Several zebrafish models have therefore been developed to provide insight into the pathogenesis of liver cancer and the related drug discovery and toxicology, and to enable the evaluation of novel small-molecule inhibitors. This review will focus on illustrative examples involving the application of zebrafish models to the study of human liver disease and HCC, through transgenesis, genome editing technology, xenografts, drug discovery, and drug-induced toxic liver injury.
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Pellagatti A, Dolatshad H, Yip BH, Valletta S, Boultwood J. Application of genome editing technologies to the study and treatment of hematological disease. Adv Biol Regul 2015; 60:122-134. [PMID: 26433620 DOI: 10.1016/j.jbior.2015.09.005] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2015] [Revised: 09/07/2015] [Accepted: 09/09/2015] [Indexed: 11/29/2022]
Abstract
Genome editing technologies have advanced significantly over the past few years, providing a fast and effective tool to precisely manipulate the genome at specific locations. The three commonly used genome editing technologies are Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas9 (CRISPR/Cas9) system. ZFNs and TALENs consist of endonucleases fused to a DNA-binding domain, while the CRISPR/Cas9 system uses guide RNAs to target the bacterial Cas9 endonuclease to the desired genomic location. The double-strand breaks made by these endonucleases are repaired in the cells either by non-homologous end joining, resulting in the introduction of insertions/deletions, or, if a repair template is provided, by homology directed repair. The ZFNs, TALENs and CRISPR/Cas9 systems take advantage of these repair mechanisms for targeted genome modification and have been successfully used to manipulate the genome in human cells. These genome editing tools can be used to investigate gene function, to discover new therapeutic targets, and to develop disease models. Moreover, these genome editing technologies have great potential in gene therapy. Here, we review the latest advances in the application of genome editing technology to the study and treatment of hematological disorders.
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Affiliation(s)
- Andrea Pellagatti
- Bloodwise Molecular Haematology Unit, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, and NIHR Biomedical Research Centre, Oxford, UK.
| | - Hamid Dolatshad
- Bloodwise Molecular Haematology Unit, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, and NIHR Biomedical Research Centre, Oxford, UK
| | - Bon Ham Yip
- Bloodwise Molecular Haematology Unit, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, and NIHR Biomedical Research Centre, Oxford, UK
| | - Simona Valletta
- Bloodwise Molecular Haematology Unit, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, and NIHR Biomedical Research Centre, Oxford, UK
| | - Jacqueline Boultwood
- Bloodwise Molecular Haematology Unit, Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, and NIHR Biomedical Research Centre, Oxford, UK.
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Nicholson SA, Moyo B, Arbuthnot PB. Progress and prospects of engineered sequence-specific DNA modulating technologies for the management of liver diseases. World J Hepatol 2015; 7:859-873. [PMID: 25937863 PMCID: PMC4411528 DOI: 10.4254/wjh.v7.i6.859] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/11/2014] [Revised: 12/16/2014] [Accepted: 01/20/2015] [Indexed: 02/06/2023] Open
Abstract
Liver diseases are one of the leading causes of mortality in the world. The hepatic illnesses, which include inherited metabolic disorders, hemophilias and viral hepatitides, are complex and currently difficult to treat. The maturation of gene therapy has heralded new avenues for developing effective intervention for these diseases. DNA modification using gene therapy is now possible and available technology may be exploited to achieve long term therapeutic benefit. The ability to edit DNA sequences specifically is of paramount importance to advance gene therapy for application to liver diseases. Recent development of technologies that allow for this has resulted in rapid advancement of gene therapy to treat several chronic illnesses. Improvements in application of derivatives of zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs), homing endonucleases (HEs) and clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR associated (Cas) systems have been particularly important. These sequence-specific technologies may be used to modify genes permanently and also to alter gene transcription for therapeutic purposes. This review describes progress in development of ZFPs, TALEs, HEs and CRISPR/Cas for application to treating liver diseases.
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33
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Nomura W. [Application and potential of genome engineering by artificial enzymes]. YAKUGAKU ZASSHI 2015; 135:405-14. [PMID: 25759050 DOI: 10.1248/yakushi.14-00240-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
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.
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Affiliation(s)
- Wataru Nomura
- Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University
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34
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Osakabe Y, Osakabe K. Genome editing with engineered nucleases in plants. PLANT & CELL PHYSIOLOGY 2015; 56:389-400. [PMID: 25416289 DOI: 10.1093/pcp/pcu170] [Citation(s) in RCA: 118] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/18/2023]
Abstract
Numerous examples of successful 'genome editing' now exist. Genome editing uses engineered nucleases as powerful tools to target specific DNA sequences to edit genes precisely in the genomes of both model and crop plants, as well as a variety of other organisms. The DNA-binding domains of zinc finger (ZF) proteins were the first to be used as genome editing tools, in the form of designed ZF nucleases (ZFNs). More recently, transcription activator-like effector nucleases (TALENs), as well as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, which utilizes RNA-DNA interactions, have proved useful. A key step in genome editing is the generation of a double-stranded DNA break that is specific to the target gene. This is achieved by custom-designed endonucleases, which enable site-directed mutagenesis via a non-homologous end-joining (NHEJ) repair pathway and/or gene targeting via homologous recombination (HR) to occur efficiently at specific sites in the genome. This review provides an overview of recent advances in genome editing technologies in plants, and discusses how these can provide insights into current plant molecular biology research and molecular breeding technology.
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Affiliation(s)
- Yuriko Osakabe
- RIKEN Center for Sustainable Resource Science, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074 Japan
| | - Keishi Osakabe
- Center for Collaboration among Agriculture, Industry and Commerce, The University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima, 770-8503 Japan
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35
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Abstract
The tools for genome engineering have become very powerful and accessible over the last several years. CRISPR/Cas nucleases, TALENs and ZFNs can all be designed to produce highly specific double-strand breaks in chromosomal DNA. These breaks are processed by cellular DNA repair machinery leading to localized mutations and to intentional sequence replacements. Because these repair processes are common to essentially all organisms, the targetable nucleases have been applied successfully to a wide range of animals, plants, and cultured cells. In each case, the mode of delivery of the nuclease, the efficiency of cleavage and the repair outcome depend on the biology of the particular system being addressed. These reagents are being used to introduce favorable characteristics into organisms of economic significance, and the prospects for enhancing human gene therapy appear very bright.
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Affiliation(s)
- Dana Carroll
- Department of Biochemistry, School of Medicine, University of Utah, Emma Eccles Jones Medical Research Building, Rm 4520, 15N. Medical Drive East, Salt Lake City, UT, 84112-5650, USA,
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36
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Ul Ain Q, Chung JY, Kim YH. Current and future delivery systems for engineered nucleases: ZFN, TALEN and RGEN. J Control Release 2014; 205:120-7. [PMID: 25553825 DOI: 10.1016/j.jconrel.2014.12.036] [Citation(s) in RCA: 71] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2014] [Revised: 12/19/2014] [Accepted: 12/24/2014] [Indexed: 12/14/2022]
Abstract
Gene therapy by engineered nucleases is a genetic intervention being investigated for curing the hereditary disorders by targeting selected genes with specific nucleotides for establishment, suppression, abolishment of a function or correction of mutation. Here, we review the fast developing technology of targeted genome engineering using site specific programmable nucleases zinc finger nucleases (ZFNs), transcription activator like nucleases (TALENs) and cluster regulatory interspaced short palindromic repeat/CRISPR associated proteins (CRISPR/Cas) based RNA-guided DNA endonucleases (RGENs) and their different characteristics including pros and cons of genome modifications by these nucleases. We have further discussed different types of delivery methods to induce gene editing, novel development in genetic engineering other than nucleases and future prospects.
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Affiliation(s)
- Qurrat Ul Ain
- Department of Bioengineering, Institute for Bioengineering and Biopharmaceutical Research, BK 21 Plus Future Biopharmaceutical Human Resources Training and Research Team, Hanyang University, 133-791 Seoul, Republic of Korea
| | - Jee Young Chung
- Department of Bioengineering, Institute for Bioengineering and Biopharmaceutical Research, BK 21 Plus Future Biopharmaceutical Human Resources Training and Research Team, Hanyang University, 133-791 Seoul, Republic of Korea
| | - Yong-Hee Kim
- Department of Bioengineering, Institute for Bioengineering and Biopharmaceutical Research, BK 21 Plus Future Biopharmaceutical Human Resources Training and Research Team, Hanyang University, 133-791 Seoul, Republic of Korea.
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37
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Kuhn H, Sahu B, Rapireddy S, Ly DH, Frank-Kamenetskii MD. Sequence specificity at targeting double-stranded DNA with a γ-PNA oligomer modified with guanidinium G-clamp nucleobases. ARTIFICIAL DNA, PNA & XNA 2014; 1:45-53. [PMID: 21687526 DOI: 10.4161/adna.1.1.12444] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/24/2010] [Revised: 05/19/2010] [Accepted: 05/24/2010] [Indexed: 11/19/2022]
Abstract
γ-PNA, a new class of peptide nucleic acids, promises to overcome previous sequence limitations of double-stranded DNA (dsDNA) targeting with PNA. To check the potential of γ-PNA, we have synthesized a biotinylated, pentadecameric γ-PNA of mixed sequence carrying three guanidinium G-clamp nucleobases. We have found that strand invasion reactions of the γ-PNA oligomer to its fully complementary target within dsDNA occurs with significantly higher binding rates than to targets containing single mismatches. Association of the PNA oligomer to mismatched targets does not go to completion but instead reaches a stationary level at or below 60%, even at conditions of very low ionic strength. Initial binding rates to both matched and mismatched targets experience a steep decrease with increasing salt concentration. We demonstrate that a linear DNA target fragment with the correct target sequence can be purified from DNA mixtures containing mismatched target or unrelated genomic DNA by affinity capture with streptavidin-coated magnetic beads. Similarly, supercoiled plasmid DNA is obtained with high purity from an initial sample mixture that included a linear DNA fragment with the fully complementary sequence. Based on the results obtained in this study we believe that γ-PNA has a great potential for specific targeting of chosen duplex DNA sites in a sequence-unrestricted fashion.
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Affiliation(s)
- Heiko Kuhn
- Center for Advanced Biotechnology; Department of Biomedical Engineering; Boston University; Boston, MA USA
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38
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Grimmer MR, Stolzenburg S, Ford E, Lister R, Blancafort P, Farnham PJ. Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation. Nucleic Acids Res 2014; 42:10856-68. [PMID: 25122745 PMCID: PMC4176344 DOI: 10.1093/nar/gku708] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
Artificial transcription factors (ATFs) and genomic nucleases based on a DNA binding platform consisting of multiple zinc finger domains are currently being developed for clinical applications. However, no genome-wide investigations into their binding specificity have been performed. We have created six-finger ATFs to target two different 18 nt regions of the human SOX2 promoter; each ATF is constructed such that it contains or lacks a super KRAB domain (SKD) that interacts with a complex containing repressive histone methyltransferases. ChIP-seq analysis of the effector-free ATFs in MCF7 breast cancer cells identified thousands of binding sites, mostly in promoter regions; the addition of an SKD domain increased the number of binding sites ∼5-fold, with a majority of the new sites located outside of promoters. De novo motif analyses suggest that the lack of binding specificity is due to subsets of the finger domains being used for genomic interactions. Although the ATFs display widespread binding, few genes showed expression differences; genes repressed by the ATF-SKD have stronger binding sites and are more enriched for a 12 nt motif. Interestingly, epigenetic analyses indicate that the transcriptional repression caused by the ATF-SKD is not due to changes in active histone modifications.
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Affiliation(s)
- Matthew R Grimmer
- Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90089, USA Integrated Genetics and Genomics, University of California-Davis, Davis, CA 95616, USA
| | - Sabine Stolzenburg
- Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA School of Anatomy, Physiology and Human Biology, M309, The University of Western Australia, Crawley, WA 6009, Australia
| | - Ethan Ford
- Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Perth, WA 6009, Australia
| | - Ryan Lister
- Australian Research Council Centre of Excellence in Plant Energy Biology, The University of Western Australia, Perth, WA 6009, Australia
| | - Pilar Blancafort
- Department of Pharmacology, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA School of Anatomy, Physiology and Human Biology, M309, The University of Western Australia, Crawley, WA 6009, Australia Cancer Epigenetics Group, Harry Perkins Institute of Medical Research, Nedlands, WA 6008, Australia
| | - Peggy J Farnham
- Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90089, USA
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Wijshake T, Baker DJ, van de Sluis B. Endonucleases: new tools to edit the mouse genome. Biochim Biophys Acta Mol Basis Dis 2014; 1842:1942-1950. [PMID: 24794718 DOI: 10.1016/j.bbadis.2014.04.020] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2014] [Revised: 04/16/2014] [Accepted: 04/18/2014] [Indexed: 12/26/2022]
Abstract
Mouse transgenesis has been instrumental in determining the function of genes in the pathophysiology of human diseases and modification of genes by homologous recombination in mouse embryonic stem cells remains a widely used technology. However, this approach harbors a number of disadvantages, as it is time-consuming and quite laborious. Over the last decade a number of new genome editing technologies have been developed, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas). These systems are characterized by a designed DNA binding protein or RNA sequence fused or co-expressed with a non-specific endonuclease, respectively. The engineered DNA binding protein or RNA sequence guides the nuclease to a specific target sequence in the genome to induce a double strand break. The subsequent activation of the DNA repair machinery then enables the introduction of gene modifications at the target site, such as gene disruption, correction or insertion. Nuclease-mediated genome editing has numerous advantages over conventional gene targeting, including increased efficiency in gene editing, reduced generation time of mutant mice, and the ability to mutagenize multiple genes simultaneously. Although nuclease-driven modifications in the genome are a powerful tool to generate mutant mice, there are concerns about off-target cleavage, especially when using the CRISPR/Cas system. Here, we describe the basic principles of these new strategies in mouse genome manipulation, their inherent advantages, and their potential disadvantages compared to current technologies used to study gene function in mouse models. This article is part of a Special Issue entitled: From Genome to Function.
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Affiliation(s)
- Tobias Wijshake
- Molecular Genetics, University of Groningen, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands
| | - Darren J Baker
- Department of Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, 200 First St SW, Rochester, MN 55905, USA
| | - Bart van de Sluis
- Molecular Genetics, University of Groningen, University Medical Center Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.
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40
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Abstract
Programmable nucleases - including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)-Cas (CRISPR-associated) system - enable targeted genetic modifications in cultured cells, as well as in whole animals and plants. The value of these enzymes in research, medicine and biotechnology arises from their ability to induce site-specific DNA cleavage in the genome, the repair (through endogenous mechanisms) of which allows high-precision genome editing. However, these nucleases differ in several respects, including their composition, targetable sites, specificities and mutation signatures, among other characteristics. Knowledge of nuclease-specific features, as well as of their pros and cons, is essential for researchers to choose the most appropriate tool for a range of applications.
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Affiliation(s)
- Hyongbum Kim
- Graduate School of Biomedical Science and Engineering, and College of Medicine, Hanyang University, Wangsimni-ro 222, Sungdong-gu, Seoul 133-791, South Korea
| | - Jin-Soo Kim
- 1] Center for Genome Engineering, Institute for Basic Science, Gwanak-ro 1, Gwanak-gu, Seoul 151-747, South Korea. [2] Department of Chemistry, Seoul National University, Gwanak-ro 1, Gwanak-gu, Seoul 151-747, South Korea
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41
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Johnson LM, Du J, Hale CJ, Bischof S, Feng S, Chodavarapu RK, Zhong X, Marson G, Pellegrini M, Segal DJ, Patel DJ, Jacobsen SE. SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation. Nature 2014; 507:124-128. [PMID: 24463519 PMCID: PMC3963826 DOI: 10.1038/nature12931] [Citation(s) in RCA: 243] [Impact Index Per Article: 22.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2013] [Accepted: 12/02/2013] [Indexed: 12/19/2022]
Abstract
RNA-directed DNA methylation in Arabidopsis thaliana depends on the upstream synthesis of 24-nucleotide small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) and downstream synthesis of non-coding transcripts by Pol V. Pol V transcripts are thought to interact with siRNAs which then recruit DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to methylate DNA. The SU(VAR)3-9 homologues SUVH2 and SUVH9 act in this downstream step but the mechanism of their action is unknown. Here we show that genome-wide Pol V association with chromatin redundantly requires SUVH2 and SUVH9. Although SUVH2 and SUVH9 resemble histone methyltransferases, a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-adenosyl methionine (SAM)-binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins. SUVH2 and SUVH9 both contain SRA (SET- and RING-ASSOCIATED) domains capable of binding methylated DNA, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of DNA METHYLTRANSFERASE 1 (MET1) causes loss of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. Furthermore, tethering SUVH9 [corrected] with a zinc finger to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. These results indicate that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of plant genomes for epigenetic silencing.
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MESH Headings
- Arabidopsis/enzymology
- Arabidopsis/genetics
- Arabidopsis Proteins/chemistry
- Arabidopsis Proteins/genetics
- Arabidopsis Proteins/metabolism
- Binding Sites/genetics
- Biocatalysis
- Chromatin/chemistry
- Chromatin/genetics
- Chromatin/metabolism
- Crystallography, X-Ray
- DNA (Cytosine-5-)-Methyltransferases/genetics
- DNA (Cytosine-5-)-Methyltransferases/metabolism
- DNA Methylation/genetics
- DNA-Binding Proteins/chemistry
- DNA-Binding Proteins/metabolism
- DNA-Directed RNA Polymerases/metabolism
- Flowers/growth & development
- Gene Expression Regulation, Plant
- Gene Silencing
- Genome, Plant/genetics
- Histone-Lysine N-Methyltransferase/chemistry
- Histone-Lysine N-Methyltransferase/metabolism
- Models, Molecular
- Mutation/genetics
- Phenotype
- Protein Structure, Tertiary
- Protein Transport
- RNA, Plant/biosynthesis
- RNA, Plant/genetics
- RNA, Plant/metabolism
- RNA, Small Interfering/biosynthesis
- RNA, Small Interfering/genetics
- RNA, Small Interfering/metabolism
- Transcription, Genetic
- Zinc Fingers
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Affiliation(s)
- Lianna M. Johnson
- Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA
| | - Jiamu Du
- Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA
| | - Christopher J. Hale
- Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA
| | - Sylvain Bischof
- Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA
| | - Suhua Feng
- Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA
- Howard Hughes Medical Institute, University of California at Los Angeles, Los Angeles, CA 90095, USA
| | - Ramakrishna K. Chodavarapu
- Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA
| | - Xuehua Zhong
- Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA
| | - Giuseppe Marson
- Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA
| | - Matteo Pellegrini
- Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA
| | - David J. Segal
- Genome Center and Department of Biochemistry and Molecular Medicine, University of California at Davis, Davis, CA 95616, USA
| | - Dinshaw J. Patel
- Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA
| | - Steven E. Jacobsen
- Department of Molecular, Cell and Developmental Biology, University of California at Los Angeles, Los Angeles, CA 90095, USA
- Howard Hughes Medical Institute, University of California at Los Angeles, Los Angeles, CA 90095, USA
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42
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Abstract
Current technology enables the production of highly specific genome modifications with excellent efficiency and specificity. Key to this capability are targetable DNA cleavage reagents and cellular DNA repair pathways. The break made by these reagents can produce localized sequence changes through inaccurate nonhomologous end joining (NHEJ), often leading to gene inactivation. Alternatively, user-provided DNA can be used as a template for repair by homologous recombination (HR), leading to the introduction of desired sequence changes. This review describes three classes of targetable cleavage reagents: zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas RNA-guided nucleases (RGNs). As a group, these reagents have been successfully used to modify genomic sequences in a wide variety of cells and organisms, including humans. This review discusses the properties, advantages, and limitations of each system, as well as the specific considerations required for their use in different biological systems.
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Affiliation(s)
- Dana Carroll
- Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112;
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43
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Pillay LM, Selland LG, Fleisch VC, Leighton PLA, Cheng CS, Famulski JK, Ritzel RG, March LD, Wang H, Allison WT, Waskiewicz AJ. Evaluating the mutagenic activity of targeted endonucleases containing a Sharkey FokI cleavage domain variant in zebrafish. Zebrafish 2013; 10:353-64. [PMID: 23781947 DOI: 10.1089/zeb.2012.0832] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Synthetic targeted endonucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have recently emerged as powerful tools for targeted mutagenesis, especially in organisms that are not amenable to embryonic stem cell manipulation. Both ZFNs and TALENs consist of DNA-binding arrays that are fused to the nonspecific FokI nuclease domain. In an effort to improve targeted endonuclease mutagenesis efficiency, we enhanced their catalytic activity using the Sharkey FokI nuclease domain variant. All constructs tested display increased DNA cleavage activity in vitro. We demonstrate that one out of four ZFN arrays containing the Sharkey FokI variant exhibits a dramatic increase in mutagenesis frequency in vivo in zebrafish. The other three ZFNs exhibit no significant alteration of activity in vivo. Conversely, we demonstrate that TALENs containing the Sharkey FokI variant exhibit absent or severely reduced in vivo mutagenic activity in zebrafish. Notably, Sharkey ZFNs and TALENs do not generate increased toxicity-related defects or mortality. Our results present Sharkey ZFNs as an effective alternative to conventional ZFNs, but advise against the use of Sharkey TALENs.
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Affiliation(s)
- Laura M Pillay
- Department of Biological Sciences, University of Alberta , Edmonton, Canada
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44
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Wilson KA, McEwen AE, Pruett-Miller SM, Zhang J, Kildebeck EJ, Porteus MH. Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification. MOLECULAR THERAPY. NUCLEIC ACIDS 2013; 2:e88. [PMID: 23632390 PMCID: PMC3650245 DOI: 10.1038/mtna.2013.13] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/17/2012] [Accepted: 02/05/2013] [Indexed: 11/08/2022]
Abstract
Recent studies have shown that zinc finger nucleases (ZFNs) are powerful reagents for making site-specific genomic modifications. The generic structure of these enzymes includes a ZF DNA-binding domain and nuclease domain (Fn) are separated by an amino acid "linker" and cut genomic DNA at sites that have a generic structure (site1)-(spacer)-(site2) where the "spacer" separates the two binding sites. In this work, we compare the activity of ZFNs with different linkers on target sites with different spacer lengths. We found those nucleases with linkers' lengths of 2 or 4 amino acid (aa) efficiently cut at target sites with 5 or 6 base pair (bp) spacers, and that those ZFNs with a 5-aa linker length efficiently cut target sites with 6 or 7 bp spacers. In addition, we demonstrate that the Oligomerized Pool ENgineering (OPEN) platform used for making three-fingered ZF proteins (ZFPs) can be modified to incorporate modular assembly fingers (including those recognizing ANNs, CNNs, and TNNs) and we were able to generate nucleases that efficiently cut cognate target sites. The ability to use module fingers in the OPEN platform at target sites of 5-7 bp spacer lengths increases the probability of finding a ZFN target site to 1 in 4 bp. These findings significantly expand the range of sites that can be potentially targeted by these custom-engineered proteins.Molecular Therapy - Nucleic Acids (2013) 2, e88; doi:10.1038/mtna.2013.13; published online 30 April 2013.
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Affiliation(s)
- Kimberly A Wilson
- Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA
- Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Abbye E McEwen
- Department of Medicine, Division of Pulmonary and Critical Care, Northwestern University, Chicago, Illinois, USA
| | - Shondra M Pruett-Miller
- Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA
- Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Jiuli Zhang
- Department of Biological Sciences, Florida Atlantic University, Boca Raton, Florida, USA
| | - Eric J Kildebeck
- Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA
- Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Matthew H Porteus
- Department of Pediatrics, Stanford University, Stanford, California, USA
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45
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Skretas G, Kolisis FN. Combinatorial approaches for inverse metabolic engineering applications. Comput Struct Biotechnol J 2013; 3:e201210021. [PMID: 24688681 PMCID: PMC3962077 DOI: 10.5936/csbj.201210021] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2012] [Revised: 02/11/2013] [Accepted: 02/17/2013] [Indexed: 11/22/2022] Open
Abstract
Traditional metabolic engineering analyzes biosynthetic and physiological pathways, identifies bottlenecks, and makes targeted genetic modifications with the ultimate goal of increasing the production of high-value products in living cells. Such efforts have led to the development of a variety of organisms with industrially relevant properties. However, there are a number of cellular phenotypes important for research and the industry for which the rational selection of cellular targets for modification is not easy or possible. In these cases, strain engineering can be alternatively carried out using “inverse metabolic engineering”, an approach that first generates genetic diversity by subjecting a population of cells to a particular mutagenic process, and then utilizes genetic screens or selections to identify the clones exhibiting the desired phenotype. Given the availability of an appropriate screen for a particular property, the success of inverse metabolic engineering efforts usually depends on the level and quality of genetic diversity which can be generated. Here, we review classic and recently developed combinatorial approaches for creating such genetic diversity and discuss the use of these methodologies in inverse metabolic engineering applications.
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Affiliation(s)
- Georgios Skretas
- Institute of Biology, Medicinal Chemistry and Biotechnology, National Hellenic Research Foundation, Athens, Greece
| | - Fragiskos N Kolisis
- Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens - Zografou Campus, Athens, Greece
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46
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Zhu C, Gupta A, Hall VL, Rayla AL, Christensen RG, Dake B, Lakshmanan A, Kuperwasser C, Stormo GD, Wolfe SA. Using defined finger-finger interfaces as units of assembly for constructing zinc-finger nucleases. Nucleic Acids Res 2013; 41:2455-65. [PMID: 23303772 PMCID: PMC3575815 DOI: 10.1093/nar/gks1357] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
Zinc-finger nucleases (ZFNs) have been used for genome engineering in a wide variety of organisms; however, it remains challenging to design effective ZFNs for many genomic sequences using publicly available zinc-finger modules. This limitation is in part because of potential finger–finger incompatibility generated on assembly of modules into zinc-finger arrays (ZFAs). Herein, we describe the validation of a new set of two-finger modules that can be used for building ZFAs via conventional assembly methods or a new strategy—finger stitching—that increases the diversity of genomic sequences targetable by ZFNs. Instead of assembling ZFAs based on units of the zinc-finger structural domain, our finger stitching method uses units that span the finger–finger interface to ensure compatibility of neighbouring recognition helices. We tested this approach by generating and characterizing eight ZFAs, and we found their DNA-binding specificities reflected the specificities of the component modules used in their construction. Four pairs of ZFNs incorporating these ZFAs generated targeted lesions in vivo, demonstrating that stitching yields ZFAs with robust recognition properties.
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Affiliation(s)
- Cong Zhu
- Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Genetics, Washington University School of Medicine, St Louis, MO, USA 63108 and Molecular Oncology Research Institute (MORI), Tufts University School of Medicine, Boston, MA, USA 02111
| | - Ankit Gupta
- Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Genetics, Washington University School of Medicine, St Louis, MO, USA 63108 and Molecular Oncology Research Institute (MORI), Tufts University School of Medicine, Boston, MA, USA 02111
| | - Victoria L. Hall
- Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Genetics, Washington University School of Medicine, St Louis, MO, USA 63108 and Molecular Oncology Research Institute (MORI), Tufts University School of Medicine, Boston, MA, USA 02111
| | - Amy L. Rayla
- Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Genetics, Washington University School of Medicine, St Louis, MO, USA 63108 and Molecular Oncology Research Institute (MORI), Tufts University School of Medicine, Boston, MA, USA 02111
| | - Ryan G. Christensen
- Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Genetics, Washington University School of Medicine, St Louis, MO, USA 63108 and Molecular Oncology Research Institute (MORI), Tufts University School of Medicine, Boston, MA, USA 02111
| | - Benjamin Dake
- Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Genetics, Washington University School of Medicine, St Louis, MO, USA 63108 and Molecular Oncology Research Institute (MORI), Tufts University School of Medicine, Boston, MA, USA 02111
| | - Abirami Lakshmanan
- Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Genetics, Washington University School of Medicine, St Louis, MO, USA 63108 and Molecular Oncology Research Institute (MORI), Tufts University School of Medicine, Boston, MA, USA 02111
| | - Charlotte Kuperwasser
- Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Genetics, Washington University School of Medicine, St Louis, MO, USA 63108 and Molecular Oncology Research Institute (MORI), Tufts University School of Medicine, Boston, MA, USA 02111
| | - Gary D. Stormo
- Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Genetics, Washington University School of Medicine, St Louis, MO, USA 63108 and Molecular Oncology Research Institute (MORI), Tufts University School of Medicine, Boston, MA, USA 02111
| | - Scot A. Wolfe
- Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA 01605, Department of Genetics, Washington University School of Medicine, St Louis, MO, USA 63108 and Molecular Oncology Research Institute (MORI), Tufts University School of Medicine, Boston, MA, USA 02111
- *To whom correspondence should be addressed. Tel: +1 508 856 3953; Fax: +1 508 856 5460;
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47
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Engineered Zinc Finger Nucleases for Targeted Genome Editing. SITE-DIRECTED INSERTION OF TRANSGENES 2013. [DOI: 10.1007/978-94-007-4531-5_5] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/17/2023]
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48
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Abstract
Recent advances in genome engineering provide newfound control over a plant's genetic material. It is now possible for most bench scientists to alter DNA in living plant cells in a variety of ways, including introducing specific nucleotide substitutions in a gene that change a protein's amino acid sequence, deleting genes or chromosomal segments, and inserting foreign DNA at precise genomic locations. Such targeted DNA sequence modifications are enabled by sequence-specific nucleases that create double-strand breaks in the genomic loci to be altered. The repair of the breaks, through either homologous recombination or nonhomologous end joining, can be controlled to achieve the desired sequence modification. Genome engineering promises to advance basic plant research by linking DNA sequences to biological function. Further, genome engineering will enable plants' biosynthetic capacity to be harnessed to produce the many agricultural products required by an expanding world population.
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Affiliation(s)
- Daniel F Voytas
- Department of Genetics, Cell Biology, and Development and Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA.
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49
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Fu F, Voytas DF. Zinc Finger Database (ZiFDB) v2.0: a comprehensive database of C₂H₂ zinc fingers and engineered zinc finger arrays. Nucleic Acids Res 2012. [PMID: 23203887 PMCID: PMC3531203 DOI: 10.1093/nar/gks1167] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/05/2022] Open
Abstract
ZiFDB (Zinc Finger Database, http://zifdb.msi.umn.edu) is a web-accessible database that houses information on individual C2H2 zinc fingers (ZFs) and engineered zinc finger arrays (ZFAs). ZiFDB serves as a resource for biologists interested in engineering ZFAs for use as sequence-specific DNA-binding reagents. Here, we describe four new features of ZiFDB: (i) the database allows users to input new ZFs and ZFAs; (ii) a shadow database temporarily stores user-submitted data, pending approval by the database curator and subsequent loading into the persistent database; (iii) ZiFDB contains 181 Context-Dependent Assembly (CoDA) ZFAs, which were generated by this newly described ZFA engineering platform; and (iv) the database also now contains 319 F1F2 CoDA units and 334 F2F3 CoDA units that can be used to construct CoDA arrays. In total, the new release of ZiFDB contains 1226 ZFs and 1123 ZFAs.
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Affiliation(s)
- Fengli Fu
- Department of Genetics, University of Minnesota, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455, USA
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50
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Zhang W, Guo Y, Zhang C, Ji H, Meng W, Wang D, Li X, Mao Q, Xia H. Rescue the failed half-ZFN by a sensitive mammalian cell-based luciferase reporter system. PLoS One 2012; 7:e45169. [PMID: 23028823 PMCID: PMC3445457 DOI: 10.1371/journal.pone.0045169] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2012] [Accepted: 08/14/2012] [Indexed: 11/30/2022] Open
Abstract
ZFN technology is a powerful research tool and has been used for genome editing in cells lines, animals and plants. The generation of functional ZFNs for particular targets in mammalian genome is still challenging for an average research group. The modular-assembly method is relatively fast, easy-to-practice but has a high failure rate. Some recent studies suggested that a ZFP with low binding activity might be able to form a working ZFN pair with another binding active half-ZFP. In order to unveil the potential ZFP candidates among those with low binding activities, this paper established a highly sensitive mammalian cell-based transcriptional reporter system to assess the DNA binding activities of ZFPs by inserting multiple copies of ZFN target sequence fragment (TSF) of an interested gene (e. g., hPGRN or hVEGF). Our results showed that this system increased the screening sensitivity up to 50-fold and markedly amplified the differences in the binding activities between different ZFPs. We also found that the targeted chromosomal gene repair efficiency of each hPGRN or hVEGF ZFN pair was in proportion with the combination of the binding activities of the ZFL (Left zinc finger) and ZFR (Right zinc finger). A hPGRN ZFR with low binding ability was able to form a biological active ZFN if combined with a hPGRN ZFL with relatively high binding ability. Lastly, site-specific genome editing by hPGRN ZFNs generated by this system was confirmed by sequencing, and the PGRN knock-out cell line showed significantly decreased cell growth compared with the control. Our system will provide a valuable tool for further optimizing the nucleases with regard to specificity and cytotoxicity.
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Affiliation(s)
- Weifeng Zhang
- Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi, People’s Republic of China
| | - Yuanxu Guo
- Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi, People’s Republic of China
| | - Chen Zhang
- Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi, People’s Republic of China
| | - Haiyan Ji
- Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi, People’s Republic of China
| | - Wenpeng Meng
- Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi, People’s Republic of China
| | - Dongyang Wang
- Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi, People’s Republic of China
| | - Xing Li
- Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi, People’s Republic of China
| | - Qinwen Mao
- Departmet of Pathology, Northwestern University Feinberg School of Medicine Chicago, Chicago, Illinois, United States of America
- * E-mail: (QM); (HX)
| | - Haibin Xia
- Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, Xi’an, Shaanxi, People’s Republic of China
- * E-mail: (QM); (HX)
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