TALEN-mediated genome editing: prospects and perspectives

2014 ◽  
Vol 462 (1) ◽  
pp. 15-24 ◽  
Author(s):  
David A. Wright ◽  
Ting Li ◽  
Bing Yang ◽  
Martin H. Spalding
Keyword(s):  
Genome Editing ◽  
Double Strand Breaks ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Strand Breaks ◽  
Natural Processes ◽  
Current State ◽  
A Genome ◽  
Dna Dsbs ◽  
Repair Mechanisms

Genome editing is the practice of making predetermined and precise changes to a genome by controlling the location of DNA DSBs (double-strand breaks) and manipulating the cell's repair mechanisms. This technology results from harnessing natural processes that have taken decades and multiple lines of inquiry to understand. Through many false starts and iterative technology advances, the goal of genome editing is just now falling under the control of human hands as a routine and broadly applicable method. The present review attempts to define the technique and capture the discovery process while following its evolution from meganucleases and zinc finger nucleases to the current state of the art: TALEN (transcription-activator-like effector nuclease) technology. We also discuss factors that influence success, technical challenges and future prospects of this quickly evolving area of study and application.

scholarly journals Simultaneous precise editing of multiple genes in human cells

2019 ◽  
Vol 47 (19) ◽  
pp. e116-e116 ◽  
Author(s):  
Stephan Riesenberg ◽  
Manjusha Chintalapati ◽  
Dominik Macak ◽  
Philipp Kanis ◽  
Tomislav Maricic ◽  
...  
Keyword(s):  
Genome Editing ◽  
Kinase Inhibitor ◽  
Double Strand Breaks ◽  
End Joining ◽  
Nucleotide Substitutions ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
A Genome ◽  
Dependent Protein ◽  
Non Homologous End Joining

Abstract When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.

scholarly journals In Vivo Genome Editing as a Therapeutic Approach

2018 ◽  
Vol 19 (9) ◽  
pp. 2721 ◽  
Author(s):  
Beatrice Ho ◽  
Sharon Loh ◽  
Woon Chan ◽  
Boon Soh
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Genetic Diseases ◽  
Gene Mutations ◽  
Genetic Mutation ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Therapeutic Benefits ◽  
A Genome

Genome editing has been well established as a genome engineering tool that enables researchers to establish causal linkages between genetic mutation and biological phenotypes, providing further understanding of the genetic manifestation of many debilitating diseases. More recently, the paradigm of genome editing technologies has evolved to include the correction of mutations that cause diseases via the use of nucleases such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs), and more recently, Cas9 nuclease. With the aim of reversing disease phenotypes, which arise from somatic gene mutations, current research focuses on the clinical translatability of correcting human genetic diseases in vivo, to provide long-term therapeutic benefits and potentially circumvent the limitations of in vivo cell replacement therapy. In this review, in addition to providing an overview of the various genome editing techniques available, we have also summarized several in vivo genome engineering strategies that have successfully demonstrated disease correction via in vivo genome editing. The various benefits and challenges faced in applying in vivo genome editing in humans will also be discussed.

scholarly journals Genome Editing: A New Horizon for Oral and Craniofacial Research

2018 ◽  
Vol 98 (1) ◽  
pp. 36-45 ◽  
Author(s):  
N. Yu ◽  
J. Yang ◽  
Y. Mishina ◽  
W.V. Giannobile
Keyword(s):  
Genome Editing ◽  
Genetic Manipulation ◽  
Zinc Finger Nucleases ◽  
Biological Research ◽  
Transcription Activator ◽  
Developmental Defects ◽  
Genetic Manipulations ◽  
Research Fields ◽  
Repair Mechanisms ◽  
Conducting Research

Precise and efficient genetic manipulations have enabled researchers to understand gene functions in disease and development, providing a platform to search for molecular cures. Over the past decade, the unprecedented advancement of genome editing techniques has revolutionized the biological research fields. Early genome editing strategies involved many naturally occurring nucleases, including meganucleases, zinc finger nucleases, and transcription activator-like effector-based nucleases. More recently, the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nucleases (CRISPR/Cas) system has greatly enriched genetic manipulation methods in conducting research. Those nucleases generate double-strand breaks in the target gene sequences and then utilize DNA repair mechanisms to permit precise yet versatile genetic manipulations. The oral and craniofacial field harbors a plethora of diseases and developmental defects that require genetic models that can exploit these genome editing techniques. This review provides an overview of the genome editing techniques, particularly the CRISPR/Cas9 technique, for the oral and craniofacial research community. We also discuss the details about the emerging applications of genome editing in oral and craniofacial biology.

scholarly journals Multiplex Genome-Editing Technologies for Revolutionizing Plant Biology and Crop Improvement

2021 ◽  
Vol 12 ◽  
Author(s):  
Mohamed Abdelrahman ◽  
Zheng Wei ◽  
Jai S. Rohila ◽  
Kaijun Zhao
Keyword(s):  
Genome Editing ◽  
Crop Improvement ◽  
Plant Molecular Biology ◽  
Plant Biology ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Simultaneous Generation ◽  
Multiple Loci ◽  
A Genome ◽  
Improvement Programs

Multiplex genome-editing (MGE) technologies are recently developed versatile bioengineering tools for modifying two or more specific DNA loci in a genome with high precision. These genome-editing tools have greatly increased the feasibility of introducing desired changes at multiple nucleotide levels into a target genome. In particular, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) [CRISPR/Cas] system-based MGE tools allow the simultaneous generation of direct mutations precisely at multiple loci in a gene or multiple genes. MGE is enhancing the field of plant molecular biology and providing capabilities for revolutionizing modern crop-breeding methods as it was virtually impossible to edit genomes so precisely at the single base-pair level with prior genome-editing tools, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Recently, researchers have not only started using MGE tools to advance genome-editing applications in certain plant science fields but also have attempted to decipher and answer basic questions related to plant biology. In this review, we discuss the current progress that has been made toward the development and utilization of MGE tools with an emphasis on the improvements in plant biology after the discovery of CRISPR/Cas9. Furthermore, the most recent advancements involving CRISPR/Cas applications for editing multiple loci or genes are described. Finally, insights into the strengths and importance of MGE technology in advancing crop-improvement programs are presented.

scholarly journals A genome editing primer for the hematologist

Blood ◽  
2016 ◽  
Vol 127 (21) ◽  
pp. 2525-2535 ◽  
Author(s):  
Megan D. Hoban ◽  
Daniel E. Bauer
Keyword(s):  
Genome Editing ◽  
Genetic Alterations ◽  
Double Strand Breaks ◽  
Blood Disorders ◽  
Site Specific ◽  
Strand Breaks ◽  
Epigenome Editing ◽  
Genome Modification ◽  
A Genome ◽  
Experimental Hematology

Abstract Gene editing enables the site-specific modification of the genome. These technologies have rapidly advanced such that they have entered common use in experimental hematology to investigate genetic function. In addition, genome editing is becoming increasingly plausible as a treatment modality to rectify genetic blood disorders and improve cellular therapies. Genome modification typically ensues from site-specific double-strand breaks and may result in a myriad of outcomes. Even single-strand nicks and targeted biochemical modifications that do not permanently alter the DNA sequence (epigenome editing) may be powerful instruments. In this review, we examine the various technologies, describe their advantages and shortcomings for engendering useful genetic alterations, and consider future prospects for genome editing to impact hematology.

Bestimmung der DNA-Schädigung in vitro

2010 ◽  
Vol 49 (S 01) ◽  
pp. S64-S68
Author(s):  
E. Dikomey
Keyword(s):  
Dna Damage ◽  
Cell Lines ◽  
Chromosome Aberrations ◽  
Genetic Factors ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Cell Inactivation ◽  
Repair Mechanisms ◽  
Break Repair

SummaryIonising irradiation acts primarily via induction of DNA damage, among which doublestrand breaks are the most important lesions. These lesions may lead to lethal chromosome aberrations, which are the main reason for cell inactivation. Double-strand breaks can be repaired by several different mechanisms. The regulation of these mechanisms appears be fairly different for normal and tumour cells. Among different cell lines capacity of doublestrand break repair varies by only few percents and is known to be determined mostly by genetic factors. Knowledge about doublestrand break repair mechanisms and their regulation is important for the optimal application of ionising irradiation in medicine.

scholarly journals CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement

2020 ◽  
Author(s):  
Anindya Bandyopadhyay ◽  
Nagesh Kancharla ◽  
vivek javalkote ◽  
santanu dasgupta ◽  
Thomas Brutnell
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Temperature Stability ◽  
Double Strand Breaks ◽  
Plant Genome ◽  
Strand Breaks ◽  
Guide Rna ◽  
Versatile Tool ◽  
Type V ◽  
Global Population

Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA (tracrRNA), targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.

scholarly journals Estimation of Rearrangement Break Rates Across the Genome

2018 ◽  
Author(s):  
Christopher Hann-Soden ◽  
Ian Holmes ◽  
John W. Taylor
Keyword(s):  
Neurospora Crassa ◽  
Interval Graph ◽  
Double Strand Breaks ◽  
Genomic Rearrangements ◽  
Strand Breaks ◽  
Minimum Cover ◽  
A Genome ◽  
History Of ◽  
Cover Problem ◽  
Special Case

Genomic rearrangements provide an important source of variation, but reconstructing the history of rearrangements often has many solutions. We answer the question of where rearrangements occur by solving the simpler problem of estimating the rate of double-strand breaks at every site in a genome. This problem is a special case of the minimum cover problem for an interval graph. We implement this method as a Python program, BRAG, and use it to estimate break rates in the genome of Neurospora crassa. We find that more frequent rearrangement in the subtelomeres facilitates the evolution of novel genes.

scholarly journals Live-cell chromosome dynamics in Arabidopsis thaliana reveals increased chromatin mobility in response to DNA damage

2021 ◽  
Author(s):  
Anis Meschichi ◽  
Adrien Sicard ◽  
Frédéric Pontvianne ◽  
Svenja Reeck ◽  
Stefanie Rosa
Keyword(s):  
Dna Damage ◽  
Arabidopsis Thaliana ◽  
Genome Instability ◽  
High Mobility ◽  
Double Strand Breaks ◽  
Nuclear Space ◽  
Strand Breaks ◽  
Homologous Sequences ◽  
Damage Response ◽  
Repair Mechanisms

Double-strand breaks (DSBs) are a particularly deleterious type of DNA damage potentially leading to translocations and genome instability. Homologous recombination (HR) is a conservative repair pathway in which intact homologous sequences are used as a template for repair. How damaged DNA molecules search for homologous sequences in the crowded space of the cell nucleus is, however, still poorly understood, especially in plants. Here, we measured global chromosome and DSB site mobility, in Arabidopsis thaliana, by tracking the motion of specific loci using the lacO/LacI tagging system and two GFP-tagged HR regulators, RAD51 and RAD54. We observed an increase in chromatin mobility upon the induction of DNA damage, specifically at the S/G2 phases of the cell cycle. Importantly, this increase in mobility was lost on sog1-1 mutant, a central transcription factor of the DNA damage response (DDR), indicating that repair mechanisms actively regulate chromatin mobility upon DNA damage. Interestingly, we observed that DSB sites show remarkably high mobility levels at the early HR stage. Subsequently, a drastic decrease of DSB mobility is observed, which seems to be associated to the relocation of DSBs to the nucleus periphery. Altogether, our data suggest that changes in chromatin mobility are triggered in response to DNA damage, and that this may act as a mechanism to enhance the physical search within the nuclear space to locate a homologous template during homology-directed DNA repair.

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