scholarly journals Efficient Assembly and Verification of ZFNs and TALENs for Modifying Porcine ApoE gene

2015 ◽  
Author(s):  
Xu Huarong ◽  
Li Tuo ◽  
Guo Yang ◽  
Li Hongfei ◽  
Wang Ling ◽  
...  
Keyword(s):  
Genome Engineering ◽  
High Efficiency ◽  
Disease Model ◽  
Apoe Gene ◽  
Zinc Finger Nucleases ◽  
Dna Binding Domain ◽  
Transcription Activator ◽  
Synthetic Nucleases ◽  
Cleavage Domain ◽  
Apoe Gene Polymorphism

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are powerful tools for genome engineering. These synthetic nucleases are assembled with programmable, sequence-specific DNA-binding domain and a non-specific FokI cleavage domain. Apolipoprotein E (ApoE) gene polymorphism is associated with cardiovascular outcomes, including ischaemic stroke and coronary heart disease (CHD). So the objective of this study is to create mutations of AopE gene by ZFNs and TALENs technology. Here, we used the Context-dependent assembly (CoDA) method to design and screen ZFNs specifically targeting with ApoE gene. The targeted cleavage capacity of these ZFNs was validated in yeast system and HEK 293T cells. Meanwhile, an efficient assembled TALENs to target ApoE gene in HEK 293T cells was as a control. The results showed that both ZFNs and TALENs worked on ApoE gene with similar high-efficiency cleavage capability. The result would provide efficient methods for genome editing, so as to get disease model for gene therapy for the further study.

scholarly journals Various Aspects of a Gene Editing System—CRISPR–Cas9

2020 ◽  
Vol 21 (24) ◽  
pp. 9604
Author(s):  
Edyta Janik ◽  
Marcin Niemcewicz ◽  
Michal Ceremuga ◽  
Lukasz Krzowski ◽  
Joanna Saluk-Bijak ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Editing ◽  
Genome Engineering ◽  
High Efficiency ◽  
Adaptive Immune System ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Guide Rna ◽  
Adaptive Immune ◽  
Cas9 Protein

The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR–Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR–Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR–Cas9.

Genome Editing Toolbox

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. SCI-11-SCI-11
Author(s):  
Andrew M. Scharenberg
Keyword(s):  
Genome Engineering ◽  
Gene Knockout ◽  
Zinc Finger Nucleases ◽  
Practical Implementation ◽  
Homing Endonucleases ◽  
Gene Repair ◽  
Dna Breaks ◽  
Transcription Activator ◽  
Advisory Committees ◽  
Core Technology

Abstract Nucleases capable of making targeted breaks in genomic DNA are a core technology required for genome engineering, an emerging field of technology for making precise alterations in cellular genomes. Over the past ten years, four major platforms have emerged for generation of nucleases able to make targeted DNA breaks with a high degree of efficiency and specificity: homing endonucleases, zinc finger nucleases, transcription activator-like (TAL) effector nucleases, and RNA-guided nucleases. This talk will cover the biochemistry and platform-specific attributes of each type of nuclease, along with evolution/improvements in nucleases and related technologies and aspects of the practical implementation of nuclease technology for gene knockout and gene repair in primary hematopoietic cells. Disclosures Scharenberg: Pregenen Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cellectis therapeutics: Consultancy.

scholarly journals Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants

2019 ◽  
Vol 20 (15) ◽  
pp. 3719 ◽  
Author(s):  
Zahra Hajiahmadi ◽  
Ali Movahedi ◽  
Hui Wei ◽  
Dawei Li ◽  
Yasin Orooji ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Guide Rna ◽  
Short Palindromic Repeat ◽  
External Forces ◽  
Time And Energy ◽  
Reduced Time ◽  
Target Effects

The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.

scholarly journals Modern Trends in Plant Genome Editing: An Inclusive Review of the CRISPR/Cas9 Toolbox

2019 ◽  
Vol 20 (16) ◽  
pp. 4045 ◽  
Author(s):  
Ali Razzaq ◽  
Fozia Saleem ◽  
Mehak Kanwal ◽  
Ghulam Mustafa ◽  
Sumaira Yousaf ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Crop Improvement ◽  
Crop Plants ◽  
Targeted Mutagenesis ◽  
Plant Genome ◽  
Zinc Finger Nucleases ◽  
Base Substitution ◽  
Transcription Activator ◽  
Abiotic Stressors

Increasing agricultural productivity via modern breeding strategies is of prime interest to attain global food security. An array of biotic and abiotic stressors affect productivity as well as the quality of crop plants, and it is a primary need to develop crops with improved adaptability, high productivity, and resilience against these biotic/abiotic stressors. Conventional approaches to genetic engineering involve tedious procedures. State-of-the-art OMICS approaches reinforced with next-generation sequencing and the latest developments in genome editing tools have paved the way for targeted mutagenesis, opening new horizons for precise genome engineering. Various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases (MNs) have enabled plant scientists to manipulate desired genes in crop plants. However, these approaches are expensive and laborious involving complex procedures for successful editing. Conversely, CRISPR/Cas9 is an entrancing, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for targeted mutagenesis, including single base substitution, multiplex gene editing, gene knockouts, and regulation of gene transcription in plants. Thus, CRISPR/Cas9-based genome editing has demonstrated great potential for crop improvement but regulation of genome-edited crops is still in its infancy. Here, we extensively reviewed the availability of CRISPR/Cas9 genome editing tools for plant biotechnologists to target desired genes and its vast applications in crop breeding research.

scholarly journals Genome targeting by hybrid Flp-TAL recombinases

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Eugenia Voziyanova ◽  
Feng Li ◽  
Riddhi Shah ◽  
Yuri Voziyanov
Keyword(s):  
Genome Engineering ◽  
Functional Modules ◽  
Dna Binding Domain ◽  
Transcription Activator ◽  
Vector Sequence ◽  
Site Specific ◽  
Hek 293 ◽  
293 Cells ◽  
Genome Manipulation ◽  
Hybrid Enzymes

Abstract Genome engineering is a rapidly evolving field that benefits from the availability of different tools that can be used to perform genome manipulation tasks. We describe here the development of the Flp-TAL recombinases that can target genomic FRT-like sequences in their native chromosomal locations. Flp-TAL recombinases are hybrid enzymes that are composed of two functional modules: a variant of site-specific tyrosine recombinase Flp, which can have either narrow or broad target specificity, and the DNA-binding domain of the transcription activator-like effector, TAL. In Flp-TAL, the TAL module is responsible for delivering and stabilizing the Flp module onto the desired genomic FRT-like sequence where the Flp module mediates recombination. We demonstrate the functionality of the Flp-TAL recombinases by performing integration and deletion experiments in human HEK-293 cells. In the integration experiments we targeted a vector to three genomic FRT-like sequences located in the β-globin locus. In the deletion experiments we excised ~ 15 kilobases of DNA that contained a fragment of the integrated vector sequence and the neighboring genome sequence. On average, the efficiency of the integration and deletion reactions was about 0.1% and 20%, respectively.

scholarly journals TALENs—an indispensable tool in the era of CRISPR: a mini review

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Anuradha Bhardwaj ◽  
Vikrant Nain
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
High Specificity ◽  
Zinc Finger Nucleases ◽  
Main Body ◽  
Transcription Activator ◽  
Scientific World ◽  
Dna Modifications ◽  
Pros And Cons ◽  
Indispensable Tool

Abstract Background Genome of an organism has always fascinated life scientists. With the discovery of restriction endonucleases, scientists were able to make targeted manipulations (knockouts) in any gene sequence of any organism, by the technique popularly known as genome engineering. Though there is a range of genome editing tools, but this era of genome editing is dominated by the CRISPR/Cas9 tool due to its ease of design and handling. But, when it comes to clinical applications, CRISPR is not usually preferred. In this review, we will elaborate on the structural and functional role of designer nucleases with emphasis on TALENs and CRISPR/Cas9 genome editing system. We will also present the unique features of TALENs and limitations of CRISPRs which makes TALENs a better genome editing tool than CRISPRs. Main body Genome editing is a robust technology used to make target specific DNA modifications in the genome of any organism. With the discovery of robust programmable endonucleases-based designer gene manipulating tools such as meganucleases (MN), zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein (CRISPR/Cas9), the research in this field has experienced a tremendous acceleration giving rise to a modern era of genome editing with better precision and specificity. Though, CRISPR-Cas9 platform has successfully gained more attention in the scientific world, TALENs and ZFNs are unique in their own ways. Apart from high-specificity, TALENs are proven to target the mitochondrial DNA (mito-TALEN), where gRNA of CRISPR is difficult to import. This review talks about genome editing goals fulfilled by TALENs and drawbacks of CRISPRs. Conclusions This review provides significant insights into the pros and cons of the two most popular genome editing tools TALENs and CRISPRs. This mini review suggests that, TALENs provides novel opportunities in the field of therapeutics being highly specific and sensitive toward DNA modifications. In this article, we will briefly explore the special features of TALENs that makes this tool indispensable in the field of synthetic biology. This mini review provides great perspective in providing true guidance to the researchers working in the field of trait improvement via genome editing.

scholarly journals Manipulating and elucidating mitochondrial gene expression with engineered proteins

2019 ◽  
Vol 375 (1790) ◽  
pp. 20190185 ◽  
Author(s):  
Christopher P. Wallis ◽  
Louis H. Scott ◽  
Aleksandra Filipovska ◽  
Oliver Rackham
Keyword(s):  
Genome Engineering ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mitochondrial Gene ◽  
Zinc Finger Nucleases ◽  
Mitochondrial Gene Expression ◽  
Transcription Activator ◽  
Theme Issue ◽  
Unusual Structure ◽  
Mitochondrial Genotype

Many conventional, modern genome engineering tools cannot be used to study mitochondrial genetics due to the unusual structure and physiology of the mitochondrial genome. Here, we review a number of newly developed, synthetic biology-based approaches for altering levels of mutant mammalian mitochondrial DNA and mitochondrial RNAs, including transcription activator-like effector nucleases, zinc finger nucleases and engineered RNA-binding proteins. These approaches allow researchers to manipulate and visualize mitochondrial processes and may provide future therapeutics. This article is part of the theme issue ‘Linking the mitochondrial genotype to phenotype: a complex endeavour’.

scholarly journals Targeted genome editing by lentiviral protein transduction of zinc-finger and TAL-effector nucleases

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yujia Cai ◽  
Rasmus O Bak ◽  
Jacob Giehm Mikkelsen
Keyword(s):  
Zinc Finger ◽  
Genome Engineering ◽  
Effective Means ◽  
Zinc Finger Nucleases ◽  
Protein Transduction ◽  
Gene Correction ◽  
Transcription Activator ◽  
Homology Directed Repair ◽  
Target Locus ◽  
Programmable Nucleases

Future therapeutic use of engineered site-directed nucleases, like zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), relies on safe and effective means of delivering nucleases to cells. In this study, we adapt lentiviral vectors as carriers of designer nuclease proteins, providing efficient targeted gene disruption in vector-treated cell lines and primary cells. By co-packaging pairs of ZFN proteins with donor RNA in ‘all-in-one’ lentiviral particles, we co-deliver ZFN proteins and the donor template for homology-directed repair leading to targeted DNA insertion and gene correction. Comparative studies of ZFN activity in a predetermined target locus and a known nearby off-target locus demonstrate reduced off-target activity after ZFN protein transduction relative to conventional delivery approaches. Additionally, TALEN proteins are added to the repertoire of custom-designed nucleases that can be delivered by protein transduction. Altogether, our findings generate a new platform for genome engineering based on efficient and potentially safer delivery of programmable nucleases.

scholarly journals Fast but not furious: a streamlined selection method for genome-edited cells

2021 ◽  
Vol 4 (6) ◽  
pp. e202101051
Author(s):  
Haribaskar Ramachandran ◽  
Soraia Martins ◽  
Zacharias Kontarakis ◽  
Jean Krutmann ◽  
Andrea Rossi
Keyword(s):  
Cell Sorting ◽  
Genome Engineering ◽  
High Efficiency ◽  
Genetically Modified ◽  
Human Fibroblasts ◽  
Cell Types ◽  
Ease Of Use ◽  
Selection Marker ◽  
Transcription Activator ◽  
Knock Out

In the last decade, transcription activator-like effector nucleases and CRISPR-based genome engineering have revolutionized our approach to biology. Because of their high efficiency and ease of use, the development of custom knock-out and knock-in animal or cell models is now within reach for almost every laboratory. Nonetheless, the generation of genetically modified cells often requires a selection step, usually achieved by antibiotics or fluorescent markers. The choice of the selection marker is based on the available laboratory resources, such as cell types, and parameters such as time and cost should also be taken into consideration. Here, we present a new and fast strategy called magnetic-activated genome-edited cell sorting, to select genetically modified cells based on the ability to magnetically sort surface antigens (i.e., tCD19) present in Cas9-positive cells. By using magnetic-activated genome-edited cell sorting, we successfully generated and isolated genetically modified human-induced pluripotent stem cells, primary human fibroblasts, SH-SY5Y neuroblast-like cells, HaCaT and HEK 293T cells. Our strategy expands the genome editing toolbox by offering a fast, cheap, and an easy to use alternative to the available selection methods.

Sign in / Sign up

Export Citation Format

Share Document