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.

Homing endonucleases: DNA scissors on a mission

Genome ◽  
2012 ◽  
Vol 55 (8) ◽  
pp. 553-569 ◽  
Author(s):  
Mohamed Hafez ◽  
Georg Hausner
Keyword(s):  
Zinc Finger Nucleases ◽  
Homing Endonucleases ◽  
Gene Repair ◽  
Transcription Activator ◽  
Group I ◽  
Gene Knockouts ◽  
Or Gene ◽  
Triplex Forming Oligonucleotide ◽  
Self Splicing ◽  
Specific Sequences

Buried within the genomes of many microorganisms are genetic elements that encode rare-cutting homing endonucleases that assist in the mobility of the elements that encode them, such as the self-splicing group I and II introns and in some cases inteins. There are several different families of homing endonucleases and their ability to initiate and target specific sequences for lateral transfers makes them attractive reagents for gene targeting. Homing endonucleases have been applied in promoting DNA modification or genome editing such as gene repair or “gene knockouts”. This review examines the categories of homing endonucleases that have been described so far and their possible applications to biotechnology. Strategies to engineer homing endonucleases to alter target site specificities will also be addressed. Alternatives to homing endonucleases such as zinc finger nucleases, transcription activator-like effector nucleases, triplex forming oligonucleotide nucleases, and targetrons are also briefly discussed.

scholarly journals Update of Regulatory Options of New Breeding Techniques and Biosafety Approaches among Selected Countries: A Review

2020 ◽  
pp. 18-35
Author(s):  
Silas Obukosia ◽  
Olalekan Akinbo ◽  
Woldeyesus Sinebo ◽  
Moussa Savadogo ◽  
Samuel Timpo ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genetically Modified Organisms ◽  
Zinc Finger Nucleases ◽  
Intermediate Step ◽  
Homing Endonucleases ◽  
Transcription Activator ◽  
Associated Proteins ◽  
Genomic Editing ◽  
New Breeding Techniques ◽  
Breeding Techniques

A new set of breeding techniques, referred to as New Breeding Techniques developed in the last two decades have potential for enhancing improved productivity in crop and animal breeding globally. These include site directed nucleases based genomic editing procedures-CRISPR and Cas associated proteins, Zinc Finger Nucleases, Meganucleases/Homing Endonucleases and Transcription- Activator Like-Effector Nucleases for genome editing and other technologies including- Oligonucleotide-Directed Mutagenesis, Cisgenesis and intragenesis, RNA-Dependent DNA methylation; Transgrafting, Agroinfiltration, Reverse breeding. There are ongoing global debates on whether the processes of and products emerging from these technologies should be regulated as genetically modified organisms or approved as conventional products. Decisions on whether to regulate as GMOs are based both on understanding of the molecular basis of their development and if the GMO intermediate step was used. For example- cisgenesis, can be developed using Agrobacterium tumefaciens methods of transformation, a process used by GMO but if the selection is properly conducted the intermediate GMO elements will be eliminated and the final product will be identical to the conventionally developed crops. Others like Site Directed Nuclease 3 are regulated as GMOs in countries such as United State of America, Canada, European Union, Argentina, Australia. Progress in genome editing research, testing of genome edited bacterial blight resistant rice, development of Guidelines for regulating new breeding techniques or genome editing in Africa is also covered with special reference to South Africa, Kenya and Nigeria. Science- and evidence-based approach to regulation of new breeding techniques among regulators and policy makers should be strongly supported.

scholarly journals CRISPR-Cas Technology: Emerging Applications in Clinical Microbiology and Infectious Diseases

2021 ◽  
Vol 14 (11) ◽  
pp. 1171
Author(s):  
Sahar Serajian ◽  
Ehsan Ahmadpour ◽  
Sonia M. Rodrigues Oliveira ◽  
Maria de Lourdes Pereira ◽  
Siamak Heidarzadeh
Keyword(s):  
Infectious Diseases ◽  
Clinical Microbiology ◽  
Gene Editing ◽  
Zinc Finger Nucleases ◽  
Homing Endonucleases ◽  
Transcription Activator ◽  
Practical Applications ◽  
Novel Technologies ◽  
Resistant Strains ◽  
Drug Resistant Strains

Through the years, many promising tools for gene editing have been developed including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-associated protein 9 (Cas9), and homing endonucleases (HEs). These novel technologies are now leading new scientific advancements and practical applications at an inimitable speed. While most work has been performed in eukaryotes, CRISPR systems also enable tools to understand and engineer bacteria. The increase in the number of multi-drug resistant strains highlights a necessity for more innovative approaches to the diagnosis and treatment of infections. CRISPR has given scientists a glimmer of hope in this area that can provide a novel tool to fight against antimicrobial resistance. This system can provide useful information about the functions of genes and aid us to find potential targets for antimicrobials. This paper discusses the emerging use of CRISPR-Cas systems in the fields of clinical microbiology and infectious diseases with a particular emphasis on future prospects.

31 PRODUCTION EFFICIENCY OF GENE KNOCKOUT PIGS USING GENOME EDITING AND SOMATIC CELL CLONING

2015 ◽  
Vol 27 (1) ◽  
pp. 108
Author(s):  
H. Matsunari ◽  
M. Watanabe ◽  
K. Nakano ◽  
A. Uchikura ◽  
Y. Asano ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Genome Editing ◽  
Production Efficiency ◽  
Gene Knockout ◽  
Genetically Modified ◽  
Zinc Finger Nucleases ◽  
International Institute ◽  
Induced Mutations ◽  
Transcription Activator

Genome editing technologies have been used as a powerful strategy for the generation of genetically modified pigs. We previously developed genetically modified clone pigs with organogenesis-disabled phenotypes, as well as pigs exhibiting diseases with similar features to those of humans. Here, we report the production efficiency of various gene knockout cloned pigs from somatic cells that were genetically modified using zinc finger nucleases (ZFN) or transcription activator-like effector nucleases (TALEN). The ZFN- or TALEN-encoding mRNAs, which targeted 7 autosomal or X-linked genes, were introduced into porcine fetal fibroblast cells using electroporation. Clonal cell populations carrying induced mutations were selected after limiting dilution. The targeted portion of the genes was amplified using PCR, followed by sequencing and mutation analysis. Among the collected knockout cell colonies, cells showing good proliferation and morphology were selected and used for somatic cell nuclear transfer (SCNT). In vitro-matured oocytes were obtained from porcine cumulus-oocyte complexes cultured in NCSU23-based medium and were used to obtain recipient oocytes for SCNT after enucleation. SCNT was performed as reported previously (Matsunari et al. 2008). The cloned embryos were cultured for 7 days in porcine zygote medium (PZM)-5 to assess their developmental ability. Cloned embryos were transplanted into the oviduct or uterus of oestrus-synchronized recipient gilts to evaluate their competence to develop to fetuses or piglets. Cloned embryos reconstructed with 7 types of knockout cells showed equal development to blastocysts compared with those derived from the wild-type cells (54.5–83.3% v. 60.7%). Our data (Table 1) demonstrated that the reconstructed embryos derived from knockout cells could efficiently give rise to cloned offspring regardless of the type of genome editing methodology (i.e. ZFN or TALEN). Table 1.Production efficiency of gene knockout cloned pigs using genome editing This study was supported by JST, ERATO, the Nakauchi Stem Cell and Organ Regeneration Project, JST, CREST, Meiji University International Institute for Bio-Resource Research (MUIIBR), and JSPS KAKENHI Grant Number 26870630.

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 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 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.

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