scholarly journals Principles of Genetic Engineering

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 291 ◽  
Author(s):  
Thomas M. Lanigan ◽  
Huira C. Kopera ◽  
Thomas L. Saunders
Keyword(s):  
Homologous Recombination ◽  
Genetic Engineering ◽  
Dna Sequences ◽  
Cultured Cells ◽  
Embryonic Stem ◽  
Zinc Finger Nucleases ◽  
Chromosome Break ◽  
Transcription Activator ◽  
Random Integration ◽  
Dna Insertion

Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.

Role of stem cells in large animal genetic engineering in the TALENs–CRISPR era

2014 ◽  
Vol 26 (1) ◽  
pp. 65 ◽  
Author(s):  
Ki-Eun Park ◽  
Bhanu Prakash V. L. Telugu
Keyword(s):  
Stem Cells ◽  
Genetic Engineering ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Zinc Finger Nucleases ◽  
Large Animal ◽  
Prime Model ◽  
Transcription Activator

The establishment of embryonic stem cells (ESCs) and gene targeting technologies in mice has revolutionised the field of genetics. The relative ease with which genes can be knocked out, and exogenous sequences introduced, has allowed the mouse to become the prime model for deciphering the genetic code. Not surprisingly, the lack of authentic ESCs has hampered the livestock genetics field and has forced animal scientists into adapting alternative technologies for genetic engineering. The recent discovery of the creation of induced pluripotent stem cells (iPSCs) by upregulation of a handful of reprogramming genes has offered renewed enthusiasm to animal geneticists. However, much like ESCs, establishing authentic iPSCs from the domestic animals is still beset with problems, including (but not limited to) the persistent expression of reprogramming genes and the lack of proven potential for differentiation into target cell types both in vitro and in vivo. Site-specific nucleases comprised of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulated interspaced short palindromic repeats (CRISPRs) emerged as powerful genetic tools for precisely editing the genome, usurping the need for ESC-based genetic modifications even in the mouse. In this article, in the aftermath of these powerful genome editing technologies, the role of pluripotent stem cells in livestock genetics is discussed.

Concepts and tools for gene editing

2017 ◽  
Vol 29 (1) ◽  
pp. 1 ◽  
Author(s):  
Santiago Josa ◽  
Davide Seruggia ◽  
Almudena Fernández ◽  
Lluis Montoliu
Keyword(s):  
Dna Sequences ◽  
Gene Editing ◽  
Embryonic Stem ◽  
Zinc Finger Nucleases ◽  
Technological Advancement ◽  
Transcription Activator ◽  
Genetic Modifications ◽  
Associated Proteins ◽  
At Will ◽  
Cas Gene

Gene editing is a relatively recent concept in the molecular biology field. Traditional genetic modifications in animals relied on a classical toolbox that, aside from some technical improvements and additions, remained unchanged for many years. Classical methods involved direct delivery of DNA sequences into embryos or the use of embryonic stem cells for those few species (mice and rats) where it was possible to establish them. For livestock, the advent of somatic cell nuclear transfer platforms provided alternative, but technically challenging, approaches for the genetic alteration of loci at will. However, the entire landscape changed with the appearance of different classes of genome editors, from initial zinc finger nucleases, to transcription activator-like effector nucleases and, most recently, with the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas). Gene editing is currently achieved by CRISPR–Cas-mediated methods, and this technological advancement has boosted our capacity to generate almost any genetically altered animal that can be envisaged.

236 GENE TARGETING USING ZINC FINGER NUCLEASES (ZFN) IN THE PORCINE FIBROBLAST

2012 ◽  
Vol 24 (1) ◽  
pp. 230
Author(s):  
S. Kim ◽  
J. W. Kim ◽  
S. M. Lee ◽  
J. H. Kim ◽  
M. J. Kang
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Zinc Finger ◽  
Marker Gene ◽  
Somatic Cells ◽  
Embryonic Stem ◽  
Domestic Animals ◽  
Zinc Finger Nucleases ◽  
Exogenous Dna ◽  
Targeting Vector

Gene targeting is a genetic technique that utilises homologous recombination between an engineered exogenous DNA fragment and the endogenous genome of an animal. In domestic animals, gene targeting has provided an important tool for producing knockout pigs for the α1,3-galactosyltransferase gene (GGTA1) to use in xenotransplantation. The frequency of homologous recombination is a critical parameter for the success of gene targeting. The efficiency of homologous recombination in somatic cells is lower than that in mouse embryonic stem cells. The application of gene targeting to somatic cells has been limited by its low efficiency. Recently, knockout rat and mouse were generated by introducing nonhomologus end joining (NHE)-mediated deletion or insertion at the target site using zinc-finger nucleases (ZFN). Therefore, the development of effective knockout and knock-in techniques in domestic animals is very important in biomedical research. In this study, we investigated homologous recombination events at the cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene locus using ZFN in porcine primary fibroblast. The CMAH-targeted ZFN plasmid and mRNA were purchased from Sigma-Aldrich (St Louis, MO, USA). Porcine ear fibroblasts cells were obtained from a 10-day-old male Chicago miniature pigs. The fibroblasts were cultured in DMEM containing 15% fetal bovine serum, 1 × nonessential amino acids, 1 × sodium pyruvate, 10–4 M β-mercaptoethanol, 100 unit mL–1 penicillin and 100 μg mL–1 streptomycin. The cells were trypsinized and resuspended at a concentration of 1.25 × 107 cells mL–1 in F10 nutrient mixture. Four hundred microliters of the cell suspension was electroporated in a 4-mm cuvette with 4 pulses of 1 ms duration using 400V capacitive discharges using the CMAH neo targeting vector and ZFN plasmid or RNA. The CMAH neo targeting vector consists of the neomycin resistance gene (neo) as a positive selectable marker gene, 789-bp 5′ arm and 763-bp 3′ arm from exon 8 of CMAH gene. After selection of G-418, PCR analysis was performed using 64 colonies transfected with ZFN plasmid and 48 colonies transfected with ZFN RNA. As a result, 19 positive colonies were identified in colonies transfected with ZFN plasmid and 15 colonies were identified in colonies transfected with ZFN RNA. The targeting efficiency was 29.7 and 31.6% in the colonies transfected with ZFN plasmid and ZFN RNA, respectively. To our knowledge, this study provides the first evidence that the efficiency of gene targeting using ZFN was higher than that of conventional gene targeting in the porcine fibroblast. These cell lines may be used in production of CMAH knockouts for xenotransplantation.

scholarly journals THE JOURNEY OF CRISPR-CAS9 FROM BACTERIAL DEFENSE MECHANISM TO A GENE EDITING TOOL IN BOTH ANIMALS AND PLANTS

2020 ◽  
Vol 2020 (1) ◽  
Author(s):  
T Tahir ◽  
Q Ali ◽  
MS Rashid ◽  
A Malik
Keyword(s):  
Immune System ◽  
Genetic Engineering ◽  
Success Rate ◽  
Genome Editing ◽  
Zinc Finger ◽  
Gene Editing ◽  
Defense Mechanism ◽  
Zinc Finger Nucleases ◽  
Transcription Activator

Today we can use multiple of endonucleases for genome editing which has become very important and used in number of applications. We use sequence specific molecular scissors out of which, most important are mega nucleases, zinc finger nucleases, TALENS (Transcription Activator Like-Effector Nucleases) and CRISPR-Cas9 which is currently the most famous due to a number of reasons, they are cheap, easy to build, very specific in nature and their success rate in plants and animals is also high. Who knew that one day these CRISPR discovered as a part of immune system of bacteria will be this much worthwhile in the field of genetic engineering? This review interprets the science behind their mechanism and how several advancements were made with the passage of time to make them more efficient for the assigned job.

scholarly journals Gene editing and CRISPR in the clinic: current and future perspectives

2020 ◽  
Vol 40 (4) ◽  
Author(s):  
Matthew P. Hirakawa ◽  
Raga Krishnakumar ◽  
Jerilyn A. Timlin ◽  
James P. Carney ◽  
Kimberly S. Butler
Keyword(s):  
Clinical Trials ◽  
Genome Editing ◽  
Dna Sequences ◽  
Gene Editing ◽  
Ex Vivo ◽  
Scientific Basis ◽  
Current Status ◽  
Zinc Finger Nucleases ◽  
Transcription Activator

Abstract Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.

scholarly journals Empowering of reproductive health of farm animals through genome editing technology

2021 ◽  
Vol 2 ◽  
pp. 4
Author(s):  
Seema Dua ◽  
Kamlesh Kumari Bajwa ◽  
Atul Prashar ◽  
Sonu Bansal ◽  
Madhuri Beniwal ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dna Sequences ◽  
Nuclear Dna ◽  
Genetic Material ◽  
Underlying Mechanism ◽  
Farm Animals ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Minimal Impact ◽  
Livestock Productivity

To cater the exponential growth of human population, need to improve food production and quality through modern biotechnology with limited recourses in a way that has minimal impact on the environment. The selective breeding and genomic selection have attended the momentum gain in livestock productivity. Recent advancement in genome-editing technologies offers exciting prospects for the production of healthy and prolific livestock. Genome editing involves altering genetic material by manipulation, addition, or removal of certain deoxyribonucleic acid (DNA) sequences at a specific locus in a way that does not occur naturally. The major genome editors are zinc finger nucleases, transcription-activator-like endonucleases, and clustered regularly interspaced short palindromic repeats associated protein nine systems which are proficient of cutting the nuclear DNA precisely at a predetermined position. This review provides an update on the use of genome editing systems to modify the genes related to reproduction of farm animal vis-à-vis human, update knowledge on the underlying mechanism and discusses new opportunities to produce genetically modified farm animals.

Modern biotechnology tools and biosecurity in the Middle East

2021 ◽  
Vol 16 (11) ◽  
pp. 155-163
Author(s):  
Alsubki Roua
Keyword(s):  
Middle East ◽  
Genetic Engineering ◽  
Genome Editing ◽  
Middle Eastern ◽  
Strand Break ◽  
Biological Weapons ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Human Genomes ◽  
Modern Biotechnology

The global health system is under a constant threat from microbial outbreaks. The innovation in genetic engineering has created an existential threat to national, regional and international security. This threat, that can edit microbial or human genomes, requires global attention. In the current review, a comprehensive literature search was conducted using PubMed, SCOPUS and Google Scholar to identify literature discussing modern biotechnology tools as well as relevance to biosafety in the Middle east region. This review was undertaken to provide an overview of biological threats due to advancements in genetic engineering, making it possible to insert or delete specific genes to increase the virulence of particular microbes. These pathogens or other toxic factors can be multiplied by technology, creating new biological weapons. Genome editing technologies including meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector (TALE)-nucleases (TALENs) and recently discovered clustered regularly interspaced short palindromic repeats (CRISPR/Cas) induce a double strand break at specific DNA target site. Genome editing technologies lead to an irreversible and permanent alteration of the genetic code and therefore, can inevitably result in security risks. Vulnerabilities in Middle Eastern laboratories raise the prospect of high levels of pathogenic microbes potentially creating a weakness in the diagnosis and monitoring of epidemics. Furthermore, the lack of regional legislation to regulate biosafety and biosecurity may lead to biological threat at the regional level.

scholarly journals Application of genome-editing systems to enhance available pig resources for agriculture and biomedicine

2020 ◽  
Vol 32 (2) ◽  
pp. 40
Author(s):  
Kiho Lee ◽  
Kayla Farrell ◽  
Kyungjun Uh
Keyword(s):  
Genetic Engineering ◽  
Genome Editing ◽  
Direct Injection ◽  
Somatic Cells ◽  
Cost Effective ◽  
Genetically Engineered ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Developmental Defects ◽  
Site Specific

Traditionally, genetic engineering in the pig was a challenging task. Genetic engineering of somatic cells followed by somatic cell nuclear transfer (SCNT) could produce genetically engineered (GE) pigs carrying site-specific modifications. However, due to difficulties in engineering the genome of somatic cells and developmental defects associated with SCNT, a limited number of GE pig models were reported. Recent developments in genome-editing tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system, have markedly changed the effort and time required to produce GE pig models. The frequency of genetic engineering in somatic cells is now practical. In addition, SCNT is no longer essential in producing GE pigs carrying site-specific modifications, because direct injection of genome-editing systems into developing embryos introduces targeted modifications. To date, the CRISPR/Cas9 system is the most convenient, cost-effective, timely and commonly used genome-editing technology. Several applicable biomedical and agricultural pig models have been generated using the CRISPR/Cas9 system. Although the efficiency of genetic engineering has been markedly enhanced with the use of genome-editing systems, improvements are still needed to optimally use the emerging technology. Current and future advances in genome-editing strategies will have a monumental effect on pig models used in agriculture and biomedicine.

Evaluating and Enhancing Target Specificity of Gene-Editing Nucleases and Deaminases

2019 ◽  
Vol 88 (1) ◽  
pp. 191-220 ◽  
Author(s):  
Daesik Kim ◽  
Kevin Luk ◽  
Scot A. Wolfe ◽  
Jin-Soo Kim
Keyword(s):  
Dna Sequences ◽  
Gene Editing ◽  
Genomic Rearrangements ◽  
Zinc Finger Nucleases ◽  
Collateral Damage ◽  
Transcription Activator ◽  
Genome Wide ◽  
Target Sites ◽  
Programmable Nucleases ◽  
Target Activity

Programmable nucleases and deaminases, which include zinc-finger nucleases, transcription activator-like effector nucleases, CRISPR RNA-guided nucleases, and RNA-guided base editors, are now widely employed for the targeted modification of genomes in cells and organisms. These gene-editing tools hold tremendous promise for therapeutic applications. Importantly, these nucleases and deaminases may display off-target activity through the recognition of near-cognate DNA sequences to their target sites, resulting in collateral damage to the genome in the form of local mutagenesis or genomic rearrangements. For therapeutic genome-editing applications with these classes of programmable enzymes, it is essential to measure and limit genome-wide off-target activity. Herein, we discuss the key determinants of off-target activity for these systems. We describe various cell-based and cell-free methods for identifying genome-wide off-target sites and diverse strategies that have been developed for reducing the off-target activity of programmable gene-editing enzymes.

scholarly journals Can pseudocomplementary peptide nucleic acid nucleases (pcPNANs) be a new tool for genetic engineering?

2014 ◽  
Author(s):  
Penghui Shi
Keyword(s):  
Nucleic Acid ◽  
Genetic Engineering ◽  
Peptide Nucleic Acid ◽  
Cleavage Site ◽  
New Technologies ◽  
Selection Procedure ◽  
Zinc Finger Nucleases ◽  
Biological Research ◽  
Target Sequence ◽  
Transcription Activator

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of biological research. Although these technologies have begun to enable targeted genome modifications, there remains a need for new technologies that are scalable, affordable, and easy to engineer. In this paper, we propose a new tool for genetic engineering, the pseudocomplementary peptide nucleic acid nucleases (pcPNANs), which are composed of a pseudocomplementary PNA (pcPNA) specific for a DNA target sequence, a FokI nuclease cleavage domain and a nuclear localization signal. pcPNANs may induce targeted DNA double-strand breaks that activate DNA damage response pathways and enable custom alterations. Their cleavage-site is determined by simple Watson-Crick rule, and thus pcPNANs for aimed cleavage of genomes can be straightforwardly designed and synthesized without any selection procedure. Accordingly, the cleavage-site and site-specificity are freely chosen by changing the sequences and the lengths of pcPNA strands. We believe that the potentiality of pcPNAN as a new tool for genetic engineering will be confirmed in the future.

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