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Trends in Biotechnology Jul 2013Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are redefining the boundaries of... (Review)
Review
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. These chimeric nucleases are composed of programmable, sequence-specific DNA-binding modules linked to a nonspecific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific genomic locations. Here, we review achievements made possible by site-specific nuclease technologies and discuss applications of these reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future prospects for the field, including the emergence of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases.
Topics: Biological Therapy; Clustered Regularly Interspaced Short Palindromic Repeats; Deoxyribonucleases; Genetic Engineering; Molecular Biology; Recombinant Proteins; Zinc Fingers
PubMed: 23664777
DOI: 10.1016/j.tibtech.2013.04.004 -
Genes Mar 2020Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination... (Review)
Review
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.
Topics: Animals; CRISPR-Cas Systems; Gene Targeting; Gene Transfer Techniques; Genetic Engineering; Humans
PubMed: 32164255
DOI: 10.3390/genes11030291 -
Science (New York, N.Y.) Aug 2018The diversity, modularity, and efficacy of CRISPR-Cas systems are driving a biotechnological revolution. RNA-guided Cas enzymes have been adopted as tools to manipulate... (Review)
Review
The diversity, modularity, and efficacy of CRISPR-Cas systems are driving a biotechnological revolution. RNA-guided Cas enzymes have been adopted as tools to manipulate the genomes of cultured cells, animals, and plants, accelerating the pace of fundamental research and enabling clinical and agricultural breakthroughs. We describe the basic mechanisms that set the CRISPR-Cas toolkit apart from other programmable gene-editing technologies, highlighting the diverse and naturally evolved systems now functionalized as biotechnologies. We discuss the rapidly evolving landscape of CRISPR-Cas applications, from gene editing to transcriptional regulation, imaging, and diagnostics. Continuing functional dissection and an expanding landscape of applications position CRISPR-Cas tools at the cutting edge of nucleic acid manipulation that is rewriting biology.
Topics: Animals; Bacterial Proteins; CRISPR-Associated Protein 9; CRISPR-Cas Systems; DNA; Endonucleases; Gene Editing; Genetic Engineering; Humans; Molecular Imaging; Plants; RNA; RNA, Guide, CRISPR-Cas Systems; Transcription, Genetic
PubMed: 30166482
DOI: 10.1126/science.aat5011 -
Transgenic Research Jun 2022To bridge the gap between organ demand and supply, xenotransplantation has long been considered as a realistic option for end-stage organ failure. Early this year this... (Review)
Review
To bridge the gap between organ demand and supply, xenotransplantation has long been considered as a realistic option for end-stage organ failure. Early this year this promise became reality for David Bennett Sr., the first patient whose own failing heart was replaced with a xeno-pig heart. To get here has been a rollercoaster ride of physiological hurdles seemingly impossible to overcome, technological breakthroughs and ethical and safety concerns. It started in 1984, with Stephanie Fae Beauclair, also known as baby Fae, receiving a baboon heart, which allowed her to survive for another 30 days. For ethical reasons primate work was soon abandoned in favour of the pig. But increased phylogenetic distance also brought with it an increased immunological incompatibility. It has been the development of ever more sophisticated genetic engineering tools, which brought down the physiological barriers, enabled humanisation of porcine organs and helped addressing safety concerns. This renewed the confidence in xenotransplantation, brought new funding opportunities and resulted finally in the first in human trial.
Topics: Animals; Genetic Engineering; Humans; Phylogeny; Primates; Swine; Transplantation, Heterologous
PubMed: 35545691
DOI: 10.1007/s11248-022-00306-w -
Oncotarget Aug 2016Cancer is caused by a series of alterations in genome and epigenome mostly resulting in activation of oncogenes or inactivation of cancer suppressor genes. Genetic... (Review)
Review
Cancer is caused by a series of alterations in genome and epigenome mostly resulting in activation of oncogenes or inactivation of cancer suppressor genes. Genetic engineering has become pivotal in the treatment of cancer and other genetic diseases, especially the formerly-niche use of clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9. In defining its superior use, we have followed the recent advances that have been made in producing CRISPR/Cas9 as a therapy of choice. We also provide important genetic mutations where CRISPRs can be repurposed to create adaptive immunity to fight carcinomas and edit genetic mutations causing it. Meanwhile, challenges to CRISPR technology are also discussed with emphasis on ability of pathogens to evolve against CRISPRs. We follow the recent developments on the function of CRISPRs with different carriers which can efficiently deliver it to target cells; furthermore, analogous technologies are also discussed along CRISPRs, including zinc-finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs). Moreover, progress in clinical applications of CRISPR therapeutics is reviewed; in effect, patients can have lower morbidity and/or mortality from the therapeutic method with least possible side-effects.
Topics: Clustered Regularly Interspaced Short Palindromic Repeats; Genetic Diseases, Inborn; Genetic Engineering; Genetic Therapy; Humans; Neoplasms
PubMed: 27250031
DOI: 10.18632/oncotarget.9646 -
Molekuliarnaia Biologiia 2016"Molecular cloning" meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful... (Review)
Review
"Molecular cloning" meaning creation of recombinant DNA molecules has impelled advancement throughout life sciences. DNA manipulation has become easy due to powerful tools showing exponential growth in applications and sophistication of recombinant DNA technology. Cloning genes has become simple what led to an explosion in the understanding of gene function by seamlessly stitching together multiple DNA fragments or by the use of swappable gene cassettes, maximizing swiftness and litheness. A novel archetype might materialize in the near future with synthetic biology techniques that will facilitate quicker assembly and iteration of DNA clones, accelerating the progress of gene therapy vectors, recombinant protein production processes and new vaccines by in vitro chemical synthesis of any in silico-specified DNA construct. The advent of innovative cloning techniques has opened the door to more refined applications such as identification and mapping of epigenetic modifications and high-throughput assembly of combinatorial libraries. In this review, we will examine the major breakthroughs in cloning techniques and their applications in various areas of biological research that have evolved mainly due to easy construction of novel expression systems.
Topics: Cloning, Molecular; DNA, Recombinant; Genetic Engineering; Genetic Vectors
PubMed: 27028806
DOI: 10.7868/S0026898416010134 -
Cell Jan 2016Three years ago, scientists reported that CRISPR technology can enable precise and efficient genome editing in living eukaryotic cells. Since then, the method has taken... (Review)
Review
Three years ago, scientists reported that CRISPR technology can enable precise and efficient genome editing in living eukaryotic cells. Since then, the method has taken the scientific community by storm, with thousands of labs using it for applications from biomedicine to agriculture. Yet, the preceding 20-year journey--the discovery of a strange microbial repeat sequence; its recognition as an adaptive immune system; its biological characterization; and its repurposing for genome engineering--remains little known. This Perspective aims to fill in this backstory--the history of ideas and the stories of pioneers--and draw lessons about the remarkable ecosystem underlying scientific discovery.
Topics: Adaptive Immunity; Animals; Archaea; Bacteria; Biomedical Research; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Genetic Engineering; Haloferax mediterranei; History, 20th Century; History, 21st Century; Humans; Laboratory Personnel
PubMed: 26771483
DOI: 10.1016/j.cell.2015.12.041 -
Nature Microbiology Dec 2022Jumbo phages such as Pseudomonas aeruginosa ФKZ have potential as antimicrobials and as a model for uncovering basic phage biology. Both pursuits are currently limited...
Jumbo phages such as Pseudomonas aeruginosa ФKZ have potential as antimicrobials and as a model for uncovering basic phage biology. Both pursuits are currently limited by a lack of genetic engineering tools due to a proteinaceous 'phage nucleus' structure that protects from DNA-targeting CRISPR-Cas tools. To provide reverse-genetics tools for DNA jumbo phages from this family, we combined homologous recombination with an RNA-targeting CRISPR-Cas13a enzyme and used an anti-CRISPR gene (acrVIA1) as a selectable marker. We showed that this process can insert foreign genes, delete genes and add fluorescent tags to genes in the ФKZ genome. Fluorescent tagging of endogenous gp93 revealed that it is ejected with the phage DNA while deletion of the tubulin-like protein PhuZ surprisingly had only a modest impact on phage burst size. Editing of two other phages that resist DNA-targeting CRISPR-Cas systems was also achieved. RNA-targeting Cas13a holds great promise for becoming a universal genetic editing tool for intractable phages, enabling the systematic study of phage genes of unknown function.
Topics: Bacteriophages; CRISPR-Cas Systems; Gene Editing; Genetic Engineering; RNA
PubMed: 36316452
DOI: 10.1038/s41564-022-01243-4 -
International Journal of Medical... 2018Results obtained from completed and on-going clinical studies indicate huge therapeutic potential of stem cell-based therapy in the treatment of degenerative, autoimmune... (Review)
Review
Results obtained from completed and on-going clinical studies indicate huge therapeutic potential of stem cell-based therapy in the treatment of degenerative, autoimmune and genetic disorders. However, clinical application of stem cells raises numerous ethical and safety concerns. In this review, we provide an overview of the most important ethical issues in stem cell therapy, as a contribution to the controversial debate about their clinical usage in regenerative and transplantation medicine. We describe ethical challenges regarding human embryonic stem cell (hESC) research, emphasizing that ethical dilemma involving the destruction of a human embryo is a major factor that may have limited the development of hESC-based clinical therapies. With previous derivation of induced pluripotent stem cells (iPSCs) this problem has been overcome, however current perspectives regarding clinical translation of iPSCs still remain. Unlimited differentiation potential of iPSCs which can be used in human reproductive cloning, as a risk for generation of genetically engineered human embryos and human-animal chimeras, is major ethical issue, while undesired differentiation and malignant transformation are major safety issues. Although clinical application of mesenchymal stem cells (MSCs) has shown beneficial effects in the therapy of autoimmune and chronic inflammatory diseases, the ability to promote tumor growth and metastasis and overestimated therapeutic potential of MSCs still provide concerns for the field of regenerative medicine. This review offers stem cell scientists, clinicians and patient's useful information and could be used as a starting point for more in-depth analysis of ethical and safety issues related to clinical application of stem cells.
Topics: Animals; Biomedical Research; Cell Culture Techniques; Cell Differentiation; Cell Transplantation; Chimera; Embryo, Mammalian; Genetic Engineering; Genetic Therapy; Human Embryonic Stem Cells; Humans; Induced Pluripotent Stem Cells; Mesenchymal Stem Cell Transplantation; Regenerative Medicine
PubMed: 29333086
DOI: 10.7150/ijms.21666 -
Molecular Therapy : the Journal of the... Apr 2007Since the turn of the nineteenth century, when their existence was first recognized, viruses have attracted considerable interest as possible agents of tumor... (Review)
Review
Since the turn of the nineteenth century, when their existence was first recognized, viruses have attracted considerable interest as possible agents of tumor destruction. Early case reports emphasized regression of cancers during naturally acquired virus infections, providing the basis for clinical trials where body fluids containing human or animal viruses were used to transmit infections to cancer patients. Most often the viruses were arrested by the host immune system and failed to impact tumor growth, but sometimes, in immunosuppressed patients, infection persisted and tumors regressed, although morbidity as a result of the infection of normal tissues was unacceptable. With the advent of rodent models and new methods for virus propagation, there were numerous attempts through the 1950s and 1960s to force the evolution of viruses with greater tumor specificity, but success was limited and many researchers abandoned the field. Technology employing reverse genetics later brought about a renewal of interest in virotherapy that allowed the generation of more potent, tumor-specific oncolytics. Here, examination of early oncolytic virotherapy before genetic engineering serves to highlight tremendous advances, yet also hints at ways to penetrate host immune defenses, a significant remaining challenge in modern virotherapy research.
Topics: Animals; Genetic Engineering; History, 19th Century; History, 20th Century; History, 21st Century; Humans; Neoplasms; Oncolytic Virotherapy; Oncolytic Viruses; Virology
PubMed: 17299401
DOI: 10.1038/sj.mt.6300108