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Prospects in Transgenic Technology 2020
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Prospects in Transgenic Technology 2020

A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Technologies and Resources for Genetics".

Deadline for manuscript submissions: closed (29 February 2020) | Viewed by 39382

Special Issue Editors

Dr. Wojtek Auerbach

E-Mail Website
Guest Editor
1. Embryonic Stem Cells Technologies, Regeneron Pharmaceuticals, Tarrytown, NY 10591, USA
2. President International Society for Transgenic Technologies
Interests: gene editing technologies; animal models of human genetic diseases; embryonic stem cells
Dr. Jan Parker-Thornburg

E-Mail Website
Guest Editor
Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
Interests: genetic engineering of animals; CRISPR/Cas9 technology; assisted reproductive technologies; mouse modeling

Special Issue Information

Dear colleagues,

With the advent of gene editing technologies, the ability to manipulate genomes has become amenable to nearly all species. Gene modification protocols that lead to new ways of generating genetically engineered model systems are being developed at an exponential pace, with new techniques reported almost daily. With this technology, we may be able to easily generate models of diseases affecting both humans and other animals, increase food production worldwide, and perform gene therapy to alleviate disease, among the many possibilities. However, success should be based on an understanding of when to use newer vs. established technologies. Scientists should be aware of both expected and unexpected consequences of the particular method chosen for gene modification. Analyses and assessments that take into account such consequences are critical for acceptance by society, as is an ethical approach to determining what uses such technology can have.

This Special Issue of Genes welcomes original research manuscripts and review papers that address the science and ethics of gene modification. Topics of interest include, but are not limited to:

  • New methods and protocols using gene editing technology;
  • Research describing previously unforeseen caveats with gene modification;
  • Gene modification research in unconventional animal models or exotic species;
  • The use of gene editing to enhance agrarian species for food production;
  • Gene editing used for gene therapy;
  • Ethical considerations associated with gene modification, particularly in humans;
  • How gene editing affects the 3Rs of animal use;
  • Methods of public outreach and education used for gene modification acceptance.

Dr. Wojtek Auerbach
Dr. Jan Parker-Thornburg
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Genes is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Gene editing technologies
  • Gene modification
  • Genome engineering
  • Transgenic technology
  • Models of disease
  • Food production
  • Gene therapy
  • CRISPR-Cas9
  • Nucleases
  • Ethics

Published Papers (4 papers)

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Research

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25 pages, 5282 KiB  
Article
Rapid Evaluation of CRISPR Guides and Donors for Engineering Mice
by Elena McBeath, Jan Parker-Thornburg, Yuka Fujii, Neeraj Aryal, Chad Smith, Marie-Claude Hofmann, Jun-ichi Abe and Keigi Fujiwara
Genes 2020, 11(6), 628; https://doi.org/10.3390/genes11060628 - 08 Jun 2020
Cited by 5 | Viewed by 5811
Abstract
Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) [...] Read more.
Although the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) technique has dramatically lowered the cost and increased the speed of generating genetically engineered mice, success depends on using guide RNAs and donor DNAs which direct efficient knock-out (KO) or knock-in (KI). By Sanger sequencing DNA from blastocysts previously injected with the same CRISPR components intended to produce the engineered mice, one can test the effectiveness of different guide RNAs and donor DNAs. We describe in detail here a simple, rapid (three days), inexpensive protocol, for amplifying DNA from blastocysts to determine the results of CRISPR point mutation KIs. Using it, we show that (1) the rate of KI seen in blastocysts is similar to that seen in mice for a given guide RNA/donor DNA pair, (2) a donor complementary to the variable portion of a guide integrated in a more all-or-none fashion, (3) donor DNAs can be used simultaneously to integrate two different mutations into the same locus, and (4) by placing silent mutations about every 6 to 10 bp between the Cas9 cut site and the desired mutation(s), the desired mutation(s) can be incorporated into genomic DNA over 30 bp away from the cut at the same high efficiency as close to the cut. Full article
(This article belongs to the Special Issue Prospects in Transgenic Technology 2020)
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Review

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21 pages, 547 KiB  
Review
Principles of Genetic Engineering
by Thomas M. Lanigan, Huira C. Kopera and Thomas L. Saunders
Genes 2020, 11(3), 291; https://doi.org/10.3390/genes11030291 - 10 Mar 2020
Cited by 42 | Viewed by 20463
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Prospects in Transgenic Technology 2020)
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17 pages, 1381 KiB  
Review
Embryo-Based Large Fragment Knock-in in Mammals: Why, How and What’s Next
by Steven Erwood and Bin Gu
Genes 2020, 11(2), 140; https://doi.org/10.3390/genes11020140 - 29 Jan 2020
Cited by 8 | Viewed by 4149
Abstract
Endonuclease-mediated genome editing technologies, most notably CRISPR/Cas9, have revolutionized animal genetics by allowing for precise genome editing directly through embryo manipulations. As endonuclease-mediated model generation became commonplace, large fragment knock-in remained one of the most challenging types of genetic modification. Due to their [...] Read more.
Endonuclease-mediated genome editing technologies, most notably CRISPR/Cas9, have revolutionized animal genetics by allowing for precise genome editing directly through embryo manipulations. As endonuclease-mediated model generation became commonplace, large fragment knock-in remained one of the most challenging types of genetic modification. Due to their unique value in biological and biomedical research, however, a diverse range of technological innovations have been developed to achieve efficient large fragment knock-in in mammalian animal model generation, with a particular focus on mice. Here, we first discuss some examples that illustrate the importance of large fragment knock-in animal models and then detail a subset of the recent technological advancements that have allowed for efficient large fragment knock-in. Finally, we envision the future development of even larger fragment knock-ins performed in even larger animal models, the next step in expanding the potential of large fragment knock-in in animal models. Full article
(This article belongs to the Special Issue Prospects in Transgenic Technology 2020)
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Other

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14 pages, 1083 KiB  
Perspective
Rewriting Human History and Empowering Indigenous Communities with Genome Editing Tools
by Keolu Fox, Kartik Lakshmi Rallapalli and Alexis C. Komor
Genes 2020, 11(1), 88; https://doi.org/10.3390/genes11010088 - 12 Jan 2020
Cited by 9 | Viewed by 8093
Abstract
Appropriate empirical-based evidence and detailed theoretical considerations should be used for evolutionary explanations of phenotypic variation observed in the field of human population genetics (especially Indigenous populations). Investigators within the population genetics community frequently overlook the importance of these criteria when associating observed [...] Read more.
Appropriate empirical-based evidence and detailed theoretical considerations should be used for evolutionary explanations of phenotypic variation observed in the field of human population genetics (especially Indigenous populations). Investigators within the population genetics community frequently overlook the importance of these criteria when associating observed phenotypic variation with evolutionary explanations. A functional investigation of population-specific variation using cutting-edge genome editing tools has the potential to empower the population genetics community by holding “just-so” evolutionary explanations accountable. Here, we detail currently available precision genome editing tools and methods, with a particular emphasis on base editing, that can be applied to functionally investigate population-specific point mutations. We use the recent identification of thrifty mutations in the CREBRF gene as an example of the current dire need for an alliance between the fields of population genetics and genome editing. Full article
(This article belongs to the Special Issue Prospects in Transgenic Technology 2020)
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