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      Bioethical issues in genome editing by CRISPR-Cas9 technology

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          Abstract

          Genome editing technologies have led to fundamental changes in genetic science. Among them, CRISPR-Cas9 technology particularly stands out due to its advantages such as easy handling, high accuracy, and low cost. It has made a quick introduction in fields related to humans, animals, and the environment, while raising difficult questions, applications, concerns, and bioethical issues to be discussed. Most concerns stem from the use of CRISPR-Cas9 to genetically alter human germline cells and embryos (called germline genome editing). Germline genome editing leads to serial bioethical issues, such as the occurrence of undesirable changes in the genome, from whom and how informed consent is obtained, and the breeding of the human species (eugenics). However, the bioethical issues that CRISPR-Cas9 technology could cause in the environment, agriculture and livestock should also not be forgotten. In order for CRISPR-Cas9 to be used safely in all areas and to solve potential issues, worldwide legislation should be prepared, taking into account the opinions of both life and social scientists, policy makers, and all other stakeholders of the sectors, and CRISPR-Cas9 applications should be implemented according to such legislations. However, these controls should not restrict scientific freedom. Here, various applications of CRISPR-Cas9 technology, especially in medicine and agriculture, are described and ethical issues related to genome editing using CRISPR-Cas9 technology are discussed. The social and bioethical concerns in relation to human beings, other organisms, and the environment are addressed.

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          Most cited references71

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          CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.

          Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate of Staphylococcus epidermidis harbors a CRISPR spacer that matches the nickase gene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation in S. epidermidis. Insertion of a self-splicing intron into nickase blocks interference despite the reconstitution of the target sequence in the spliced mRNA, which indicates that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.
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            In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9.

            Probing gene function in the mammalian brain can be greatly assisted with methods to manipulate the genome of neurons in vivo. The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) can be used to edit single or multiple genes in replicating eukaryotic cells, resulting in frame-shifting insertion/deletion (indel) mutations and subsequent protein depletion. Here, we delivered SpCas9 and guide RNAs using adeno-associated viral (AAV) vectors to target single (Mecp2) as well as multiple genes (Dnmt1, Dnmt3a and Dnmt3b) in the adult mouse brain in vivo. We characterized the effects of genome modifications in postmitotic neurons using biochemical, genetic, electrophysiological and behavioral readouts. Our results demonstrate that AAV-mediated SpCas9 genome editing can enable reverse genetic studies of gene function in the brain.
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              Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells

              DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate their roles and genomic targets. Disruption of DNMT3A or DNMT3B individually, as well as of both enzymes in tandem, creates viable, pluripotent cell lines with distinct effects on their DNA methylation landscape as assessed by whole-genome bisulfite sequencing. Surprisingly, in contrast to mouse, deletion of DNMT1 resulted in rapid cell death in human ESCs. To overcome the immediate lethality, we generated a doxycycline (DOX) responsive tTA-DNMT1* rescue line and readily obtained homozygous DNMT1 mutant lines. However, DOX-mediated repression of the exogenous DNMT1* initiates rapid, global loss of DNA methylation, followed by extensive cell death. Our data provide a comprehensive characterization of DNMT mutant ESCs, including single base genome-wide maps of their targets.
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                Author and article information

                Journal
                Turk J Biol
                Turk. J. Biol
                Turkish Journal of Biology
                The Scientific and Technological Research Council of Turkey
                1300-0152
                1303-6092
                2020
                02 April 2020
                : 44
                : 2
                : 110-120
                Affiliations
                [1 ] Tissue Engineering, Biomaterials and Nanobiotechnology Laboratory, Ankara University Faculty of Science,Ankara University Biotechnology Institute, Ankara University Stem Cell Institute, Ankara Turkey
                [2 ] Biovalda Health Technologies, Inc., Ankara Turkey
                Author notes
                * To whom correspondence should be addressed. E-mail: elcinmurat@ 123456gmail.com

                CONFLICT OF INTEREST:

                The third author is the founder and shareholder of Biovalda Health Technologies, Inc. (Ankara, Turkey). The authors declare no competing financial interests in relation to this article. The authors alone are responsible for the content and writing of the paper.

                Author information
                https://orcid.org/0000-0002-8994-9444
                https://orcid.org/0000-0003-4674-6556
                https://orcid.org/0000-0003-1037-0825
                Article
                10.3906/biy-1912-52
                7129066
                32256147
                de585ce2-faec-4991-8c5d-1dcac7d3a6c1
                Copyright © 2020 The Author(s)

                This article is distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use and redistribution provided that the original author and source are credited.

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                Article

                genome editing,crispr-cas9 technology,bioethical issues,bioethics

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