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Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing
In contrast to genome editing, which introduces genetic changes at the DNA level, disrupting or editing gene transcripts provides a distinct approach to perturbing a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulating RNA, we first...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125684/ https://www.ncbi.nlm.nih.gov/pubmed/29860393 http://dx.doi.org/10.1093/nar/gky433 |
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author | Jing, Xinyun Xie, Bingran Chen, Longxian Zhang, Niubing Jiang, Yiyi Qin, Hang Wang, Hongbing Hao, Pei Yang, Sheng Li, Xuan |
author_facet | Jing, Xinyun Xie, Bingran Chen, Longxian Zhang, Niubing Jiang, Yiyi Qin, Hang Wang, Hongbing Hao, Pei Yang, Sheng Li, Xuan |
author_sort | Jing, Xinyun |
collection | PubMed |
description | In contrast to genome editing, which introduces genetic changes at the DNA level, disrupting or editing gene transcripts provides a distinct approach to perturbing a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulating RNA, we first implemented a member of the type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a), in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. This approach was shown to knock down targeted endogenous gene transcripts with different efficiencies. Second, we engineered an RNA editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human adenosine deaminase acting on RNA type 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized system parameters using a dual-fluorescence reporter and demonstrated the utility of the system in editing randomly selected endogenous gene transcripts. We further used it to restore the transposition of retrotransposon Tf1 mutants in fission yeast, providing a potential novel toolset for retrovirus manipulation and interference. |
format | Online Article Text |
id | pubmed-6125684 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-61256842018-09-11 Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing Jing, Xinyun Xie, Bingran Chen, Longxian Zhang, Niubing Jiang, Yiyi Qin, Hang Wang, Hongbing Hao, Pei Yang, Sheng Li, Xuan Nucleic Acids Res Methods Online In contrast to genome editing, which introduces genetic changes at the DNA level, disrupting or editing gene transcripts provides a distinct approach to perturbing a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulating RNA, we first implemented a member of the type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a), in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. This approach was shown to knock down targeted endogenous gene transcripts with different efficiencies. Second, we engineered an RNA editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human adenosine deaminase acting on RNA type 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized system parameters using a dual-fluorescence reporter and demonstrated the utility of the system in editing randomly selected endogenous gene transcripts. We further used it to restore the transposition of retrotransposon Tf1 mutants in fission yeast, providing a potential novel toolset for retrovirus manipulation and interference. Oxford University Press 2018-09-06 2018-05-31 /pmc/articles/PMC6125684/ /pubmed/29860393 http://dx.doi.org/10.1093/nar/gky433 Text en © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Jing, Xinyun Xie, Bingran Chen, Longxian Zhang, Niubing Jiang, Yiyi Qin, Hang Wang, Hongbing Hao, Pei Yang, Sheng Li, Xuan Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing |
title | Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing |
title_full | Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing |
title_fullStr | Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing |
title_full_unstemmed | Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing |
title_short | Implementation of the CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing |
title_sort | implementation of the crispr-cas13a system in fission yeast and its repurposing for precise rna editing |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6125684/ https://www.ncbi.nlm.nih.gov/pubmed/29860393 http://dx.doi.org/10.1093/nar/gky433 |
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