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An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes)

BACKGROUND: The CRISPR/Cas system is a powerful genome editing tool that enables targeted genome modifications in various organisms. In medaka (Oryzias latipes), targeted mutagenesis with small insertions and deletions using this system have become a robust technique and are now widely used. However...

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Autores principales: Murakami, Yu, Ansai, Satoshi, Yonemura, Akari, Kinoshita, Masato
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500998/
https://www.ncbi.nlm.nih.gov/pubmed/28694996
http://dx.doi.org/10.1186/s40851-017-0071-x
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author Murakami, Yu
Ansai, Satoshi
Yonemura, Akari
Kinoshita, Masato
author_facet Murakami, Yu
Ansai, Satoshi
Yonemura, Akari
Kinoshita, Masato
author_sort Murakami, Yu
collection PubMed
description BACKGROUND: The CRISPR/Cas system is a powerful genome editing tool that enables targeted genome modifications in various organisms. In medaka (Oryzias latipes), targeted mutagenesis with small insertions and deletions using this system have become a robust technique and are now widely used. However, to date there have been only a small number of reports on targeted gene integration using this system. We thus sought in the present study to identify factors that enhance the efficiency of targeted gene integration events in medaka. RESULTS: We show that longer homology arms (ca. 500 bp) and linearization of circular donor plasmids by cleavage with bait sequences enhances the efficiency of targeted integration of plasmids in embryos. A new bait sequence, BaitD, which we designed and selected by in silico screening, achieved the highest efficiency of the targeted gene integration in vivo. Using this system, donor plasmids integrated precisely at target sites and were efficiently transmitted to progeny. We also report that the genotype of F(2) siblings, obtained by mating of individuals harboring two different colors of fluorescent protein genes (e.g. GFP and RFP) in the same locus, can be easily and rapidly determined non-invasively by visual observations alone. CONCLUSION: We report that the efficiency of targeted gene integration can be enhanced by using donor vectors with longer homologous arms and linearization using a highly active bait system in medaka. These findings may contribute to the establishment of more efficient systems for targeted gene integration in medaka and other fish species. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40851-017-0071-x) contains supplementary material, which is available to authorized users.
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spelling pubmed-55009982017-07-10 An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes) Murakami, Yu Ansai, Satoshi Yonemura, Akari Kinoshita, Masato Zoological Lett Research Article BACKGROUND: The CRISPR/Cas system is a powerful genome editing tool that enables targeted genome modifications in various organisms. In medaka (Oryzias latipes), targeted mutagenesis with small insertions and deletions using this system have become a robust technique and are now widely used. However, to date there have been only a small number of reports on targeted gene integration using this system. We thus sought in the present study to identify factors that enhance the efficiency of targeted gene integration events in medaka. RESULTS: We show that longer homology arms (ca. 500 bp) and linearization of circular donor plasmids by cleavage with bait sequences enhances the efficiency of targeted integration of plasmids in embryos. A new bait sequence, BaitD, which we designed and selected by in silico screening, achieved the highest efficiency of the targeted gene integration in vivo. Using this system, donor plasmids integrated precisely at target sites and were efficiently transmitted to progeny. We also report that the genotype of F(2) siblings, obtained by mating of individuals harboring two different colors of fluorescent protein genes (e.g. GFP and RFP) in the same locus, can be easily and rapidly determined non-invasively by visual observations alone. CONCLUSION: We report that the efficiency of targeted gene integration can be enhanced by using donor vectors with longer homologous arms and linearization using a highly active bait system in medaka. These findings may contribute to the establishment of more efficient systems for targeted gene integration in medaka and other fish species. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s40851-017-0071-x) contains supplementary material, which is available to authorized users. BioMed Central 2017-07-06 /pmc/articles/PMC5500998/ /pubmed/28694996 http://dx.doi.org/10.1186/s40851-017-0071-x Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Murakami, Yu
Ansai, Satoshi
Yonemura, Akari
Kinoshita, Masato
An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes)
title An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes)
title_full An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes)
title_fullStr An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes)
title_full_unstemmed An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes)
title_short An efficient system for homology-dependent targeted gene integration in medaka (Oryzias latipes)
title_sort efficient system for homology-dependent targeted gene integration in medaka (oryzias latipes)
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500998/
https://www.ncbi.nlm.nih.gov/pubmed/28694996
http://dx.doi.org/10.1186/s40851-017-0071-x
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