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Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants

Gene targeting (GT) enables precise genome modification—e.g., the introduction of base substitutions—using donor DNA as a template. Combined with clean excision of the selection marker used to select GT cells, GT is expected to become a standard, generally applicable, base editing system. Previously...

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Autores principales: Ohtsuki, Namie, Kizawa, Keiko, Mori, Akiko, Nishizawa-Yokoi, Ayako, Komatsuda, Takao, Yoshida, Hitoshi, Hayakawa, Katsuyuki, Toki, Seiichi, Saika, Hiroaki
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8525353/
https://www.ncbi.nlm.nih.gov/pubmed/34713238
http://dx.doi.org/10.3389/fgeed.2020.617713
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author Ohtsuki, Namie
Kizawa, Keiko
Mori, Akiko
Nishizawa-Yokoi, Ayako
Komatsuda, Takao
Yoshida, Hitoshi
Hayakawa, Katsuyuki
Toki, Seiichi
Saika, Hiroaki
author_facet Ohtsuki, Namie
Kizawa, Keiko
Mori, Akiko
Nishizawa-Yokoi, Ayako
Komatsuda, Takao
Yoshida, Hitoshi
Hayakawa, Katsuyuki
Toki, Seiichi
Saika, Hiroaki
author_sort Ohtsuki, Namie
collection PubMed
description Gene targeting (GT) enables precise genome modification—e.g., the introduction of base substitutions—using donor DNA as a template. Combined with clean excision of the selection marker used to select GT cells, GT is expected to become a standard, generally applicable, base editing system. Previously, we demonstrated marker excision via a piggyBac transposon from GT-modified loci in rice. However, piggyBac-mediated marker excision has the limitation that it recognizes only the sequence TTAA. Recently, we proposed a novel and universal precise genome editing system consisting of GT with subsequent single-strand annealing (SSA)-mediated marker excision, which has, in principle, no limitation of target sequences. In this study, we introduced base substitutions into the microRNA miR172 target site of the OsCly1 gene—an ortholog of the barley Cleistogamy1 gene involved in cleistogamous flowering. To ensure efficient SSA, the GT vector harbors 1.2-kb overlapped sequences at both ends of a selection marker. The frequency of positive–negative selection-mediated GT using the vector with overlapped sequences was comparable with that achieved using vectors for piggyBac-mediated marker excision without overlapped sequences, with the frequency of SSA-mediated marker excision calculated as ~40% in the T(0) generation. This frequency is thought to be adequate to produce marker-free cells, although it is lower than that achieved with piggyBac-mediated marker excision, which approaches 100%. To date, introduction of precise substitutions in discontinuous multiple bases of a targeted gene using base editors and the prime editing system based on CRISPR/Cas9 has been quite difficult. Here, using GT and our SSA-mediated marker excision system, we succeeded in the precise base substitution not only of single bases but also of artificial discontinuous multiple bases in the miR172 target site of the OsCly1 gene. Precise base substitution of miRNA target sites in target genes using this precise genome editing system will be a powerful tool in the production of valuable crops with improved traits.
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spelling pubmed-85253532021-10-27 Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants Ohtsuki, Namie Kizawa, Keiko Mori, Akiko Nishizawa-Yokoi, Ayako Komatsuda, Takao Yoshida, Hitoshi Hayakawa, Katsuyuki Toki, Seiichi Saika, Hiroaki Front Genome Ed Genome Editing Gene targeting (GT) enables precise genome modification—e.g., the introduction of base substitutions—using donor DNA as a template. Combined with clean excision of the selection marker used to select GT cells, GT is expected to become a standard, generally applicable, base editing system. Previously, we demonstrated marker excision via a piggyBac transposon from GT-modified loci in rice. However, piggyBac-mediated marker excision has the limitation that it recognizes only the sequence TTAA. Recently, we proposed a novel and universal precise genome editing system consisting of GT with subsequent single-strand annealing (SSA)-mediated marker excision, which has, in principle, no limitation of target sequences. In this study, we introduced base substitutions into the microRNA miR172 target site of the OsCly1 gene—an ortholog of the barley Cleistogamy1 gene involved in cleistogamous flowering. To ensure efficient SSA, the GT vector harbors 1.2-kb overlapped sequences at both ends of a selection marker. The frequency of positive–negative selection-mediated GT using the vector with overlapped sequences was comparable with that achieved using vectors for piggyBac-mediated marker excision without overlapped sequences, with the frequency of SSA-mediated marker excision calculated as ~40% in the T(0) generation. This frequency is thought to be adequate to produce marker-free cells, although it is lower than that achieved with piggyBac-mediated marker excision, which approaches 100%. To date, introduction of precise substitutions in discontinuous multiple bases of a targeted gene using base editors and the prime editing system based on CRISPR/Cas9 has been quite difficult. Here, using GT and our SSA-mediated marker excision system, we succeeded in the precise base substitution not only of single bases but also of artificial discontinuous multiple bases in the miR172 target site of the OsCly1 gene. Precise base substitution of miRNA target sites in target genes using this precise genome editing system will be a powerful tool in the production of valuable crops with improved traits. Frontiers Media S.A. 2021-01-12 /pmc/articles/PMC8525353/ /pubmed/34713238 http://dx.doi.org/10.3389/fgeed.2020.617713 Text en Copyright © 2021 Ohtsuki, Kizawa, Mori, Nishizawa-Yokoi, Komatsuda, Yoshida, Hayakawa, Toki and Saika. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Genome Editing
Ohtsuki, Namie
Kizawa, Keiko
Mori, Akiko
Nishizawa-Yokoi, Ayako
Komatsuda, Takao
Yoshida, Hitoshi
Hayakawa, Katsuyuki
Toki, Seiichi
Saika, Hiroaki
Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants
title Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants
title_full Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants
title_fullStr Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants
title_full_unstemmed Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants
title_short Precise Genome Editing in miRNA Target Site via Gene Targeting and Subsequent Single-Strand-Annealing-Mediated Excision of the Marker Gene in Plants
title_sort precise genome editing in mirna target site via gene targeting and subsequent single-strand-annealing-mediated excision of the marker gene in plants
topic Genome Editing
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8525353/
https://www.ncbi.nlm.nih.gov/pubmed/34713238
http://dx.doi.org/10.3389/fgeed.2020.617713
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