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Efficient deletion of multiple circle RNA loci by CRISPR‐Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice

CRISPR‐Cas9 is an emerging genome editing tool for reverse genetics in plants. However, its application for functional study of non‐coding RNAs in plants is still at its infancy. Despite being a major class of non‐coding RNAs, the biological roles of circle RNAs (circRNAs) remain largely unknown in...

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Autores principales: Zhou, Jianping, Yuan, Mingzhu, Zhao, Yuxin, Quan, Quan, Yu, Dong, Yang, Han, Tang, Xu, Xin, Xuhui, Cai, Guangze, Qian, Qian, Qi, Yiping, Zhang, Yong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196656/
https://www.ncbi.nlm.nih.gov/pubmed/33440058
http://dx.doi.org/10.1111/pbi.13544
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author Zhou, Jianping
Yuan, Mingzhu
Zhao, Yuxin
Quan, Quan
Yu, Dong
Yang, Han
Tang, Xu
Xin, Xuhui
Cai, Guangze
Qian, Qian
Qi, Yiping
Zhang, Yong
author_facet Zhou, Jianping
Yuan, Mingzhu
Zhao, Yuxin
Quan, Quan
Yu, Dong
Yang, Han
Tang, Xu
Xin, Xuhui
Cai, Guangze
Qian, Qian
Qi, Yiping
Zhang, Yong
author_sort Zhou, Jianping
collection PubMed
description CRISPR‐Cas9 is an emerging genome editing tool for reverse genetics in plants. However, its application for functional study of non‐coding RNAs in plants is still at its infancy. Despite being a major class of non‐coding RNAs, the biological roles of circle RNAs (circRNAs) remain largely unknown in plants. Previous plant circRNA studies have focused on identification and annotation of putative circRNAs, with their functions largely uninvestigated by genetic approaches. Here, we applied a multiplexed CRISPR‐Cas9 strategy to efficiently acquire individual null mutants for four circRNAs in rice. We showed each of these rice circRNA loci (Os02circ25329, Os06circ02797, Os03circ00204 and Os05circ02465) can be deleted at 10% or higher efficiency in both protoplasts and stable transgenic T0 lines. Such high efficiency deletion enabled the generation of circRNA null allele plants without the CRISPR‐Cas9 transgene in the T1 generation. Characterization of the mutants reveals these circRNAs’ participation in salt stress response during seed germination and in particular the Os05circ02465 null mutant showed high salt tolerance. Notably, the seedlings of the Os06circ02797 mutant showed rapid growth phenotype after seed germination with the seedlings containing higher chlorophyll A/B content. Further molecular and computational analyses suggested a circRNA–miRNA–mRNA regulatory network where Os06circ02797 functions to bind and sequester OsMIR408, an important and conserved microRNA in plants. This study not only presents genetic evidence for the first time in plants that certain circRNAs may serve as sponges to negatively regulate miRNAs, a phenomenon previously demonstrated in mammalian cells, but also provides important insights for improving agronomic traits through gene editing of circRNA loci in crops.
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spelling pubmed-81966562021-06-15 Efficient deletion of multiple circle RNA loci by CRISPR‐Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice Zhou, Jianping Yuan, Mingzhu Zhao, Yuxin Quan, Quan Yu, Dong Yang, Han Tang, Xu Xin, Xuhui Cai, Guangze Qian, Qian Qi, Yiping Zhang, Yong Plant Biotechnol J Research Articles CRISPR‐Cas9 is an emerging genome editing tool for reverse genetics in plants. However, its application for functional study of non‐coding RNAs in plants is still at its infancy. Despite being a major class of non‐coding RNAs, the biological roles of circle RNAs (circRNAs) remain largely unknown in plants. Previous plant circRNA studies have focused on identification and annotation of putative circRNAs, with their functions largely uninvestigated by genetic approaches. Here, we applied a multiplexed CRISPR‐Cas9 strategy to efficiently acquire individual null mutants for four circRNAs in rice. We showed each of these rice circRNA loci (Os02circ25329, Os06circ02797, Os03circ00204 and Os05circ02465) can be deleted at 10% or higher efficiency in both protoplasts and stable transgenic T0 lines. Such high efficiency deletion enabled the generation of circRNA null allele plants without the CRISPR‐Cas9 transgene in the T1 generation. Characterization of the mutants reveals these circRNAs’ participation in salt stress response during seed germination and in particular the Os05circ02465 null mutant showed high salt tolerance. Notably, the seedlings of the Os06circ02797 mutant showed rapid growth phenotype after seed germination with the seedlings containing higher chlorophyll A/B content. Further molecular and computational analyses suggested a circRNA–miRNA–mRNA regulatory network where Os06circ02797 functions to bind and sequester OsMIR408, an important and conserved microRNA in plants. This study not only presents genetic evidence for the first time in plants that certain circRNAs may serve as sponges to negatively regulate miRNAs, a phenomenon previously demonstrated in mammalian cells, but also provides important insights for improving agronomic traits through gene editing of circRNA loci in crops. John Wiley and Sons Inc. 2021-01-28 2021-06 /pmc/articles/PMC8196656/ /pubmed/33440058 http://dx.doi.org/10.1111/pbi.13544 Text en © 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd. https://creativecommons.org/licenses/by/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Articles
Zhou, Jianping
Yuan, Mingzhu
Zhao, Yuxin
Quan, Quan
Yu, Dong
Yang, Han
Tang, Xu
Xin, Xuhui
Cai, Guangze
Qian, Qian
Qi, Yiping
Zhang, Yong
Efficient deletion of multiple circle RNA loci by CRISPR‐Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice
title Efficient deletion of multiple circle RNA loci by CRISPR‐Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice
title_full Efficient deletion of multiple circle RNA loci by CRISPR‐Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice
title_fullStr Efficient deletion of multiple circle RNA loci by CRISPR‐Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice
title_full_unstemmed Efficient deletion of multiple circle RNA loci by CRISPR‐Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice
title_short Efficient deletion of multiple circle RNA loci by CRISPR‐Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice
title_sort efficient deletion of multiple circle rna loci by crispr‐cas9 reveals os06circ02797 as a putative sponge for osmir408 in rice
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8196656/
https://www.ncbi.nlm.nih.gov/pubmed/33440058
http://dx.doi.org/10.1111/pbi.13544
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