Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice
Abstract Background Application of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits. Results We report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRI...
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BMC
2019-11-01
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Series: | BMC Plant Biology |
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Online Access: | http://link.springer.com/article/10.1186/s12870-019-2131-1 |
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author | Wen Xu Wei Song Yongxing Yang Ying Wu Xinxin Lv Shuang Yuan Ya Liu Jinxiao Yang |
author_facet | Wen Xu Wei Song Yongxing Yang Ying Wu Xinxin Lv Shuang Yuan Ya Liu Jinxiao Yang |
author_sort | Wen Xu |
collection | DOAJ |
description | Abstract Background Application of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits. Results We report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture. Conclusions We have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible. |
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institution | Directory Open Access Journal |
issn | 1471-2229 |
language | English |
last_indexed | 2024-12-22T00:14:57Z |
publishDate | 2019-11-01 |
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series | BMC Plant Biology |
spelling | doaj.art-eeaa5c7a69904823998c4a1b4e6c12112022-12-21T18:45:20ZengBMCBMC Plant Biology1471-22292019-11-0119111010.1186/s12870-019-2131-1Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in riceWen Xu0Wei Song1Yongxing Yang2Ying Wu3Xinxin Lv4Shuang Yuan5Ya Liu6Jinxiao Yang7Beijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing Academy of Agriculture & Forestry SciencesBeijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing Academy of Agriculture & Forestry SciencesBeijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing Academy of Agriculture & Forestry SciencesBeijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing Academy of Agriculture & Forestry SciencesBeijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing Academy of Agriculture & Forestry SciencesBeijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing Academy of Agriculture & Forestry SciencesBeijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing Academy of Agriculture & Forestry SciencesBeijing Key Laboratory of Maize DNA Fingerprinting and Molecular Breeding, Beijing Academy of Agriculture & Forestry SciencesAbstract Background Application of the CRISPR/Cas9 system or its derived base editors enables targeted genome modification, thereby providing a programmable tool to exploit gene functions and to improve crop traits. Results We report that PmCDA1 is much more efficient than rAPOBEC1 when fused to CRISPR/Cas9 nickase for the conversion of cytosine (C) to thymine (T) in rice. Three high-fidelity SpCas9 variants, eSpCas9(1.1), SpCas9-HF2 and HypaCas9, were engineered to serve with PmCDA1 (pBEs) as C-to-T base editors. These three high-fidelity editors had distinct multiplex-genome editing efficiencies. To substantially improve their base-editing efficiencies, a tandemly arrayed tRNA-modified single guide RNA (sgRNA) architecture was applied. The efficiency of eSpCas9(1.1)-pBE was enhanced up to 25.5-fold with an acceptable off-target effect. Moreover, two- to five-fold improvement was observed for knock-out mutation frequency by these high-fidelity Cas9s under the direction of the tRNA-modified sgRNA architecture. Conclusions We have engineered a diverse toolkit for efficient and precise genome engineering in rice, thus making genome editing for plant research and crop improvement more flexible.http://link.springer.com/article/10.1186/s12870-019-2131-1CRISPR/Cas9Base editingHigh-fidelity Cas9 variantstRNA-sgRNAOff-target effect |
spellingShingle | Wen Xu Wei Song Yongxing Yang Ying Wu Xinxin Lv Shuang Yuan Ya Liu Jinxiao Yang Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice BMC Plant Biology CRISPR/Cas9 Base editing High-fidelity Cas9 variants tRNA-sgRNA Off-target effect |
title | Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice |
title_full | Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice |
title_fullStr | Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice |
title_full_unstemmed | Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice |
title_short | Multiplex nucleotide editing by high-fidelity Cas9 variants with improved efficiency in rice |
title_sort | multiplex nucleotide editing by high fidelity cas9 variants with improved efficiency in rice |
topic | CRISPR/Cas9 Base editing High-fidelity Cas9 variants tRNA-sgRNA Off-target effect |
url | http://link.springer.com/article/10.1186/s12870-019-2131-1 |
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