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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Main Authors: Wen Xu, Wei Song, Yongxing Yang, Ying Wu, Xinxin Lv, Shuang Yuan, Ya Liu, Jinxiao Yang
Format: Article
Language:English
Published: BMC 2019-11-01
Series:BMC Plant Biology
Subjects:
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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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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