A CRISPR/Cas9 toolkit for multiplex genome editing in plants

BMC Plant Biology

Methodology Article

Li Dong¹  Hai-Yan Zhang¹  Zhi-Ping Wang¹  Chun-Yan Han¹  Hui-Li Xing¹  Qi-Jun Chen¹  Bing Liu¹  Xue-Chen Wang¹ 

[1] State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, 100193, Beijing, China;

关键词: CRISPR/Cas9;    Genome editing;    Multiple gene mutations;    Assembly of multiple gRNAs;   

DOI  :  10.1186/s12870-014-0327-y

 received in 2014-10-09, accepted in 2014-11-06,  发布年份 2014

来源: Springer

【 摘 要 】

BackgroundTo accelerate the application of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/ CRISPR-associated protein 9) system to a variety of plant species, a toolkit with additional plant selectable markers, more gRNA modules, and easier methods for the assembly of one or more gRNA expression cassettes is required.ResultsWe developed a CRISPR/Cas9 binary vector set based on the pGreen or pCAMBIA backbone, as well as a gRNA (guide RNA) module vector set, as a toolkit for multiplex genome editing in plants. This toolkit requires no restriction enzymes besides BsaI to generate final constructs harboring maize-codon optimized Cas9 and one or more gRNAs with high efficiency in as little as one cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to exhibit high efficiency and specificity. More importantly, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic seedlings of the T1 generation. Moreover, the multiple-gene mutations could be inherited by the next generation.ConclusionsWe developed a toolkit that facilitates transient or stable expression of the CRISPR/Cas9 system in a variety of plant species, which will facilitate plant research, as it enables high efficiency generation of mutants bearing multiple gene mutations.

【 授权许可】

Unknown   
© Xing et al.; licensee BioMed Central Ltd. 2014. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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.