Efficient multiplex mutagenesis by RNA-guided Cas9 and its use in the characterization of regulatory elements in the AGAMOUS gene
- Background The efficiency of multiplex editing in plants by the RNA-guided Cas9 system is limited by efficient introduction of its components into the genome and by their activity. The possibility of introducing large fragment deletions by RNA-guided Cas9 tool provides the potential to study the function of any DNA region of interest in its ‘endogenous’ environment. Results Here, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable large-fragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that thisBackground The efficiency of multiplex editing in plants by the RNA-guided Cas9 system is limited by efficient introduction of its components into the genome and by their activity. The possibility of introducing large fragment deletions by RNA-guided Cas9 tool provides the potential to study the function of any DNA region of interest in its ‘endogenous’ environment. Results Here, an RNA-guided Cas9 system was optimized to enable efficient multiplex editing in Arabidopsis thaliana. We demonstrate the flexibility of our system for knockout of multiple genes, and to generate heritable large-fragment deletions in the genome. As a proof of concept, the function of part of the second intron of the flower development gene AGAMOUS in Arabidopsis was studied by generating a Cas9-free mutant plant line in which part of this intron was removed from the genome. Further analysis revealed that deletion of this intron fragment results 40 % decrease of AGAMOUS gene expression without changing the splicing of the gene which indicates that this regulatory region functions as an activator of AGAMOUS gene expression. Conclusions Our modified RNA-guided Cas9 system offers a versatile tool for the functional dissection of coding and non-coding DNA sequences in plants.…
Author details: | Wenhao Yan, Dijun ChenORCiDGND, Kerstin KaufmannORCiD |
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DOI: | https://doi.org/10.1186/s13007-016-0125-7 |
ISSN: | 1746-4811 |
Title of parent work (English): | Plant methods |
Publisher: | BioMed Central |
Place of publishing: | London |
Publication type: | Article |
Language: | English |
Year of first publication: | 2016 |
Publication year: | 2016 |
Publishing institution: | Universität Potsdam |
Release date: | 2016/05/24 |
Tag: | Germline transmission; Large fragment deletion; Multiplex mutagenesis; RNA-guided Cas9 |
Volume: | 12 |
First page: | 1 |
Last Page: | 9 |
Funding institution: | Universität Potsdam, Publikationsfonds |
Funding number: | PA 2016_20 |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
DDC classification: | 5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie |
5 Naturwissenschaften und Mathematik / 58 Pflanzen (Botanik) / 580 Pflanzen (Botanik) | |
Peer review: | Referiert |
Grantor: | Publikationsfonds der Universität Potsdam |
Publishing method: | Open Access |
License (German): | CC-BY - Namensnennung 4.0 International |
External remark: | Zweitveröffentlichung in der Schriftenreihe Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe ; 224 |