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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 largefragment 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 largefragment 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.show moreshow less

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Metadaten
Author:Wenhao Yan, Dijun Chen, Kerstin Kaufmann
URN:urn:nbn:de:kobv:517-opus4-90895
Series (Serial Number):Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe (224)
Document Type:Postprint
Language:English
Date of first Publication:2016/05/24
Year of Completion:2016
Publishing Institution:Universität Potsdam
Release Date:2016/05/24
Tag:Germline transmission; Large fragment deletion; Multiplex mutagenesis; RNA-guided Cas9
Funder:Universität Potsdam, Publikationsfonds
Grant Number:PA 2016_20
Organizational units:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
5 Naturwissenschaften und Mathematik / 58 Pflanzen (Botanik) / 580 Pflanzen (Botanik)
Peer Review:Referiert
Publication Way:Open Access
Grantor:BioMed Central
Licence (German):License LogoCreative Commons - Namensnennung, 4.0 International
Notes extern:Bibliographieeintrag der Originalveröffentlichung/Quelle