TY  - JOUR
A1  - Yan, Wenhao
A1  - Chen, Dijun
A1  - Kaufmann, Kerstin
T1  - Efficient multiplex mutagenesis by RNA-guided Cas9 and its use in the characterization of regulatory elements in the AGAMOUS gene
JF  - Plant methods
N2  - 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 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.
KW  - RNA-guided Cas9
KW  - Multiplex mutagenesis
KW  - Large fragment deletion
KW  - Germline transmission
Y1  - 2016
U6  - https://doi.org/10.1186/s13007-016-0125-7
SN  - 1746-4811
VL  - 12
SP  - 1
EP  - 9
PB  - BioMed Central
CY  - London
ER  - 
TY  - JOUR
A1  - Yan, Wenhao
A1  - Chen, Dijun
A1  - Kaufmann, Kerstin
T1  - Efficient multiplex mutagenesis by RNA-guided Cas9 and its use in the characterization of regulatory elements in the AGAMOUS gene
JF  - Plant Methods
N2  - 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.
KW  - RNA-guided Cas9
KW  - Multiplex mutagenesis
KW  - Large fragment deletion
KW  - Germline transmission
Y1  - 2016
U6  - https://doi.org/10.1186/s13007-016-0125-7
SN  - 1746-4811
VL  - 12
SP  - 2381
EP  - 2389
PB  - BioMed Central
CY  - London
ER  - 
TY  - GEN
A1  - Yan, Wenhao
A1  - Chen, Dijun
A1  - Kaufmann, Kerstin
T1  - Efficient multiplex mutagenesis by RNA‑guided Cas9 and its use in the characterization of regulatory elements in the AGAMOUS gene
N2  - 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 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.
T3  - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 224 
KW  - RNA-guided Cas9
KW  - Multiplex mutagenesis
KW  - Large fragment deletion
KW  - Germline transmission
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-90895
ER  -