Efficient multiplex mutagenesis by RNA-guided Cas9 and its use in the characterization of regulatory elements in the AGAMOUS gene
- 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 |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/27118985 |
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 |
Release date: | 2020/03/22 |
Tag: | Germline transmission; Large fragment deletion; Multiplex mutagenesis; RNA-guided Cas9 |
Volume: | 12 |
Number of pages: | 9 |
First page: | 2381 |
Last Page: | 2389 |
Funding institution: | Alexander-von-Humboldt foundation; BMBF |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
Peer review: | Referiert |