Efficient multiplex mutagenesis by RNA-guided Cas9 and its use in the characterization of regulatory elements in the AGAMOUS gene. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Yan, Wenhao; Chen, Dijun; Kaufmann, Kerstin
• Is Part Of: Plant methods. Volume 12:Issue 1(2016)
• Page Start: 1
• Page End: 9
• Publication Date: 2016-12
• Subjects: RNA-guided Cas9 -- Multiplex mutagenesis -- Large fragment deletion -- Germline transmission; Botany -- Methodology -- Periodicals; 572.2
• DOI: 10.1186/s13007-016-0125-7
• Languages: English
• ISSNs: 1746-4811

Abstract

Abstract Background The efficiency of multiplex editing in plants by the RNA-guidedCas9 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-guidedCas9 tool provides the potential to study the function of any DNA region of interest in its 'endogenous' environment. Results Here, an RNA-guidedCas9 system was optimized to enable efficient multiplex editing inArabidopsis 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 geneAGAMOUS inArabidopsis 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 ofAGAMOUS gene expression without changing the splicing of the gene which indicates that this regulatory region functions as an activator ofAGAMOUS gene expression. Conclusions Our modified RNA-guidedCas9 system offers a versatile tool for the functional dissection of coding and non-coding DNA sequences in plants.