An Efficient Visual Screen for CRISPR/Cas9 Activity inArabidopsis thaliana.
The CRISPR/Cas9 system enables precision editing of the genome of the model plantArabidopsis thalianaand likely of any other organism. Tools and methods for further developing and optimizing this widespread and versatile system inArabidopsiswould hence be welcomed. Here, we designed a generic vector system that can be used to clone any sgRNA sequence in a plant T-DNA vector containing an ubiquitously expressedCas9gene. With this vector, we explored two alternative marker systems for tracking Cas9-mediated gene-editingin vivo:BIALAPHOS RESISTANCE(BAR) andGLABROUS1(GL1).BARconfers resistance to glufosinate and is widely used as a positive selection marker;GL1is required for the formation of trichomes. Reversion of a frameshift nullBARallele to a functional one by Cas9-mediated gene editing yielded a higher than expected number of plants that are resistant to glufosinate. Surprisingly, many of those plants did not display reversion of theBARgene through the germline. We hypothesize that fewBARrevertant cells in a highly chimeric plant likely provide system-wide resistance to glufosinate and thus we suggest that BAR is not suitable as marker for tracking Cas9-mediated gene-editing. Targeting theGL1gene for disruption with Cas9 provided clearly visible phenotypes of partially and completely glabrous plants. 50% of the analyzed T1 plants produced descendants with a chimeric phenotype and we could recover fully homozygous plants in the T3 generation with high efficiency. We propose that targeting ofGL1is suitable for assessing and optimizing Cas9-mediated gene-editing inArabidopsis.
Institute of Plant Biochemistry, Cluster of Excellence on Plant Science, Center for Synthetic Life Sciences, Heinrich Heine University Düsseldorf, Germany.
Florian
F
Hahn
Institute of Plant Biochemistry, Cluster of Excellence on Plant Science, Center for Synthetic Life Sciences, Heinrich Heine University Düsseldorf, Germany.
Otho
O
Mantegazza
Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin Berlin, Germany.
André
A
Greiner
Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin Berlin, Germany.
Peter
P
Hegemann
Institute of Plant Biochemistry, Cluster of Excellence on Plant Science, Center for Synthetic Life Sciences, Heinrich Heine University Düsseldorf, Germany.
Marion
M
Eisenhut
Institute of Plant Biochemistry, Cluster of Excellence on Plant Science, Center for Synthetic Life Sciences, Heinrich Heine University Düsseldorf, Germany.
Andreas P M
AP
Weber
BASTA; CRISPR/Cas9; Glabrous1; Glufosinate; gene editing; marker; trichome