AUTHOR=Hahn Florian , Mantegazza Otho , Greiner André , Hegemann Peter , Eisenhut Marion , Weber Andreas P. M. 

TITLE=An Efficient Visual Screen for CRISPR/Cas9 Activity in Arabidopsis thaliana

JOURNAL=Frontiers in Plant Science

VOLUME=8

YEAR=2017

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2017.00039

DOI=10.3389/fpls.2017.00039

ISSN=1664-462X

ABSTRACT=<p>The CRISPR/Cas9 system enables precision editing of the genome of the model plant <italic>Arabidopsis thaliana</italic> and likely of any other organism. Tools and methods for further developing and optimizing this widespread and versatile system in <italic>Arabidopsis</italic> would 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 expressed <italic>Cas9</italic> gene. With this vector, we explored two alternative marker systems for tracking Cas9-mediated gene-editing <italic>in vivo</italic>: <italic>BIALAPHOS RESISTANCE</italic> (<italic>BAR</italic>) and <italic>GLABROUS1</italic> (<italic>GL1</italic>). <italic>BAR</italic> confers resistance to glufosinate and is widely used as a positive selection marker; <italic>GL1</italic> is required for the formation of trichomes. Reversion of a frameshift null <italic>BAR</italic> allele 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 the <italic>BAR</italic> gene through the germline. We hypothesize that few <italic>BAR</italic> revertant 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 the <italic>GL1</italic> gene 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 of <italic>GL1</italic> is suitable for assessing and optimizing Cas9-mediated gene-editing in <italic>Arabidopsis</italic>.</p>