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

Abstract

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