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The CRISPR/Cas9 system has emerged as a powerful tool for gene editing in plants and beyond. We have developed a plant vector system for targeted Cas9-dependent mutagenesis of genes in up to two different target sites in Arabidopsis thaliana. This protocol describes a simple 1-week cloning procedure for a single T-DNA vector containing the genes for Cas9 and sgRNAs, as well as the detection of induced mutations in planta. The procedure can likely be adapted for other transformable plant species.

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

C-4 photosynthetic plants outperform C-3 plants in hot and arid climates. By concentrating carbon dioxide around Rubisco C-4 plants drastically reduce photorespiration. The frequency with which plants evolved C-4 photosynthesis independently challenges researchers to unravel the genetic mechanisms underlying this convergent evolutionary switch. The conversion of C-3 crops, such as rice, towards C-4 photosynthesis is a long-standing goal. Nevertheless, at the present time, in the age of synthetic biology, this still remains a monumental task, partially because the C-4 carbon-concentrating biochemical cycle spans two cell types and thus requires specialized anatomy. Here we review the advances in understanding the molecular basis and the evolution of the C-4 trait, advances in the last decades that were driven by systems biology methods. In this review we emphasise essential genetic engineering tools needed to translate our theoretical knowledge into engineering approaches. With our current molecular understanding of the biochemical C-4 pathway, we propose a simplified rational engineering model exclusively built with known C-4 metabolic components. Moreover, we discuss an alternative approach to the progressing international engineering attempts that would combine targeted mutagenesis and directed evolution.