Abstract
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.
Highlights
A growing world population facing increasingly scarce arable land would benefit from applying synthetic biology approaches to generate rationally designed plants with low effort and within short time frames (Baltes and Voytas, 2015)
The development of the CRISPR/Cas9 system, which relies on the bacterial Cas9 nuclease from Streptococcus pyogenes and a single guide RNA molecule that directs the nuclease to its specific target (Jinek et al, 2012) has greatly reduced the effort to create double-strand breaks (DSBs) at specific loci in the genome of living cells (Doudna and Charpentier, 2014)
We aimed at testing and evaluating markers for easy detection of Cas9 induced mutagenesis
Summary
A growing world population facing increasingly scarce arable land would benefit from applying synthetic biology approaches to generate rationally designed plants with low effort and within short time frames (Baltes and Voytas, 2015). DSBs are lethal for the cell and must be repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ), which are carried out by plant internal DNA break repair mechanisms (Puchta and Fauser, 2014). Both mechanisms can be exploited for targeted. The Cas system allows the creation of DSBs in virtually any gene and even in several genes simultaneously. It has already been successfully applied in many model and crop plants species (Bortesi and Fischer, 2015). Markers as indicators for the successful mutagenesis are still scarce
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