Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9

Hahn F, Eisenhut M, Mantegazza O, Weber A. Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9. Bio-protocol . 2017;7(13)

Engineering C-4 photosynthesis into C-3 chassis in the synthetic biology age

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

Generation of Targeted Knockout Mutants in Arabidopsis thaliana Using CRISPR/Cas9

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

Homology-Directed Repair of a Defective Glabrous Gene in Arabidopsis With Cas9-Based Gene Targeting

The CRISPR/Cas9 system has emerged as a powerful tool for targeted genome editing in plants and beyond. Double-strand breaks induced by the Cas9 enzyme are repaired by the cell’s own repair machinery either by the non-homologous end joining pathway or by homologous recombination (HR). While the first repair mechanism results in random mutations at the double-strand break site, HR uses the genetic information from a highly homologous repair template as blueprint for repair of the break. By offering an artificial repair template, this pathway can be exploited to introduce specific changes at a site of choice in the genome. However, frequencies of double-strand break repair by HR are very low. In this study, we compared two methods that have been reported to enhance frequencies of HR in plants. The first method boosts the repair template availability through the formation of viral replicons, the second method makes use of an in planta gene targeting (IPGT) approach. Additionally, we comparatively applied a nickase instead of a nuclease for target strand priming. To allow easy, visual detection of HR events, we aimed at restoring trichome formation in a glabrous Arabidopsis mutant by repairing a defective glabrous1 gene. Using this efficient visual marker, we were able to regenerate plants repaired by HR at frequencies of 0.12% using the IPGT approach, while both approaches using viral replicons did not yield any trichome-bearing plants

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

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 Reviewe

Surveying the Oligomeric State of Arabidopsis thaliana Chloroplasts

Blue native-PAGE (BN-PAGE) resolves protein complexes in their native state. When combined with immunoblotting, it can be used to identify the presence of high molecular weight complexes at high resolution for any protein, given a suitable antibody. To identify proteins in high molecular weight complexes on a large scale and to bypass the requirement for specific antibodies, we applied a tandem mass spectrometry (MS/MS) approach to BN-PAGE-resolved chloroplasts. Fractionation of the gel into six bands allowed identification and label-free quantification of 1000 chloroplast proteins with native molecular weight separation. Significantly, this approach achieves a depth of identification comparable with traditional shotgun proteomic analyses of chloroplasts, indicating much of the known chloroplast proteome is amenable to MS/MS identification under our fractionation scheme. By limiting the number of fractionation bands to six, we facilitate scaled-up comparative analyses, as we demonstrate with the reticulata chloroplast mutant displaying a reticulated leaf phenotype. Our comparative proteomics approach identified a candidate interacting protein of RETICULATA as well as effects on lipid remodeling proteins, amino acid metabolic enzymes, and plastid division machinery. We additionally highlight selected proteins from each sub-compartment of the chloroplast that provide novel insight on known or hypothesized protein complexes to further illustrate the utility of this approach. Our results demonstrate the high sensitivity and reproducibility of this technique, which is anticipated to be widely adaptable to other sub-cellular compartments

A set of AP2-like genes is associated with inflorescence branching and architecture in domesticated rice

Land restoration is considered to be the remedy for 21st century global challenges of land degradation. As a result, various land restoration and conservation efforts are underway at different scales. Ethiopia is one of the countries with huge investments in land restoration. Tremendous land management practices have been implemented across the country since the 1970s. However, the spatial distribution of the interventions has not been documented, and there is no systematic, quantitative evidence on whether land restoration efforts have achieved the restoration of desired ecosystem services. Therefore, we carried out a meta-analysis of peer-reviewed scientific literature related to land restoration efforts and their impacts in Ethiopia. Results show that most of the large-scale projects have been implemented in the highlands, specifically in Tigray and Amhara regions covering about 24 agro-ecological zones, and land restoration impact studies are mostly focused in the highlands but restricted in about 11 agro-ecological zones. The highest mean effect on agricultural productivity is obtained from the combination of bunds and biological interventions followed by conservation agriculture practices with 170 % and 18% increases, respectively. However, bunds alone, biological intervention alone, and terracing (Fanya Juu) reveal negative effects on productivity. The mean effect of all land restoration interventions on soil organic carbon is positive, the highest effect being from “bunds + biological” (139%) followed by exclosure (90%). Reduced soil erosion and runoff are the dominant impacts of all interventions. The results can be used to improve existing guidelines to better match land restoration options with specific desired ecosystem functions and services. While the focus of this study was on the evaluation of the impacts of land restoration efforts on selected ecosystem services, impacts on livelihood and national socio-economy have not been examined. Thus, strengthening socio-economic studies at national scale to assess the sustainability of land restoration initiatives are an essential next step