The CRISPR/Cas technology has recently become the tool of choice for targeted genome modification in plants and beyond. Although CRSIPR/Cas offers a rapid and facile way of introducing changes at genomic loci of interest, its application is associated with off-targeting, i.e. introduction of unintended mutations at off-target sites within the genome, which has been reported frequently in the mammalian field. Here we summarise the current knowledge on the precision of CRISPR/Cas in plant systems and provide a summary of state-of the-art strategies for avoiding off-target mutations, as well as unintended on-target changes, in plants. These include using natural (e.g. Cas12a) or engineered (e.g. SpCas9-HF) CRISPR/Cas nucleases characterised by higher precision, as compared to the commonly used wild type SpCas9. In addition, we discuss the usage of CRISPR/Cas nucleases in the form of ribonucleoproteins (RNPs) as an option for reducing off-targeting in plants. Finally, we conclude that the most important factor for reducing CRISPR/Cas off-targeting remains careful selection of target sequences, for which we provide an overview of available online software tools and experimental guidance.

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.

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.

Background CRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs. Results Here we present an expanded cloning toolkit that contains 103 modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts. Conclusions We believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community.

This is an open access article under the terms of the Creat ive Commo ns Attri bution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Many enzymes involved in photosynthesis possess highly conserved cysteine residues that serve as redox switches in chloroplasts. These redox switches function to activate or deactivate enzymes during light-dark transitions and have the function of fine-tuning their activities according to the intensity of light. Accordingly, many studies on chloroplast redox regulation have been conducted under the hypothesis that “fine regulation of the activities of these enzymes is crucial for efficient photosynthesis.” However, the impact of the regulatory system on plant metabolism is still unclear. To test this hypothesis, we here studied the impact of the ablation of a redox switch in chloroplast NADP-malate dehydrogenase (MDH). By genome editing, we generated a mutant plant whose MDH lacks one of its redox switches and is active even in dark conditions. Although NADPH consumption by MDH in the dark is expected to be harmful to plant growth, the mutant line did not show any phenotypic differences under standard long-day conditions. In contrast, the mutant line showed severe growth retardation under short-day or fluctuating light conditions. These results indicate that thiol-switch redox regulation of MDH activity is crucial for maintaining NADPH homeostasis in chloroplasts under these conditions.

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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.