Sucrose-responsive osmoregulation of plant cell size by a long non-coding RNA.

In plants, sugars are the key source of energy and metabolic building blocks. The systemic transport of sugars is essential for plant growth and morphogenesis. Plants evolved intricate molecular networks to effectively distribute sugars.

Regulation of gene-edited plants in Europe: from the valley of tears into the shining sun?

Some 20 years ago, the EU introduced complex regulatory rules for the growth of transgenic crops, which resulted in a de facto ban to grow these plants in fields within most European countries. With the rise of novel genome editing technologies, it has become possible to improve crops genetically in a directed way without the need for incorporation of foreign genes. Unfortunately, in 2018, the European Court of Justice ruled that such gene-edited plants are to be regulated like transgenic plants.

Protocol to efficiently induce CRISPR-Kill-mediated cell ablation in Arabidopsis thaliana.

The CRISPR-Kill system enables targeted cell ablation by inducing multiple double-strand breaks in evolutionarily conserved repetitive genomic regions. Here, we present a protocol for the application of the CRISPR-Kill system to analyze the systemic and cellular effects of targeted cell death in Arabidopsis. We describe steps for generating constitutive and inducible CRISPR-Kill lines, chemically inducing CRISPR-Cas9-mediated genome elimination, and monitoring of cell death in shoot and root apical meristems.

Deficiency of both classical and alternative end-joining pathways leads to a synergistic defect in double-strand break repair but not to an increase in homology-dependent gene targeting in Arabidopsis.

In eukaryotes, double-strand breaks (DSBs) are either repaired by homologous recombination (HR) or non-homologous end-joining (NHEJ). In somatic plant cells, HR is very inefficient. Therefore, the vast majority of DSBs are repaired by two different pathways of NHEJ.

Increasing deletion sizes and the efficiency of CRISPR/Cas9-mediated mutagenesis by SunTag-mediated TREX1 recruitment.

Previously, it has been shown that mutagenesis frequencies can be improved by directly fusing the human exonuclease TREX2 to Cas9, resulting in a strong increase in the frequency of smaller deletions at the cut site. Here, we demonstrate that, by using the SunTag system for recruitment of TREX2, the mutagenesis efficiency can be doubled in comparison to the direct fusion in Arabidopsis thaliana. Therefore, we also tested the efficiency of the system for targeted deletion formation by recruiting two other 3'-5' exonucleases, namely the human TREX1 and E.

Plant chromosome engineering - past, present and future.

Spontaneous chromosomal rearrangements (CRs) play an essential role in speciation, genome evolution and crop domestication. To be able to use the potential of CRs for breeding, plant chromosome engineering was initiated by fragmenting chromosomes by X-ray irradiation. With the rise of the CRISPR/Cas system, it became possible to induce double-strand breaks (DSBs) in a highly efficient manner at will at any chromosomal position.

Optimizing ErCas12a for efficient gene editing in Arabidopsis thaliana.

The ErCas12a nuclease, also known as MAD7, is part of a CRISPR/Cas system from Eubacterium rectale and distantly related to Cas12a nucleases. As it shares only 31% sequence homology with the commonly used AsCas12a, its intellectual property may not be covered by the granted patent rights for Cas12a nucleases. Thus, ErCas12a became an attractive alternative for practical applications.

An inducible CRISPR-Kill system for temporally controlled cell type-specific cell ablation in Arabidopsis thaliana.

The application of the CRISPR/Cas system as a biotechnological tool for genome editing has revolutionized plant biology. Recently, the repertoire was expanded by CRISPR-Kill, enabling CRISPR/Cas-mediated tissue engineering through genome elimination by tissue-specific expression. Using the Cas9 nuclease from Staphylococcus aureus (SaCas9), CRISPR-Kill relies on the induction of multiple double-strand breaks (DSBs) in conserved repetitive genome regions, such as the rDNA, causing cell death of the targeted cells.

SMC5/6 complex-mediated SUMOylation stimulates DNA-protein cross-link repair in Arabidopsis.

DNA-protein cross-links (DPCs) are highly toxic DNA lesions consisting of proteins covalently attached to chromosomal DNA. Unrepaired DPCs physically block DNA replication and transcription. Three DPC repair pathways have been identified in Arabidopsis (Arabidopsis thaliana) to date: the endonucleolytic cleavage of DNA by the structure-specific endonuclease MUS81; proteolytic degradation of the crosslinked protein by the metalloprotease WSS1A; and cleavage of the cross-link phosphodiester bonds by the tyrosyl phosphodiesterases TDP1 and TDP2.

Getting better all the time - recent progress in the development of CRISPR/Cas-based tools for plant genome engineering.

Since their first adaptation for plant genome editing, clustered regularly interspaced short palindromic repeats/CRISPR-associated system nucleases and tools have revolutionized the field. While early approaches focused on targeted mutagenesis that relies on mutagenic repair of induced double-strand breaks, newly developed tools now enable the precise induction of predefined modifications. Constant efforts to optimize these tools have led to the generation of more efficient base editors with enlarged editing windows and have enabled previously unachievable C-G transversions.

Massive crossover suppression by CRISPR-Cas-mediated plant chromosome engineering.

Recent studies have demonstrated that not only genes but also entire chromosomes can be engineered using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPER-associated protein 9 (Cas9). A major objective of applying chromosome restructuring in plant breeding is the manipulation of genetic exchange. Here we show that meiotic recombination can be suppressed in nearly the entire chromosome using chromosome restructuring.

CRISPR-Cas9-mediated chromosome engineering in Arabidopsis thaliana.

The rise of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system has made it possible to induce double-strand breaks at almost any desired target site in the genome. In plant somatic cells, double-strand breaks are predominantly repaired by the error-prone nonhomologous end-joining pathway, which can lead to mutations at the break site upon repair. So far, it had only been possible to induce genomic changes of up to a few hundred kilobases in plants utilizing this mechanism.

Using CRISPR-Kill for organ specific cell elimination by cleavage of tandem repeats.

CRISPR/Cas has been mainly used for mutagenesis through the induction of double strand breaks (DSBs) within unique protein-coding genes. Using the SaCas9 nuclease to induce multiple DSBs in functional repetitive DNA of Arabidopsis thaliana, we can now show that cell death can be induced in a controlled way. This approach, named CRISPR-Kill, can be used as tool for tissue engineering.

Nonhomologous end joining as key to CRISPR/Cas-mediated plant chromosome engineering.

Although clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)-mediated gene editing has revolutionized biology and plant breeding, large-scale, heritable restructuring of plant chromosomes is still in its infancy. Duplications and inversions within a chromosome, and also translocations between chromosomes, can now be achieved. Subsequently, genetic linkages can be broken or can be newly created.

The DNA-dependent protease AtWSS1A suppresses persistent double strand break formation during replication.

The protease WSS1A is an important factor in the repair of DNA-protein crosslinks in plants. Here we show that the loss of WSS1A leads to a reduction of 45S rDNA repeats and chromosomal fragmentation in Arabidopsis. Moreover, in the absence of any factor of the RTR (RECQ4A/TOP3α/RMI1/2) complex, which is involved in the dissolution of DNA replication intermediates, WSS1A becomes essential for viability.

The repair of topoisomerase 2 cleavage complexes in Arabidopsis.

DNA-protein crosslinks (DPCs) and DNA double-stranded breaks (DSBs), including those produced by stalled topoisomerase 2 cleavage complexes (TOP2ccs), must be repaired to ensure genome stability. The basic mechanisms of TOP2cc repair have been characterized in other eukaryotes, but we lack information for plants. Using CRISPR/Cas-induced mutants, we show that Arabidopsis thaliana has two main TOP2cc repair pathways: one is defined by TYROSYL-DNA-PHOSPHODIESTERASE 2 (TDP2), which hydrolyzes TOP2-DNA linkages, the other by the DNA-dependent protease WSS1A (a homolog of human SPARTAN/yeast weak suppressor of smt3 [Wss1]), which also functions in DPC repair.

Different DNA repair pathways are involved in single-strand break-induced genomic changes in plants.

In nature, single-strand breaks (SSBs) in DNA occur more frequently (by orders of magnitude) than double-strand breaks (DSBs). SSBs induced by the CRISPR/Cas9 nickase at a distance of 50-100 bp on opposite strands are highly mutagenic, leading to insertions/deletions (InDels), with insertions mainly occurring as direct tandem duplications. As short tandem repeats are overrepresented in plant genomes, this mechanism seems to be important for genome evolution.

CRISPR-Cas-mediated chromosome engineering for crop improvement and synthetic biology.

Plant breeding relies on the presence of genetic variation, as well as on the ability to break or stabilize genetic linkages between traits. The development of the genome-editing tool clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) has allowed breeders to induce genetic variability in a controlled and site-specific manner, and to improve traits with high efficiency. However, the presence of genetic linkages is a major obstacle to the transfer of desirable traits from wild species to their cultivated relatives.

ZYP1 is required for obligate cross-over formation and cross-over interference in .

The synaptonemal complex is a tripartite proteinaceous ultrastructure that forms between homologous chromosomes during prophase I of meiosis in the majority of eukaryotes. It is characterized by the coordinated installation of transverse filament proteins between two lateral elements and is required for wild-type levels of crossing over and meiotic progression. We have generated null mutants of the duplicated transverse filament genes and using a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis.

Novel CRISPR/Cas applications in plants: from prime editing to chromosome engineering.

In the last years, tremendous progress has been made in the development of CRISPR/Cas-mediated genome editing tools. A number of natural CRISPR/Cas nuclease variants have been characterized. Engineered Cas proteins have been developed to minimize PAM restrictions, off-side effects and temperature sensitivity.

Different functional roles of RTR complex factors in DNA repair and meiosis in Arabidopsis and tomato.

The RTR (RecQ/Top3/Rmi1) complex has been elucidated as essential for ensuring genome stability in eukaryotes. Fundamental for the dissolution of Holliday junction (HJ)-like recombination intermediates, the factors have been shown to play further, partly distinct roles in DNA repair and homologous recombination. Across all kingdoms, disruption of this complex results in characteristic phenotypes including hyper-recombination and sensitivity to genotoxins.

Efficient gene targeting in Nicotiana tabacum using CRISPR/SaCas9 and temperature tolerant LbCas12a.

Nicotiana tabacum is a non-food herb that has the potential to be utilized as bio-factory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for pre-designed genome modifications is indispensable. The development of CRISPR/Cas nucleases allows the induction of site-specific double-strand breaks to enhance homologous recombination-mediated gene targeting (GT).