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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. coli ExoI. It turns out that SunTag-mediated recruitment of TREX1 not only improved the general mutation induction efficiency slightly in comparison to TREX2, but that, more importantly, the mean size of the induced deletions was also enhanced, mainly via an increase of deletions of 25 bp or more. EcExoI also yielded a higher amount of larger deletions. However, only in the case of TREX1 and TREX2, the effect was predominately SunTag-dependent, indicating efficient target-specific recruitment. Using SunTag-mediated TREX1 recruitment at other genomic sites, we were able to obtain similar deletion patterns. Thus, we were able to develop an attractive novel editing tool that is especially useful for obtaining deletions in the range from 20 to 40 bp around the cut site. Such sizes are often required for the manipulation of cis-regulatory elements. This feature is closing an existing gap as previous approaches, based on single nucleases or paired nickases or nucleases, resulted in either shorter or longer deletions, respectively., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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. The classical (cNHEJ) pathway depends on the heterodimer KU70/KU80, while polymerase theta (POLQ) is central to the alternative (aNHEJ) pathway. Surprisingly, Arabidopsis plants are viable, even when both pathways are impaired. However, they exhibit severe growth retardation and reduced fertility. Analysis of mitotic anaphases indicates that the double mutant is characterized by a dramatic increase in chromosome fragmentation due to defective DSB repair. In contrast to the single mutants, the double mutant was found to be highly sensitive to the DSB-inducing genotoxin bleomycin. Thus, both pathways can complement for each other efficiently in DSB repair. We speculated that in the absence of both NHEJ pathways, HR might be enhanced. This would be especially attractive for gene targeting (GT) in which predefined changes are introduced using a homologous template. Unexpectedly, the polq single mutant as well as the double mutant showed significantly lower GT frequencies in comparison to wildtype plants. Accordingly, we were able to show that elimination of both NHEJ pathways does not pose an attractive approach for Agrobacterium-mediated GT. However, our results clearly indicate that a loss of cNHEJ leads to an increase in GT frequency, which is especially drastic and attractive for practical applications, in which the in planta GT strategy is used., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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. This has enabled a completely new level of predesigned chromosome engineering. The genetic linkage between specific genes can be broken by inducing chromosomal translocations. Natural inversions, which suppress genetic exchange, can be reverted for breeding. In addition, various approaches for constructing minichromosomes by downsizing regular standard A or supernumerary B chromosomes, which could serve as future vectors in plant biotechnology, have been developed. Recently, a functional synthetic centromere could be constructed. Also, different ways of genome haploidization have been set up, some based on centromere manipulations. In the future, we expect to see even more complex rearrangements, which can be combined with previously developed engineering technologies such as recombinases. Chromosome engineering might help to redefine genetic linkage groups, change the number of chromosomes, stack beneficial genes on mini cargo chromosomes, or set up genetic isolation to avoid outcrossing., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

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. The dynamic distribution of these osmotically active compounds is a handy tool for regulating cell turgor pressure, an instructive force in developmental biology. In this study, we have investigated the molecular mechanism behind the dual role of the receptor-like kinase CANAR. We functionally characterized a long non-coding RNA, CARMA, as a negative regulator of CANAR. Sugar-responsive CARMA specifically fine-tunes CANAR expression in the phloem, the route of sugar transport. Our genetic, molecular, microscopy, and biophysical data suggest that the CARMA-CANAR module controls the shoot-to-root phloem transport of sugars, allows cells to flexibly adapt to the external osmolality by appropriate water uptake, and thus adjust the size of vascular cell types during organ growth and development. Our study identifies a nexus of plant vascular tissue formation with cell internal pressure monitoring, revealing a novel functional aspect of long non-coding RNAs in developmental biology., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

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. Here, we show that in addition to spatial control by tissue-specific expression, temporal control of CRISPR-mediated cell death is feasible in Arabidopsis thaliana. We established a chemically inducible tissue-specific CRISPR-Kill system that allows the simultaneous detection of targeted cells by fluorescence markers. As proof of concept, we were able to eliminate lateral roots and ablate root stem cells. Moreover, using a multi-tissue promoter, we induced targeted cell death at defined time points in different organs at select developmental stages. Thus, using this system makes it possible to gain new insights into the developmental plasticity of certain cell types. In addition to enabling tissue engineering in plants, our system provides an invaluable tool to study the response of developing plant tissue to cell elimination through positional signaling and cell-to-cell communication., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

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. This enables the investigation of a wide range of questions in developmental plant biology. For complete details on the use and execution of this protocol, please refer to Gehrke et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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) 1-5 . A major objective of applying chromosome restructuring in plant breeding is the manipulation of genetic exchange 6 . Here we show that meiotic recombination can be suppressed in nearly the entire chromosome using chromosome restructuring. We were able to induce a heritable inversion of a >17 Mb-long chromosome fragment that contained the centromere and covered most of chromosome 2 of the Arabidopsis ecotype Col-0. Only the 2 and 0.5 Mb-long telomeric ends remained in their original orientation. In single-nucleotide polymorphism marker analysis of the offspring of crosses with the ecotype Ler-1, we detected a massive reduction of crossovers within the inverted chromosome region, coupled with a shift of crossovers to the telomeric ends. The few genetic exchanges detected within the inversion all originated from double crossovers. This not only indicates that heritable genetic exchange can occur by interstitial chromosome pairing, but also that it is restricted to the production of viable progeny., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

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. However, the editing efficiency of ErCas12a is strongly target sequence- and temperature-dependent. Therefore, optimization of the enzyme activity through protein engineering is especially attractive for its application in plants, as they are cultivated at lower temperatures. Based on the knowledge obtained from the optimization of Cas12a nucleases, we opted to improve the gene editing efficiency of ErCas12a by introducing analogous amino acid exchanges. Interestingly, neither of these mutations analogous to those in the enhanced or Ultra versions of AsCas12a resulted in significant editing enhancement of ErCas12a in Arabidopsis thaliana. However, two different mutations, V156R and K172R, in putative alpha helical structures of the enzyme showed a detectable improvement in editing. By combining these two mutations, we obtained an improved ErCas12a (imErCas12a) variant, showing several-fold increase in activity in comparison to the wild-type enzyme in Arabidopsis. This variant yields strong editing efficiencies at 22 °C which could be further increased by raising the cultivation temperature to 28 °C and even enabled editing of formerly inaccessible targets. Additionally, no enhanced off-site activity was detected. Thus, imErCas12a is an economically attractive and efficient alternative to other CRISPR/Cas systems for plant genome engineering., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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. Since then, European scientists and breeders have challenged this decision and requested a revision of this outdated law. Finally, after 5 years, the European Commission has now published a proposal on how, in the future, to regulate crops produced by new breeding technologies. The proposal tries to find a balance between the different interest groups in Europe. On one side, genetically modified plants, which cannot be discerned from their natural counterparts, will exclusively be used for food and feed and are-besides a registration step-not to be regulated at all. On the other side, plants expressing herbicide resistance are to be excluded from this regulation, a concession to the strong environmental associations and NGOs in Europe. Moreover, edited crops are to be excluded from organic farming to protect the business interests of the strong organic sector in Europe. Nevertheless, if this law passes European parliament and council, unchanged, it will present a big step forward toward establishing a more sustainable European agricultural system. Thus, it might soon be possible to develop and grow crops that are more adapted to global warming and whose cultivation will require lower amounts of pesticides. However, there is still a long way to go until the law is passed. Too often, the storm of arguments raised by the opponents, based on irrational fears of mutations and a naive understanding of nature, has fallen on fruitful ground in Europe., Competing Interests: Conflict of interestOn behalf of all authors, the corresponding author states that there is no conflict of interest., (© The Author(s) 2023.)

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. However, by combining the highly efficient Staphylococcus aureus Cas9 (SaCas9) with an egg-cell-specific promoter to facilitate heritable mutations, chromosomal rearrangements in the Mb range, such as inversion and translocations, were obtained in Arabidopsis thaliana recently. Here we describe the chromosome-engineering protocol used to generate these heritable chromosomal rearrangements in A. thaliana. The protocol is based on Agrobacterium-mediated transformation of A. thaliana with transfer DNA constructs containing SaCas9, which is driven by an egg-cell-specific promoter, and two guide RNAs that have been preselected based on their cutting efficiency. In the T1 generation, primary transformants are selected and, if required, analyzed by Droplet Digital PCR and propagated. In the following generations, junction-specific PCR screenings are carried out until plants that carry the rearrangement homozygously are identified. Using this protocol, overall rearrangement frequencies range between 0.03% and 0.5%, depending on the type of rearrangement. In total, it takes about 1 year to establish homozygous lines., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

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. If WSS1A loss is combined with loss of the classical (c) or alternative (a) nonhomologous end joining (NHEJ) pathways of double-strand break (DSB) repair, the resulting mutants show proliferation defects and enhanced chromosome fragmentation, which is especially aggravated in the absence of aNHEJ. This indicates that WSS1A is involved either in the suppression of DSB formation or in DSB repair itself. To test the latter we induced DSB by CRISPR/Cas9 at different loci in wild-type and mutant cells and analyzed their repair by deep sequencing. However, no change in the quality of the repair events and only a slight increase in their quantity was found. Thus, by removing complex DNA-protein structures, WSS1A seems to be required for the repair of replication intermediates which would otherwise be resolved into persistent DSB leading to genome instability., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

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. One way to address this issue is to create mutants with deficiencies in the meiotic recombination machinery, thereby enhancing global crossover frequencies between homologous parental chromosomes. Although this seemed to be a promising approach at first, thus far, no crossover frequencies could be enhanced in recombination-cold regions of the genome. Additionally, this approach can lead to unintended genomic instabilities due to DNA repair defects. Therefore, efforts have been undertaken to obtain predefined crossovers between homologues by inducing site-specific double-strand breaks (DSBs) in meiotic, as well as in somatic plant cells using CRISPR-Cas tools. However, this strategy has not been able to produce a substantial number of heritable homologous recombination-based crossovers. Most recently, heritable chromosomal rearrangements, such as inversions and translocations, have been obtained in a controlled way using CRISPR-Cas in plants. This approach unlocks a completely new way of manipulating genetic linkages, one in which the DSBs are induced in somatic cells, enabling the formation of chromosomal rearrangements in the megabase range, by DSB repair via non-homologous end-joining. This technology might also enable the restructuring of genomes more globally, resulting in not only the obtainment of synthetic plant chromosome, but also of novel plant species.

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. The type IA topoisomerase TOP3α has been shown as essential for viability in various animals. In contrast, in the model plant species Arabidopsis, the top3α mutant is viable. rmi1 mutants are deficient in the repair of DNA damage. Moreover, as opposed to other eukaryotes, TOP3α and RMI1 were found to be indispensable for proper meiotic progression, with mutants showing severe meiotic defects and sterility. We now established mutants of both TOP3α and RMI1 in tomato using CRISPR/Cas technology. Surprisingly, we found phenotypes that differed dramatically from those of Arabidopsis: the top3α mutants proved to be embryo-lethal, implying an essential role of the topoisomerase in tomato. In contrast, no defect in somatic DNA repair or meiosis was detectable for rmi1 mutants in tomato. This points to a differentiation of function of RTR complex partners between plant species. Our results indicate that there are relevant differences in the roles of basic factors involved in DNA repair and meiosis within dicotyledons, and thus should be taken as a note of caution when generalizing knowledge regarding basic biological processes obtained in the model plant Arabidopsis for the entire plant kingdom., (© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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. Here we describe the evolutionary conserved STRUCTURAL MAINTENANCE OF CHROMOSOMEs SMC5/6 complex as a crucial component involved in DPC repair. We identified multiple alleles of the SMC5/6 complex core subunit gene SMC6B via a forward-directed genetic screen designed to identify the factors involved in the repair of DPCs induced by the cytidine analog zebularine. We monitored plant growth and cell death in response to DPC-inducing chemicals, which revealed that the SMC5/6 complex is essential for the repair of several types of DPCs. Genetic interaction and sensitivity assays showed that the SMC5/6 complex works in parallel to the endonucleolytic and proteolytic pathways. The repair of zebularine-induced DPCs was associated with SMC5/6-dependent SUMOylation of the damage sites. Thus, we present the SMC5/6 complex as an important factor in plant DPC repair., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

CRISPR/Cas systems enable gene editing through the induction of site-specific DNA double-strand breaks (DSB). However, the nature of the induced modification highly depends on the mechanism used for DNA DSB repair. Non-homologous end joining (NHEJ)-mediated targeted mutagenesis induced by CRISPR/Cas is an already standardly applied tool, which can lead to various different kinds of mutations at a specific genomic site. Nevertheless, precise genome modification using homologous donor sequences is still challenging in plants. Applications depending on the less frequent homologous recombination (HR) require further improvements to create an attractive and efficient tool for general application in plants. Focusing on this issue, we developed the in planta gene targeting (ipGT) system, which is based on the simultaneous excision of a stably integrated, homologous donor sequence and the induction of a DSB within the target site. In recent years, several improvements were achieved enhancing gene targeting (GT) frequencies. After the successful application of Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9) for ipGT, we were able to further improve the system using Lachnospiraceae bacterium Cas12a (LbCas12a), which also enables cleavage in T-rich regions. Most recently, we tested an improved, temperature-tolerant version of LbCas12a (ttLbCas12a) for ipGT and were able to further increase GT efficiencies. Here, we describe the experimental procedure of the recently published ipGT system using ttLbCas12a in Arabidopsis thaliana in detail. © 2020 The Authors. Basic Protocol 1: Construction of CRISPR/ttLbCas12a expression vector to analyze ipGT efficiencies Basic Protocol 2: Achieving heritable GT plants., (© 2020 The Authors.)

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) technology has been applied in plant breeding mainly on genes for improving single or multiple traits 1-4 . Here we show that this technology can also be used to restructure plant chromosomes. Using the Cas9 nuclease from Staphylococcus aureus 5 , we were able to induce reciprocal translocations in the Mbp range between heterologous chromosomes in Arabidopsis thaliana. Of note, translocation frequency was about five times more efficient in the absence of the classical non-homologous end-joining pathway. Using egg-cell-specific expression of the Cas9 nuclease and consecutive bulk screening, we were able to isolate heritable events and establish lines homozygous for the translocation, reaching frequencies up to 2.5% for individual lines. Using molecular and cytological analysis, we confirmed that the chromosome-arm exchanges we obtained between chromosomes 1 and 2 and between chromosomes 1 and 5 of Arabidopsis were conservative and reciprocal. The induction of chromosomal translocations enables mimicking of genome evolution or modification of chromosomes in a directed manner, fixing or breaking genetic linkages between traits on different chromosomes. Controlled restructuring of plant genomes has the potential to transform plant breeding.

The controlled change of plant genomes by homologous recombination (HR) is still difficult to achieve. We previously developed the in planta gene targeting (ipGT) technology which depends on the simultaneous activation of the target locus by a double-strand break and the excision of the target vector. Whereas the use of SpCas9 resulted in low ipGT frequencies in Arabidopsis, we were recently able to improve the efficiency by using egg cell-specific expression of the potent but less broadly applicable SaCas9 nuclease. In this study, we now tested whether we could improve ipGT further, by either performing it in cells with enhanced intrachromosomal HR efficiencies or by the use of Cas12a, a different kind of CRISPR/Cas nuclease with an alternative cutting mechanism. We could show before that plants possess three kinds of DNA ATPase complexes, which all lead to instabilities of homologous genomic repeats if lost by mutation. As these proteins act in independent pathways, we tested ipGT in double mutants in which intrachromosomal HR is enhanced 20-80-fold. However, we were not able to obtain higher ipGT frequencies, indicating that mechanisms for gene targeting (GT) and chromosomal repeat-induced HR differ. However, using LbCas12a, the GT frequencies were higher than with SaCas9, despite a lower non-homologous end-joining (NHEJ) induction efficiency, demonstrating the particular suitability of Cas12a to induce HR. As SaCas9 has substantial restrictions due to its longer GC rich PAM sequence, the use of LbCas12a with its AT-rich PAM broadens the range of ipGT drastically, particularly when targeting in CG-deserts like promoters and introns., (© 2019 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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. Prime editors were also optimized for the application in plants and now allow to accurately induce substitutions, insertions, and deletions. Recently, great progress was made through precise restructuring of chromosomes, which enables not only the breakage or formation of genetic linkages but also the swapping of promoters., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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. Also, the order of genes on a chromosome can be changed. While natural chromosomal recombination occurs by homologous recombination during meiosis, CRISPR/Cas-mediated chromosomal rearrangements can be obtained best by harnessing nonhomologous end joining (NHEJ) pathways in somatic cells. NHEJ can be subdivided into the classical (cNHEJ) and alternative NHEJ (aNHEJ) pathways, which partially operate antagonistically. The cNHEJ pathway not only protects broken DNA ends from degradation but also suppresses the joining of previously unlinked broken ends. Hence, in the absence of cNHEJ, more inversions or translocations can be obtained which can be ascribed to the unrestricted use of the aNHEJ pathway for double-strand break (DSB) repair. In contrast to inversions or translocations, short tandem duplications can be produced by paired single-strand breaks via a Cas9 nickase. Interestingly, the cNHEJ pathway is essential for these kinds of duplications, whereas aNHEJ is required for patch insertions that can also be formed during DSB repair. As chromosome engineering has not only been accomplished in the model plant Arabidopsis (Arabidopsis thaliana) but also in the crop maize (Zea mays), we expect that this technology will soon transform the breeding process., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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. By simply exchanging the constitutive promoter of SaCas9 with cell type-specific promoters, it is possible to block organogenesis in Arabidopsis. By AP1-specific expression of CRISPR-Kill, we are able to restore the apetala1 phenotype and to specifically eliminate petals. In addition, by expressing CRISPR-Kill in root-specific pericycle cells, we are able to dramatically reduce the number and the length of lateral roots. In the future, the application of CRISPR-Kill may not only help to control development but could also be used to change the biochemical properties of plants., (© 2022. The Author(s).)

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. TDP1 and TDP2 function nonredundantly in TOP1cc repair, indicating that they act specifically on their respective stalled cleavage complexes. The nuclease METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81) plays a major role in global DPC repair and a minor role in TOP2cc repair. DSBs arise as intermediates of TOP2cc repair and are repaired by classical and alternative nonhomologous end joining (NHEJ) pathways. Double-mutant analysis indicates that "clean" DNA ends caused by TDP2 hydrolysis are mainly religated by classical NHEJ, which helps avoid mutation. In contrast, the mutagenic alternative NHEJ pathway mainly processes nonligateable DNA ends. Thus, TDP2 promotes maintenance of plant genome integrity by error-free repair of TOP2cc., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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. We investigated the distance at which paired 5'-overhanging SSBs are mutagenic and which DNA repair pathways are essential for insertion formation in Arabidopsis thaliana. We were able to detect InDel formation up to a distance of 250 bp, although with much reduced efficiency. Surprisingly, the loss of the classical nonhomologous end joining (NHEJ) pathway factors KU70 or DNA ligase 4 completely abolished tandem repeat formation. The microhomology-mediated NHEJ factor POLQ was required only for patch-like insertions, which are well-known from DSB repair as templated insertions from ectopic sites. As SSBs can also be repaired using homology, we furthermore asked whether the classical homologous recombination (HR) pathway is involved in this process in plants. The fact that RAD54 is not required for homology-mediated SSB repair demonstrates that the mechanisms for DSB- and SSB-induced HR differ in plants., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Mapping the network of proteins provides a powerful means to investigate the function of disease genes and to unravel the molecular basis of phenotypes. We present an automated informatics-aided and bioluminescence resonance energy transfer-based approach (iBRET) enabling high-confidence detection of protein-protein interactions in living mammalian cells. A screen of the ABCD1 protein, which is affected in X-linked adrenoleukodystrophy (X-ALD), against an organelle library of peroxisomal proteins demonstrated applicability of iBRET for large-scale experiments. We identified novel protein-protein interactions for ABCD1 (with ALDH3A2, DAO, ECI2, FAR1, PEX10, PEX13, PEX5, PXMP2, and PIPOX), mapped its position within the peroxisomal protein-protein interaction network, and determined that pathogenic missense variants in ABCD1 alter the interaction with selected binding partners. These findings provide mechanistic insights into pathophysiology of X-ALD and may foster the identification of new disease modifiers.

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. Both kinds of enzymes have, by now, been applied widely and efficiently in many plant species to generate either single or multiple mutations at the desired loci by multiplexing. In addition to DSB-induced mutagenesis, specifically designed CRISPR/Cas systems allow more precise gene editing, resulting not only in random mutations but also in predefined changes. Applications in plants include gene targeting by homologous recombination, base editing and, more recently, prime editing. We will evaluate these different technologies for their prospects and practical applicability in plants. In addition, we will discuss a novel application of the Cas9 nuclease in plants, enabling the induction of heritable chromosomal rearrangements, such as inversions and translocations. This technique will make it possible to change genetic linkages in a programmed way and add another level of genome engineering to the toolbox of plant breeding. Also, strategies for tissue culture free genome editing were developed, which might be helpful to overcome the transformation bottlenecks in many crops. All in all, the recent advances of CRISPR/Cas technology will help agriculture to address the challenges of the twenty-first century related to global warming, pollution and the resulting food shortage., (© 2021. The Author(s).)

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). However, the efficiency of GT is still a challenging obstacle for many crops including tobacco. Recently, studies in several plant species indicated that by replacing SpCas9 with other CRISPR/Cas-based nucleases, GT efficiencies might be enhanced considerably. Therefore, we tested SaCas9 as well as a temperature-insensitive version of LbCas12a (ttLbCas12a) for targeting the tobacco SuRB gene. At the same time, we also optimized the protocol for Agrobacterium-mediated tobacco transformation and tissue culture. In this way, we could improve GT efficiencies to up to a third of the inoculated cotyledons when using ttLbCas12a, which outperformed SaCas9 considerably. In addition, we could show that the conversion tract length of the GT reaction can be up to 606 bp long and in the majority of cases, it is longer than 250 bp. We obtained multiple heritable GT events, mostly heterozygous, but also biallelic GT events and some without T-DNA integration. Thus, we were not only able to obtain CRISPR/Cas-based heritable GT events in allotetraploid Nicotiana tabacum for the first time, but our results also indicate that ttLbCas12a might be a superior alternative for gene editing and GT in tobacco as well as in other crops., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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 Arabidopsis transverse filament genes zyp1a and zyp1b using a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis. Cytological and genetic analysis of the zyp1 null mutants reveals loss of the obligate chiasma, an increase in recombination map length by 1.3- to 1.7-fold and a virtual absence of cross-over (CO) interference, determined by a significant increase in the number of double COs. At diplotene, the numbers of HEI10 foci, a marker for Class I interference-sensitive COs, are twofold greater in the zyp1 mutant compared to wild type. The increase in recombination in zyp1 does not appear to be due to the Class II interference-insensitive COs as chiasmata were reduced by ∼52% in msh5/zyp1 compared to msh5 These data suggest that ZYP1 limits the formation of closely spaced Class I COs in Arabidopsis Our data indicate that installation of ZYP1 occurs at ASY1-labeled axial bridges and that loss of the protein disrupts progressive coalignment of the chromosome axes., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background and Aims: Chronic inflammation induces liver fibrosis, cirrhosis and potentially liver cancer. Kupffer cells modulate hepatic stellate cells by secreting immunologically active proteins as TGF-β. TGF-β promotes liver fibrosis via the activation of Sma- and Mad-related protein 3. IL-37 broadly suppresses innate and adaptive immune responses. Intracellular IL-37 interacts with Smad3. We hypothesize that IL-37 downregulates the activation of hepatic Kupffer and stellate cells and interferes with the TGF-β signaling cascade to modulate liver fibrogenesis. Methods: The role of IL-37 on liver inflammation and fibrogenesis was assessed in three mouse models as well as isolated Kupffer- and stellate cells. Serum IL-37 was tested by ELISA in a clinical cohort and correlated with liver disease severity. Results: Transgene expression of IL-37 in mice extends survival, reduces hepatic damage, expression of early markers of fibrosis and histologically assessed liver fibrosis after bile duct ligation. IL-37tg mice were protected against CCl 4 -induced liver inflammation. Colitis-associated liver inflammation and fibrosis was less severe in IL-10 knockout IL-37tg mice. Spontaneous and LPS/TGF-β-induced cytokine release and profibrogenic gene expression was lower in HSC and KC isolated from IL-37tg mice and IL-37 overexpressing, IL-1β stimulated human LX-2 stellate cells. However, administration of recombinant human IL-37 did not modulate fibrosis pathways after BDL in mice, LX2 cells or murine HSCs. In a large clinical cohort, we observed a positive correlation of serum IL-37 levels with disease severity in liver cirrhosis. Conclusions: Predominantly intracellular IL-37 downregulates liver inflammation and fibrosis. The correlation of serum IL-37 with disease severity in cirrhosis suggests its potential as a novel target modulating the course of liver fibrosis., Competing Interests: SH was employed by the company Ethris GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mountford, Effenberger, Noll-Puchta, Griessmair, Ringleb, Haas, Denk, Reiter, Mayr, Dinarello, Tilg and Bufler.)

Background: Cardiovascular magnetic resonance T1-mapping is increasingly used for tissue characterization, commonly based on Modified Look-Locker Inversion recovery (MOLLI). However, there are numerous MOLLI variants with differing normal ranges. This lack of standardization presents confusion and difficulty in inter-center comparisons, hindering widespread adoption of T1-mapping., Methods: To address this, we performed a structured literature search for native left ventricular myocardial T1-mapping in healthy humans measured using MOLLI variants at 1.5 and 3 Tesla, across scanner vendors. We then used k-means clustering to structure normal MOLLI-T1 values according to magnetic field strength, and investigated correlations between common imaging parameters: repetition time (TR), echo time (TE), flip angle (FA)., Results: We analyzed data from 2207 healthy controls in 76 independent reports. Normal MOLLI-T1 standard deviations varied by 11-fold, and dependencies on TE, TR, and FA differed between 1.5 T and 3 T, thwarting meaningful T1 standardization even within a single field strength, including the use of Z-score. However, divergent MOLLI-T1 norms may be structured using data clustering. For 1.5 T, two clusters emerged: Cluster1 1.5T : T1 = 958 ± 16 ms (n = 1280); Cluster2 1.5T : T1 = 1027 ± 19 ms (n = 386). For 3 T, three clusters emerged: Cluster1 3T : T1 = 1160 ± 21 ms (n = 330); Cluster2 3T : T1 = 1067 ± 18 ms (n = 178); Cluster3 3T : T1 = 1227 ± 19 ms (n = 41). We then propose the concept of an online calculator for assigning local norms to a known MOLLI-T1 cluster, allowing benchmarking against published norms., Conclusion: Clustered structuring allows T1 standardization of widely-divergent MOLLI variants, benchmarking local norms (usually based on smaller samples) against published norms (larger samples). This may increase confidence and quality control in method implementation, facilitating wider clinical adoption of T1-mapping., Competing Interests: Declaration of Competing Interest SKP has patent authorship rights for U.S. patent US20120078084A1. Systems and methods for shortened Look Locker inversion recovery (Sh-MOLLI) cardiac gated mapping of T1. Granted March 15, 2016. IP is managed by Oxford University Innovations; the license exclusively transferred to Siemens Healthcare. All other authors have no industry relationships relevant to the contents of this paper to disclose., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The advent of powerful site-specific nucleases, particularly the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system, which enables precise genome manipulation, has revolutionized plant breeding. Until recently, the main focus of researchers has been to simply knock-in or knock-out single genes, or to induce single base changes, but constant improvements of this technology have enabled more ambitious applications that aim to improve plant productivity or other desirable traits. One long-standing aim has been the induction of targeted chromosomal rearrangements (crossovers, inversions, or translocations). The feasibility of this technique has the potential to transform plant breeding, because natural rearrangements, like inversions, for example, typically present obstacles to the breeding process. In this way, genetic linkages between traits could be altered to combine or separate favorable and deleterious genes, respectively. In this review, we discuss recent breakthroughs in the field of chromosome engineering in plants and their potential applications in the field of plant breeding. In the future, these approaches might be applicable in shaping plant chromosomes in a directed manner, based on plant breeding needs., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Over the last years, the discovery of various natural and the development of a row of engineered CRISPR/Cas nucleases have made almost every site of plant genomes accessible for the induction of specific changes. Newly developed tools open up a wide range of possibilities for the induction of genetic variability, from changing a single bp to Mbps, and thus to fine-tune plant performance. Whereas early approaches focused on targeted mutagenesis, recently developed tools enable the induction of precise and predefined genomic modifications. The use of base editors allows the substitution of single nucleotides, whereas the use of prime editors and gene targeting methods enables the induction of larger sequence modifications from a few bases to several kbp. Recently, through CRISPR/Cas-mediated chromosome engineering, it became possible to induce heritable inversions and translocations in the Mbp range. Thus, a novel way of breaking and fixing genetic linkages has come into reach for breeders. In addition, sequence-specific recruitment of various factors involved in transcriptional and post-transcriptional regulation has been shown to provide an additional class of methods for the fine tuning of plant performance. In this review, we provide an overview of the most recent progress in the field of CRISPR/Cas-based tool development for plant genome engineering and try to evaluate the importance of these developments for breeding and biotechnological applications., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Osteoporosis and ischemic heart disease (IHD) represent important public health problems. Existing research suggests an association between the two conditions beyond that attributable to shared risk factors, with a potentially causal relationship. In this study, we tested the association of bone speed of sound (SOS) from quantitative heel ultrasound with (i) measures of arterial compliance from cardiovascular magnetic resonance (aortic distensibility [AD]); (ii) finger photoplethysmography (arterial stiffness index [ASI]); and (iii) incident myocardial infarction and IHD mortality in the UK Biobank cohort. We considered the potential mediating effect of a range of blood biomarkers and cardiometabolic morbidities and evaluated differential relationships by sex, menopause status, smoking, diabetes, and obesity. Furthermore, we considered whether associations with arterial compliance explained association of SOS with ischemic cardiovascular outcomes. Higher SOS was associated with lower arterial compliance by both ASI and AD for both men and women. The relationship was most consistent with ASI, likely relating to larger sample size available for this variable (n = 159,542 versus n = 18,229). There was no clear evidence of differential relationship by menopause, smoking, diabetes, or body mass index (BMI). Blood biomarkers appeared important in mediating the association for both men and women, but with different directions of effect and did not fully explain the observed effects. In fully adjusted models, higher SOS was associated with significantly lower IHD mortality in men, but less robustly in women. The association of SOS with ASI did not explain this observation. In conclusion, our findings support a positive association between bone and vascular health with consistent patterns of association in men and women. The underlying mechanisms are complex and appear to vary by sex. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)., (© 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).)

Chromosomal inversions are recurrent rearrangements that occur between different plant isolates or cultivars. Such inversions may underlie reproductive isolation in evolution and represent a major obstacle for classical breeding as no crossovers can be observed between inverted sequences on homologous chromosomes. The heterochromatic knob (hk4S) on chromosome 4 is the most well-known inversion of Arabidopsis. If a knob carrying accession such as Col-0 is crossed with a knob-less accession such as Ler-1, crossovers cannot be recovered within the inverted region. Our work shows that by egg-cell specific expression of the Cas9 nuclease from Staphylococcus aureus, a targeted reversal of the 1.1 Mb long hk4S-inversion can be achieved. By crossing Col-0 harbouring the rearranged chromosome 4 with Ler-1, meiotic crossovers can be restored into a region with previously no detectable genetic exchange. The strategy of somatic chromosome engineering for breaking genetic linkage has huge potential for application in plant breeding.

Development of live imaging techniques for providing information how chromatin is organized in living cells is pivotal to decipher the regulation of biological processes. Here, we demonstrate the improvement of a live imaging technique based on CRISPR/Cas9. In this approach, the sgRNA scaffold is fused to RNA aptamers including MS2 and PP7. When the dead Cas9 (dCas9) is co-expressed with chimeric sgRNA, the fluorescent coat protein-tagged for MS2 and PP7 aptamers (tdMCP-FP and tdPCP-FP) are recruited to the targeted sequence. Compared to previous work with dCas9:GFP, we show that the quality of telomere labeling was improved in transiently transformed Nicotiana benthamiana using aptamer-based CRISPR-imaging constructs. Labeling is influenced by the copy number of aptamers and less by the promoter types. The same constructs were not applicable for labeling of repeats in stably transformed plants and roots. The constant interaction of the RNP complex with its target DNA might interfere with cellular processes., (Copyright © 2020 Khosravi, Schindele, Gladilin, Dunemann, Rutten, Puchta and Houben.)

The systematic perturbation of genomes using CRISPR/Cas9 deciphers gene function at an unprecedented rate, depth and ease. Commercially available sgRNA libraries typically contain tens of thousands of pre-defined constructs, resulting in a complexity challenging to handle. In contrast, custom sgRNA libraries comprise gene sets of self-defined content and size, facilitating experiments under complex conditions such as in vivo systems. To streamline and upscale cloning of custom libraries, we present CLUE, a bioinformatic and wet-lab pipeline for the multiplexed generation of pooled sgRNA libraries. CLUE starts from lists of genes or pasted sequences provided by the user and designs a single synthetic oligonucleotide pool containing various libraries. At the core of the approach, a barcoding strategy for unique primer binding sites allows amplifying different user-defined libraries from one single oligonucleotide pool. We prove the approach to be straightforward, versatile and specific, yielding uniform sgRNA distributions in all resulting libraries, virtually devoid of cross-contaminations. For in silico library multiplexing and design, we established an easy-to-use online platform at www.crispr-clue.de. All in all, CLUE represents a resource-saving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)