Genome editing around the globe: An update on policies and perceptions.

A decade ago, the CRISPR/Cas system has been adapted for genome editing. Since then, hundreds of organisms have been altered using genome editing and discussions were raised on the regulatory status of genome edited organisms esp. crops. To date, many countries have made decisions on the regulatory status of products of genome editing, by exempting some kinds of edits from the classical GMO regulation. However, the guidance differs between countries even in the same region. Several countries are still debating the issue or are in the progress of updating guidance and regulatory systems to cover products of genome editing. The current global situation of different regulatory systems is putting a harmonized framework on genome-edited crops in the far future. In this update, we summarize the current developments in the field of regulation concerning edited crops and present a short insight into perception of genome editing in the society.

Heterologous Complementation of SPO11-1 and -2 Depends on the Splicing Pattern.

In the past, major findings in meiosis have been achieved, but questions towards the global understanding of meiosis remain concealed. In plants, one of these questions covers the need for two diverse meiotic active SPO11 proteins. In Arabidopsis and other plants, both meiotic SPO11 are indispensable in a functional form for double strand break induction during meiotic prophase I. This stands in contrast to mammals and fungi, where a single SPO11 is present and sufficient. We aimed to investigate the specific function and evolution of both meiotic SPO11 paralogs in land plants. By performing immunostaining of both SPO11-1 and -2, an investigation of the spatiotemporal localization of each SPO11 during meiosis was achieved. We further exchanged SPO11-1 and -2 in Arabidopsis and could show a species-specific function of the respective SPO11. By additional changes of regions between SPO11-1 and -2, a sequence-specific function for both the SPO11 proteins was revealed. Furthermore, the previous findings about the aberrant splicing of each SPO11 were refined by narrowing them down to a specific developmental phase. These findings let us suggest that the function of both SPO11 paralogs is highly sequence specific and that the orthologs are species specific.

Which Factors Affect the Occurrence of Off-Target Effects Caused by the Use of CRISPR/Cas: A Systematic Review in Plants.

CRISPR/Cas enables a targeted modification of DNA sequences. Despite their ease and efficient use, one limitation is the potential occurrence of associated off-target effects. This systematic review aims to answer the following research question: Which factors affect the occurrence of off-target effects caused by the use of CRISPR/Cas in plants? Literature published until March 2019 was considered for this review. Articles were screened for relevance based on pre-defined inclusion criteria. Relevant studies were subject to critical appraisal. All studies included in the systematic review were synthesized in a narrative report, but studies rated as high and medium/high validity were reported separately from studies rated as low and medium/low or unclear validity. In addition, we ran a binary logistic regression analysis to verify five factors that may affect the occurrence of off-target effects: (1) Number of mismatches (2) Position of mismatches (3) GC-content of the targeting sequence (4) Altered nuclease variants (5) Delivery methods. In total, 180 relevant articles were included in this review containing 468 studies therein. Seventy nine percentage of these studies were rated as having high or medium/high validity. Within these studies, 6,416 potential off-target sequences were assessed for the occurrence of off-target effects. Results clearly indicate that an increased number of mismatches between the on-target and potential off-target sequence steeply decreases the likelihood of off-target effects. The observed rate of off-target effects decreased from 59% when there is one mismatch between the on-target and off-target sequences toward 0% when four or more mismatches exist. In addition, mismatch/es located within the first eight nucleotides proximal to the PAM significantly decreased the occurrence of off-target effects. There is no evidence that the GC-content significantly affects off-target effects. The database regarding the impact of the nuclease variant and the delivery method is very poor as the majority of studies applied the standard nuclease SpCas9 and the CRISPR/Cas system was stably delivered in the genome. Hence, a general significant impact of these two factors on the occurrence of off-target effects cannot be proved. This identified evidence gap needs to be filled by systematic studies exploring these individual factors in sufficient numbers.

Genome Edited Crops Touch the Market: A View on the Global Development and Regulatory Environment.

Products of genome editing as the most promising "New Plant Breeding Technology" (NPBT) have made the transition from the lab to the market in a short time. Globally, research activities employing genome editing are constantly expanding and more and more plants with market-oriented traits are being developed, and companies have already released the first genome edited crops to the market. Few countries, most of which are located in the Americas, have adapted legislations to these technologies or released guidelines supporting the use of genome editing. Other countries are debating the path to come either because there is no clarity on the legal classification or due consensus is hampered by a renewed GMO debate. In recent years (2017-2020), eight countries have introduced guidelines clarifying the legal status of genome edited products and many of those are actively committed to international harmonization of their policies. In this publication we give an overview on the current and potentially future international regulatory environment and an update on plants derived by genome editing with market-oriented traits.

Questions Regarding the Implementation of EU Mutagenesis Ruling in France.

The European Commission has asked EU Member States for comments on a French law notification demanding plant varieties produced with the help of in vitro mutagenesis have to be eliminated from the national catalog of approved varieties because of missing legal authorization deemed required by genetic engineering law. Primary target are herbicide-tolerant Clearfield oilseed rape varieties. The scientific reasoning is questionable, traceability is illusive, and law enforcement is likely to be impossible.

Genome-edited plants in the field.

The application of site directed nucleases (SDN) for Genome Editing (GE) in plant breeding and research increases exponentially in the last few years. The main research so far was on 'proof of concept' studies or improvement of the precision and delivery of the SDN. Nevertheless, a reasonable amount of research is present on market-oriented applications for cash crops such as rice but also for commercially lesser interesting crops and vegetables. Reported field trials involving GE plants are scarce around the world and almost not existing in Europe. This is due to the regulatory landscape for GE plants, which is quite distinct and especially in the European Union very demanding. By far the most field trials involve GE rice varieties in the Asian area, followed up by tomato and other vegetables and crops.

GeMoMa: Homology-Based Gene Prediction Utilizing Intron Position Conservation and RNA-seq Data.

GeMoMa is a homology-based gene prediction program that predicts gene models in target species based on gene models in evolutionary related reference species. GeMoMa utilizes amino acid sequence conservation, intron position conservation, and RNA-seq data to accurately predict protein-coding transcripts. Furthermore, GeMoMa supports the combination of predictions based on several reference species allowing to transfer high-quality annotation of different reference species to a target species. Here, we present a detailed description of GeMoMa modules and the GeMoMa pipeline and how they can be used on the command line to address particular biological problems.

Detection and Identification of Genome Editing in Plants: Challenges and Opportunities.

Conventional genetic engineering techniques generate modifications in the genome via stable integration of DNA elements which do not occur naturally in this combination. Therefore, the resulting organisms and (most) products thereof can unambiguously be identified with event-specific PCR-based methods targeting the insertion site. New breeding techniques such as genome editing diversify the toolbox to generate genetic variability in plants. Several of these techniques can introduce single nucleotide changes without integrating foreign DNA and thereby generate organisms with intended phenotypes. Consequently, such organisms and products thereof might be indistinguishable from naturally occurring or conventionally bred counterparts with established analytical tools. The modifications can entirely resemble random mutations regardless of being spontaneous or induced chemically or via irradiation. Therefore, if an identification of these organisms or products thereof is demanded, a new challenge will arise for (official) seed, food, and feed testing laboratories and enforcement institutions. For detailed consideration, we distinguish between the detection of sequence alterations - regardless of their origin - the identification of the process that generated a specific modification and the identification of a genotype, i.e., an organism produced by genome editing carrying a specific genetic alteration in a known background. This article briefly reviews the existing and upcoming detection and identification strategies (including the use of bioinformatics and statistical approaches) in particular for plants developed with genome editing techniques.

Risk Assessment and Regulation of Plants Modified by Modern Biotechniques: Current Status and Future Challenges.

This review describes the current status and future challenges of risk assessment and regulation of plants modified by modern biotechniques, namely genetic engineering and genome editing. It provides a general overview of the biosafety and regulation of genetically modified plants and details different regulatory frameworks with a focus on the European situation. The environmental risk and safety assessment of genetically modified plants is explained, and aspects of toxicological assessments are discussed, especially the controversial debate in Europe on the added scientific value of untargeted animal feeding studies. Because RNA interference (RNAi) is increasingly explored for commercial applications, the risk and safety assessment of RNAi-based genetically modified plants is also elucidated. The production, detection, and identification of genome-edited plants are described. Recent applications of modern biotechniques, namely synthetic biology and gene drives, are discussed, and a short outlook on the future follows.

New Phenotypes of Potato Co-induced by Mismatch Repair Deficiency and Somatic Hybridization.

As plants are sessile they need a very efficient system for repairing damage done by external or internal mutagens to their DNA. Mismatch repair (MMR) is one of the systems that maintain genome integrity and prevent homeologous recombination. In all eukaryotes mismatches are recognized by evolutionary conserved MSH proteins often acting as heterodimers, the constant component of which is MSH2. Changes affecting the function of MSH2 gene may induce a 'mutator' phenotype and microsatellite instability (MSI), as is demonstrated in MSH2 knock-out and silenced lines of Arabidopsis thaliana. The goal of this study was to screen for 'mutator' phenotypes in somatic hybrids between potato cvs. 'Delikat' and 'Désirée' and MMR deficient Solanum chacoense transformed using antisense (AS) or dominant negative mutant (DN) AtMSH2 genes. The results demonstrate that first generation fusion hybrids have a range of morphological abnormalities caused by uniparental MMR deficiency; these mutant phenotypes include: dwarf or gigantic plants; bushiness; curled, small, large or abnormal leaves; a deterioration in chloroplast structure; small deep-purple tubers and early dehiscent flowers. Forty percent of the viable somatic hybrids planted in a greenhouse, (10 out of 25 genotypes) had mutant phenotypes accompanied by MSI. The majority of the hybrids with 'mutator' phenotypes cultured on media containing kanamycin developed roots so sustaining the presence of selectable marker gene nptII, from the initial constructs. Here for the first time, MMR deficiency combined with somatic hybridization, are used to induce new phenotypes in plants, which supports the role of MMR deficiency in increasing introgressions between two related species.

The topoisomerase 3α zinc-finger domain T1 of Arabidopsis thaliana is required for targeting the enzyme activity to Holliday junction-like DNA repair intermediates.

Topoisomerase 3α, a class I topoisomerase, consists of a TOPRIM domain, an active centre and a variable number of zinc-finger domains (ZFDs) at the C-terminus, in multicellular organisms. Whereas the functions of the TOPRIM domain and the active centre are known, the specific role of the ZFDs is still obscure. In contrast to mammals where a knockout of TOP3α leads to lethality, we found that CRISPR/Cas induced mutants in Arabidopsis are viable but show growth retardation and meiotic defects, which can be reversed by the expression of the complete protein. However, complementation with AtTOP3α missing either the TOPRIM-domain or carrying a mutation of the catalytic tyrosine of the active centre leads to embryo lethality. Surprisingly, this phenotype can be overcome by the simultaneous removal of the ZFDs from the protein. In combination with a mutation of the nuclease AtMUS81, the TOP3α knockout proved to be also embryo lethal. Here, expression of TOP3α without ZFDs, and in particular without the conserved ZFD T1, leads to only a partly complementation in root growth-in contrast to the complete protein, that restores root length to mus81-1 mutant level. Expressing the E. coli resolvase RusA in this background, which is able to process Holliday junction (HJ)-like recombination intermediates, we could rescue this root growth defect. Considering all these results, we conclude that the ZFD T1 is specifically required for targeting the topoisomerase activity to HJ like recombination intermediates to enable their processing. In the case of an inactivated enzyme, this leads to cell death due to the masking of these intermediates, hindering their resolution by MUS81.

Combining RNA-seq data and homology-based gene prediction for plants, animals and fungi.

BACKGROUND: Genome annotation is of key importance in many research questions. The identification of protein-coding genes is often based on transcriptome sequencing data, ab-initio or homology-based prediction. Recently, it was demonstrated that intron position conservation improves homology-based gene prediction, and that experimental data improves ab-initio gene prediction. RESULTS: Here, we present an extension of the gene prediction program GeMoMa that utilizes amino acid sequence conservation, intron position conservation and optionally RNA-seq data for homology-based gene prediction. We show on published benchmark data for plants, animals and fungi that GeMoMa performs better than the gene prediction programs BRAKER1, MAKER2, and CodingQuarry, and purely RNA-seq-based pipelines for transcript identification. In addition, we demonstrate that using multiple reference organisms may help to further improve the performance of GeMoMa. Finally, we apply GeMoMa to four nematode species and to the recently published barley reference genome indicating that current annotations of protein-coding genes may be refined using GeMoMa predictions. CONCLUSIONS: GeMoMa might be of great utility for annotating newly sequenced genomes but also for finding homologs of a specific gene or gene family. GeMoMa has been published under GNU GPL3 and is freely available at http://www.jstacs.de/index.php/GeMoMa .

RecQ Helicases Function in Development, DNA Repair, and Gene Targeting in Physcomitrella patens.

RecQ DNA helicases are genome surveillance proteins found in all kingdoms of life. They are characterized best in humans, as mutations in RecQ genes lead to developmental abnormalities and diseases. To better understand RecQ functions in plants we concentrated on Arabidopsis thaliana and Physcomitrella patens, the model species predominantly used for studies on DNA repair and gene targeting. Phylogenetic analysis of the six P. patens RecQ genes revealed their orthologs in humans and plants. Because Arabidopsis and P. patens differ in their RecQ4 and RecQ6 genes, reporter and deletion moss mutants were generated and gene functions studied in reciprocal cross-species and cross-kingdom approaches. Both proteins can be found in meristematic moss tissues, although at low levels and with distinct expression patterns. PpRecQ4 is involved in embryogenesis and in subsequent development as demonstrated by sterility of ΔPpRecQ4 mutants and by morphological aberrations. Additionally, ΔPpRecQ4 displays an increased sensitivity to DNA damages and an increased rate of gene targeting. Therefore, we conclude that PpRecQ4 acts as a repressor of recombination. In contrast, PpRecQ6 is not obviously important for moss development or DNA repair but does function as a potent enhancer of gene targeting.

Regulatory hurdles for genome editing: process- vs. product-based approaches in different regulatory contexts.

Novel plant genome editing techniques call for an updated legislation regulating the use of plants produced by genetic engineering or genome editing, especially in the European Union. Established more than 25 years ago and based on a clear distinction between transgenic and conventionally bred plants, the current EU Directives fail to accommodate the new continuum between genetic engineering and conventional breeding. Despite the fact that the Directive 2001/18/EC contains both process- and product-related terms, it is commonly interpreted as a strictly process-based legislation. In view of several new emerging techniques which are closer to the conventional breeding than common genetic engineering, we argue that it should be actually interpreted more in relation to the resulting product. A legal guidance on how to define plants produced by exploring novel genome editing techniques in relation to the decade-old legislation is urgently needed, as private companies and public researchers are waiting impatiently with products and projects in the pipeline. We here outline the process in the EU to develop a legislation that properly matches the scientific progress. As the process is facing several hurdles, we also compare with existing frameworks in other countries and discuss ideas for an alternative regulatory system.

Using intron position conservation for homology-based gene prediction.

Annotation of protein-coding genes is very important in bioinformatics and biology and has a decisive influence on many downstream analyses. Homology-based gene prediction programs allow for transferring knowledge about protein-coding genes from an annotated organism to an organism of interest.Here, we present a homology-based gene prediction program called GeMoMa. GeMoMa utilizes the conservation of intron positions within genes to predict related genes in other organisms. We assess the performance of GeMoMa and compare it with state-of-the-art competitors on plant and animal genomes using an extended best reciprocal hit approach. We find that GeMoMa often makes more precise predictions than its competitors yielding a substantially increased number of correct transcripts. Subsequently, we exemplarily validate GeMoMa predictions using Sanger sequencing. Finally, we use RNA-seq data to compare the predictions of homology-based gene prediction programs, and find again that GeMoMa performs well.Hence, we conclude that exploiting intron position conservation improves homology-based gene prediction, and we make GeMoMa freely available as command-line tool and Galaxy integration.

Plant genome editing by novel tools: TALEN and other sequence specific nucleases.

Genome editing technologies using sequence specific nucleases (SSNs) became a tremendously powerful and precise tool for reverse genetic approaches and applied biology. Transcription activator-like effector nucleases (TALENs) in particular, consisting of a free designable DNA binding domain and a nuclease, have been exploited today by a huge number of approaches in many different organisms. The convenience of designing the DNA binding domain and straightforward protocols for their assembly, as well as the broad number of applications in different scientific fields made it Natures method of the year 2011. TALENs act as molecular scissors by introducing double strand breaks (DSBs) to the DNA at a given location. The DSBs are subsequently repaired by the cell itself using different repair pathways such as non-homologous end joining (NHEJ) or homologous recombination (HR). These mechanisms can lead to deletions, insertions, replacements or larger chromosomal rearrangements. By offering a template DNA it is possible to channel the repair in direction of HR. In this article we review the recent findings in the field of SSN approaches with emphasis on plants.

The splicing fate of plant SPO11 genes.

Toward the global understanding of plant meiosis, it seems to be essential to decipher why all as yet sequenced plants need or at least encode for two different meiotic SPO11 genes. This is in contrast to mammals and fungi, where only one SPO11 is present. Both SPO11 in Arabidopsis thaliana are essential for the initiation of double strand breaks (DSBs) during the meiotic prophase. In nearly all eukaryotic organisms DSB induction during prophase I by SPO11 leads to meiotic DSB repair, thereby ensuring the formation of a necessary number of crossovers (CO) as physical connections between the homologous chromosomes. We aim to investigate the specific functions and evolution of both SPO11 genes in land plants. Therefore, we identified and cloned the respective orthologous genes from Brassica rapa, Carica papaya, Oryza sativa, and Physcomitrella patens. In parallel we determined the full length cDNA sequences of SPO11-1 and -2 from all of these plants by RT-PCR. During these experiments we observed that the analyzed plants exhibit a pattern of alternative splicing products of both SPO11 mRNAs. Such an aberrant splicing has previously been described for Arabidopsis and therefore seems to be conserved throughout evolution. Most of the splicing forms of SPO11-1 and -2 seem to be non-functional as they either showed intron retention (IR) or shortened exons. However, the positional distribution and number of alternative splicing events vary strongly between the different plants. The cDNAs showed in most cases premature termination codons (PTCs) due to frameshift. Nevertheless, in some cases we found alternatively spliced but functional cDNAs. These findings let us suggest that alternative splicing of SPO11 depends on the respective gene sequence and on the plant species. Therefore, this conserved mechanism might play a role concerning regulation of SPO11.

Precise plant breeding using new genome editing techniques: opportunities, safety and regulation in the EU.

Several new plant breeding techniques (NPBTs) have been developed during the last decade, and make it possible to precisely perform genome modifications in plants. The major problem, other than technical aspects, is the vagueness of regulation concerning these new techniques. Since the definition of eight NPBTs by a European expert group in 2007, there has been an ongoing debate on whether the resulting plants and their products are covered by GMO legislation. Obviously, cover by GMO legislation would severely hamper the use of NPBT, because genetically modified plants must pass a costly and time-consuming GMO approval procedure in the EU. In this review, we compare some of the NPBTs defined by the EU expert group with classical breeding techniques and conventional transgenic plants. The list of NPBTs may be shortened (or extended) during the international discussion process initiated by the Organization for Economic Co-operation and Development. From the scientific point of view, it may be argued that plants developed by NPBTs are often indistinguishable from classically bred plants and are not expected to possess higher risks for health and the environment. In light of the debate on the future regulation of NPBTs and the accumulated evidence on the biosafety of genetically modified plants that have been commercialized and risk-assessed worldwide, it may be suggested that plants modified by crop genetic improvement technologies, including genetic modification, NPBTs or other future techniques, should be evaluated according to the new trait and the resulting end product rather than the technique used to create the new plant variety.

Defining the roles of the N-terminal region and the helicase activity of RECQ4A in DNA repair and homologous recombination in Arabidopsis.

RecQ helicases are critical for the maintenance of genomic stability. The Arabidopsis RecQ helicase RECQ4A is the functional counterpart of human BLM, which is mutated in the genetic disorder Bloom's syndrome. RECQ4A performs critical roles in regulation of homologous recombination (HR) and DNA repair. Loss of RECQ4A leads to elevated HR frequencies and hypersensitivity to genotoxic agents. Through complementation studies, we were now able to demonstrate that the N-terminal region and the helicase activity of RECQ4A are both essential for the cellular response to replicative stress induced by methyl methanesulfonate and cisplatin. In contrast, loss of helicase activity or deletion of the N-terminus only partially complemented the mutant hyper-recombination phenotype. Furthermore, the helicase-deficient protein lacking its N-terminus did not complement the hyper-recombination phenotype at all. Therefore, RECQ4A seems to possess at least two different and independent sub-functions involved in the suppression of HR. By in vitro analysis, we showed that the helicase core was able to regress an artificial replication fork. Swapping of the terminal regions of RECQ4A with the closely related but functionally distinct helicase RECQ4B indicated that in contrast to the C-terminus, the N-terminus of RECQ4A was required for its specific functions in DNA repair and recombination.

Different functions for the domains of the Arabidopsis thaliana RMI1 protein in DNA cross-link repair, somatic and meiotic recombination.

Recombination intermediates, such as double Holliday junctions, can be resolved by nucleases or dissolved by the combined action of a DNA helicase and a topoisomerase. In eukaryotes, dissolution is mediated by the RTR complex consisting of a RecQ helicase, a type IA topoisomerase and the structural protein RecQ-mediated genome instability 1 (RMI1). Throughout eukaryotes, the RTR complex is involved in DNA repair and in the suppression of homologous recombination (HR) in somatic cells. Surprisingly, Arabidopsis thaliana mutants of topoisomerase 3α and RMI1 are also sterile due to extensive chromosome breakage in meiosis I, indicating that both proteins are essential for meiotic recombination in plants. AtRMI1 harbours an N-terminal DUF1767 domain and two oligosaccharide binding (OB)-fold domains. To define specific roles for these individual domains, we performed complementation experiments on Atrmi1 mutants with an AtRMI1 full-length open reading frame (ORF) or deletion constructs lacking specific domains. We show that the DUF1767 domain and the OB-fold domain 1 are both essential for the function of AtRMI1 in DNA cross-link repair as well as meiotic recombination, but partially dispensable for somatic HR suppression. The OB-fold domain 2 is not necessary for either somatic or meiotic HR, but it seems to have a minor function in DNA cross-link repair.