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.
Background: Within the last decades, genome-editing techniques such as CRISPR/Cas, TALENs, Zinc-Finger Nucleases, Meganucleases, Oligonucleotide-Directed Mutagenesis and base editing have been developed enabling a precise modification of DNA sequences. Such techniques provide options for simple, time-saving and cost-effective applications compared to other breeding techniques and hence genome editing has already been promoted for a wide range of plant species. Although the application of genome-editing induces less unintended modifications (off-targets) in the genome compared to classical mutagenesis techniques, off-target effects are a prominent point of criticism as they are supposed to cause unintended effects, e.g. genomic instability or cell death. To address these aspects, this map aims to answer the following question: What is the available evidence for the range of applications of genome-editing as a new tool for plant trait modification and the potential occurrence of associated off-target effects? This primary question will be considered by two secondary questions: One aims to overview the market-oriented traits being modified by genome-editing in plants and the other explores the occurrence of off-target effects. Methods: A literature search in nine bibliographic databases, Google Scholar, and 47 web pages of companies and governmental agencies was conducted using predefined and tested search strings in English language. Articles were screened on title/abstract and full text level for relevance based on pre-defined inclusion criteria. The relevant information of included studies were mapped using a pre-defined data extraction strategy. Besides a descriptive summary of the relevant literature, a spreadsheet containing all extracted data is provided. Results: Altogether, 555 relevant articles from journals, company web pages and web pages of governmental agencies were identified containing 1328 studies/applications of genome-editing in model plants and agricultural crops in the period January 1996 to May 2018. Most of the studies were conducted in China followed by the USA. Genomeediting was already applied in 68 different plants. Although most of the studies were basic research, 99 different market-oriented applications were identified in 28 different crops leading to plants with improved food and feed quality, agronomic value like growth characteristics or increased yield, tolerance to biotic and abiotic stress, herbicide tolerance or industrial benefits. 252 studies explored off-target effects. Most of the studies were conducted using CRISPR/ Cas. Several studies firstly investigated whether sites in the genome show similarity to the target sequence and secondly analyzed these potential off-target sites by sequencing. In around 3% of the analyzed potential off-target
Genome Editing using engineered endonuclease (GEEN) systems rapidly took over the field of plant science and plant breeding. So far, Genome Editing techniques have been applied in more than fifty different plants; including model species like Arabidopsis; main crops like rice, maize or wheat as well as economically less important crops like strawberry, peanut and cucumber. These techniques have been used for basic research as proof-of-concept or to investigate gene functions in most of its applications. However, several market-oriented traits have been addressed including enhanced agronomic characteristics, improved food and feed quality, increased tolerance to abiotic and biotic stress and herbicide tolerance. These technologies are evolving at a tearing pace and especially the field of CRISPR based Genome Editing is advancing incredibly fast. CRISPR-Systems derived from a multitude of bacterial species are being used for targeted Gene Editing and many modifications have already been applied to the existing CRISPR-Systems such as (i) alter their protospacer adjacent motif (ii) increase their specificity (iii) alter their ability to cut DNA and (iv) fuse them with additional proteins. Besides, the classical transformation system using Agrobacteria tumefaciens or Rhizobium rhizogenes, other transformation technologies have become available and additional methods are on its way to the plant sector. Some of them are utilizing solely proteins or protein-RNA complexes for transformation, making it possible to alter the genome without the use of recombinant DNA. Due to this, it is impossible that foreign DNA is being incorporated into the host genome. In this review we will present the recent developments and techniques in the field of DNA-free Genome Editing, its advantages and pitfalls and give a perspective on technologies which might be available in the future for targeted Genome Editing in plants. Furthermore, we will discuss these techniques in the light of existing– and potential future regulations.
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.
Background: Plant breeding is a developing process and breeding methods have continuously evolved over time. In recent years, genome editing techniques such as clustered regularly interspaced short palindromic repeats/CRISPR associated proteins (CRISPR/Cas), transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFN), meganucleases (MN) and oligonucleotide-directed mutagenesis (ODM) enabled a precise modification of DNA sequences in plants. Genome editing has already been applied in a wide range of plant species due to its simplicity, time saving and cost-effective application compared to earlier breeding techniques including classical mutagenesis. Although genome editing techniques induce much less unintended modifications in the genome (off-target effects) compared to classical mutagenesis techniques, off-target effects are a prominent point of criticism as they might cause genomic instability, cytotoxicity and cell death. Methods: The aim of this systematic map is to address the following primary question: "What is the available evidence for the application of genome editing as a new tool for plant trait modification and the potential occurrence of associated off-target effects"? The primary question will be considered by two secondary questions: One is aimed at the traits being modified by genome editing in plants and the other explores the occurrence of off-target effects. The systematic map will focus on model plants as well as on plants produced for agricultural production that were subjected to genome editing techniques. Academic and grey literature will be searched in English and German language. Inclusion/exclusion criteria were developed for the two secondary questions and will be applied on title/abstract and full text stage. Included studies will be catalogued in a searchable and open access database and study results will be summarized using descriptive statistics. Furthermore, the extracted data will serve as a preparatory step for further in-depth analysis, e.g. by a systematic review.
Genome editing techniques such as CRISPR/Cas, TALENs, Zinc-Finger Nucleases, Meganucleases, Oligonucleotide-Directed Mutagenesis and Base editing enable a targeted modification of DNA sequences in a site-directed manner. Despite their simple, time-saving and cost-effective use, there is an ongoing debate about their precise targeting and the extent to which off-target effects occur and matter. Off-target effects are unintended cleavage and mutations at untargeted genomic sites showing similar but not identical sequences compared to the target one. The number of research articles about genome editing in plants is increasing rapidly and evidence synthesis of these articles play an important role in controlling the rapidly expanding evidence base and contributing to evidence-based decision-making. The overall objective of this study was to systematically synthesize and evaluate the evidence on the impact of genome editing in plants. For this purpose, a multi stage conceptual approach was chosen. In a first step, a protocol was developed in order to make the review process more rigorous, transparent and well-defined as well as to minimize reviewer bias. Based on the protocol a systematic map was conducted aiming to provide a comprehensive overview on the applications of genome editing and the potential occurrence of associated off-target effects. The main findings of this map are that although most of the studies are categorized as basic research, nearly 100 market-oriented applications were identified in 28 different crops leading to plants with improved food and feed quality, agronomic value like growth characteristics or increased yield, tolerance to biotic and abiotic stress, herbicide tolerance or industrial benefits. Results further indicate that the large majority of off-target studies were conducted using CRISPR/Cas. Biased detection methods are the dominant approach to detect off-target effects, meaning that potential off-target sequences are first predicted using bioinformatics programs followed by targeted sequencing of these sites to track the occurrence of off-target effects. Off-target effects were detected in several studies. However, studies investigating potential off-target sequences are very heterogeneous in their structure and design. Based on these findings, the objective of the follow up systematic review was to identify and systematically analyze factors that may affect the occurrence of off-target effects. Results of the analysis clearly indicate that an increased number of mismatches between the on-target and potential off-target sequence significantly decreases the occurrence of off-target effects. The observed off-target rate decreases from 59% when there was one mismatch between the on-target and potential off-target sequence towards 0% when at least four mismatches exist. In addition, mismatch/es located within the first eight nucleotides proximal to the PAM significantly decrease the occurrence of off-target effects and there is a tendency that adjoined mismatches increase the likelihood...
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