Evidence synthesis on the impact of genome editing on plant breeding
von Dominik Modrzejewski
Datum der mündl. Prüfung:2020-07-15
Erschienen:2020-11-27
Betreuer:Prof. Dr. Achim Spiller
Gutachter:Dr. Ralf Wilhelm
Gutachter:Prof. Dr. Anne-Katrin Mahlein
Gutachter:Prof. Dr. Stefan Scholten
Dateien
Name:201118 Doktorarbeit_Modrzejewski_Final_Ohne ...pdf
Size:2.79Mb
Format:PDF
Zusammenfassung
Englisch
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 of off-target effects. There was no clear indication that the occurrence of off-target effects shows an obvious trend in relation to the GC-content of the protospacer sequence. Regarding the nuclease variant and the delivery method the given data base is considerably poor, as the large majority of studies applied the standard nuclease SpCas9 and the CRISPR/Cas system was stably delivered in the genome. Only a limited number of studies applied altered nuclease variants and delivery methods. Hence, a general significant impact of the nuclease variants and the delivery method on the occurrence of off-target effects cannot be proved. Based on the results of this study, in order to minimize off-target effects, it is recommended to select a target sequence which differs in at least four disjunct positions from similar genomic sequences.
Keywords: Genome Editing; CRISPR/Cas; Off-target; Systematic Map; Systematic Review; New plant breeding technique