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Generation of targeted mutagenesis in sugar beet using geminiviral replicons for delivering CRISPR/Cas systems

  • Autor/in: Eini, O., N. Schumann, M. Niessen, M. Varrelmann
  • Jahr: 2021
  • Zeitschrift: Abstracts Oral Presentations and Posters Presented at the 2021 General Meeting of the ASSBT Virtual Meeting March 1 – March 4, 2021
  • Seite/n: 80-81, DOI: 10.5274/JSBR.58.1.57


Genome editing (GE) tools like the type II clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system possess the potential to accelerate crop improvement by providing the means to modify genomes (gene disruption, correction or insertion) rapidly in a precise and predictable manner. The efficacy of the entire process, however, is critical and depends on efficient delivery of the editing components, which are the sequence-specific nucleases (SSN) and the single guide RNA (sgRNA). Geminiviruses (Family Geminiviridae) are plant viruses with circular single stranded DNA genomes that rely on host DNA replication machinery for their genome amplification. Therefore, they need to change the host cell cycle into S phase, in which DNA replication and homology-directed DNA recombination (HDR) occur. This study aims to establish targeted gene mutation in sugar beet by means of Geminivirus based plant virus vectors for the efficient delivery of SSN and sgRNA for mutagenesis in Acetlactate synthetase 1(ALS1) gene. We have prepared two geminiviral replicons (GVRs) from Beet curly top virus (BCTV-Svr) and Beet curly top Iran virus (BCTIV) by removing the virion sense genes. Their replication efficacy was compared with wild type virus. The BCTIV replicon accumulates at a lower level as compared with wild type BCTIV. However, the replicon from BCTV-Svr accumulates more efficiently in plants tissues as compared to the wild type virus. Both replicons provide a large cargo capacity up to 7 Kb which enable more efficient delivery of CRISPR/Cas components. The efficacy for targeted mutagenesis in ALS1 gene using both replicons was comparable to the conventional T-DNA system. This demonstrates application of geminiviral replicons for efficient delivery of CRISPR/Cas components into sugar beet and provides a framework for future studies in sugar beet and other plants.
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