Targeted gene deletion with Sp Cas9 and multiple guide RNAs in Arabidopsis thaliana : four are better than two
| Autor: | Jana Ordon, Niklas Kiel, Dieter Becker, Carola Kretschmer, Paul Schulze-Lefert, Johannes Stuttmann |
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| Přispěvatelé: | Max Planck Institute for Plant Breeding Research (MPIPZ), Heinrich Heine Universität Düsseldorf = Heinrich Heine University [Düsseldorf], Martin-Luther-University Halle-Wittenberg, Laboratoire d'Ecologie Microbienne de la Rhizosphère et d'Environnements Extrêmes (LEMIRE), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Julius Kühn-Institut (JKI) |
| Jazyk: | angličtina |
| Rok vydání: | 2023 |
| Předmět: | |
| Zdroj: | bioRxiv-PRE bioRxiv-PREPRINT, 2023, ⟨10.1101/2023.01.10.523375⟩ |
| DOI: | 10.1101/2023.01.10.523375⟩ |
| Popis: | BackgroundIn plant genome editing, RNA-guided nucleases such as Cas9 fromStreptococcus pyogenes(SpCas9) predominantly induce small insertions or deletions at target sites. This can be used for inactivation of protein-coding genes by frame shift mutations. However, in some cases, it may be advantageous to delete larger chromosomal segments. This is achieved by simultaneously inducing double strand breaks upstream and downstream of the fragment to be deleted. Experimental approaches for deletion induction have not been systematically evaluated.ResultsWe designed three pairs of guide RNAs for deletion of the ArabidopsisWRKY30locus (~2.2 kb). We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency ofwrky30deletions in editing experiments. Our data demonstrate that compared to one pair of guide RNAs, two pairs increase the frequency of chromosomal deletions. The exonuclease TREX2 enhanced mutation frequency at individual target sites and shifted the mutation profile towards larger deletions. However, TREX2 did not elevate the frequency of chromosomal deletions.ConclusionsMultiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal deletions, and thus simplifies the selection of corresponding mutants. Co-expression of the TREX2 exonuclease can be used as a general strategy to increase editing efficiency in Arabidopsis without obvious negative effects. |
| Databáze: | OpenAIRE |
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