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Targeted gene deletion with SpCas9 and multiple guide RNAs in Arabidopsis thaliana: four are better than two.
Ordon, Jana; Kiel, Niklas; Becker, Dieter; Kretschmer, Carola; Schulze-Lefert, Paul; Stuttmann, Johannes.
Afiliação
  • Ordon J; Department of Plant-Microbe Interactions, Max-Planck Institute for Plant Breeding Research, D50829, Cologne, Germany.
  • Kiel N; Department of Plant-Microbe Interactions, Max-Planck Institute for Plant Breeding Research, D50829, Cologne, Germany.
  • Becker D; Cluster of Excellence on Plant Sciences (CEPLAS), Max Planck Institute for Plant Breeding Research, Cologne, Germany.
  • Kretschmer C; Department of Plant-Microbe Interactions, Max-Planck Institute for Plant Breeding Research, D50829, Cologne, Germany.
  • Schulze-Lefert P; Institute for Biology, Department of Plant Genetics, Martin Luther University Halle-Wittenberg, D06120, Halle, Germany.
  • Stuttmann J; Department of Plant-Microbe Interactions, Max-Planck Institute for Plant Breeding Research, D50829, Cologne, Germany.
Plant Methods ; 19(1): 30, 2023 Mar 28.
Article em En | MEDLINE | ID: mdl-36978193
ABSTRACT

BACKGROUND:

In plant genome editing, RNA-guided nucleases such as Cas9 from Streptococcus 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 segment to be deleted. Experimental approaches for the deletion of larger chromosomal segments have not been systematically evaluated.

RESULTS:

We designed three pairs of guide RNAs for deletion of a ~ 2.2 kb chromosomal segment containing the Arabidopsis WRKY30 locus. We tested how the combination of guide RNA pairs and co-expression of the exonuclease TREX2 affect the frequency of wrky30 deletions 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 segment deletions.

CONCLUSIONS:

Multiplex editing with at least two pairs of guide RNAs (four guide RNAs in total) elevates the frequency of chromosomal segment deletions at least at the AtWRKY30 locus, 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.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Ano de publicação: 2023 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Ano de publicação: 2023 Tipo de documento: Article