An efficient multiplex approach to CRISPR/Cas9 gene editing in citrus.
CRISPR/Cas9
Citrus
Gene editing
Multiplex
Vector design
Journal
Plant methods
ISSN: 1746-4811
Titre abrégé: Plant Methods
Pays: England
ID NLM: 101245798
Informations de publication
Date de publication:
28 Sep 2024
28 Sep 2024
Historique:
received:
28
06
2024
accepted:
13
09
2024
medline:
29
9
2024
pubmed:
29
9
2024
entrez:
28
9
2024
Statut:
epublish
Résumé
CRISPR/Cas9-mediated gene editing requires high efficiency to be routinely implemented, especially in species which are laborious and slow to transform. This requirement intensifies further when targeting multiple genes simultaneously, which is required for genetic screening or more complex genome engineering. Species in the Citrus genus fall into this category. Here we describe a series of experiments with the collective aim of improving multiplex gene editing in the Carrizo citrange cultivar using tRNA-based sgRNA arrays. We evaluate a range of promoters for their efficacy in such experiments and achieve significant improvements by optimizing the expression of both the Cas9 endonuclease and the sgRNA array. In the case of the former we find the UBQ10 or RPS5a promoters from Arabidopsis driving the zCas9i endonuclease variant useful for achieving high levels of editing. The choice of promoter expressing the sgRNA array also had a large impact on gene editing efficiency across multiple targets. In this respect Pol III promoters perform especially well, but we also demonstrate that the UBQ10 and ES8Z promoters from Arabidopsis are robust alternatives. Ultimately, this study provides a quantitative insight into CRISPR/Cas9 vector design that has practical application in the simultaneous editing of multiple genes in Citrus, and potentially other eudicot plant species.
Identifiants
pubmed: 39342225
doi: 10.1186/s13007-024-01274-4
pii: 10.1186/s13007-024-01274-4
doi:
Types de publication
Journal Article
Langues
eng
Pagination
148Subventions
Organisme : National Institute of Food and Agriculture
ID : 2020-70029-33160
Organisme : National Institute of Food and Agriculture
ID : 2023-70029-41277
Organisme : National Institute of Food and Agriculture
ID : 2023-70029-41277
Organisme : National Institute of Food and Agriculture
ID : 2020-70029-33160
Organisme : National Institute of Food and Agriculture
ID : 2020-70029-33160
Organisme : National Institute of Food and Agriculture
ID : 2020-70029-33160
Organisme : Citrus Research Board
ID : 5200-166
Organisme : Citrus Research Board
ID : 5200-166
Informations de copyright
© 2024. The Author(s).
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