An optimised CRISPR/Cas9 protocol to create targeted mutations in homoeologous genes and an efficient genotyping protocol to identify edited events in wheat.
CRISPR/Cas9
Gene editing in homoeologous genes
Genotyping
Transgenic plants
Wheat
sgRNA
Journal
Plant methods
ISSN: 1746-4811
Titre abrégé: Plant Methods
Pays: England
ID NLM: 101245798
Informations de publication
Date de publication:
2019
2019
Historique:
received:
20
06
2019
accepted:
03
10
2019
entrez:
2
11
2019
pubmed:
2
11
2019
medline:
2
11
2019
Statut:
epublish
Résumé
Targeted genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been applied in a large number of plant species. Using a gene-specific single guide RNA (sgRNA) and the CRISPR/Cas9 system, small editing events such as deletions of few bases can be obtained. However larger deletions are required for some applications. In addition, identification and characterization of edited events can be challenging in plants with complex genomes, such as wheat. In this study, we used the CRISPR/Cas9 system and developed a protocol that yielded high number of large deletions employing a pair of co-expressed sgRNA to target the same gene. The protocol was validated by targeting three genes, Co-expressed pairs of sgRNA targeting single genes in conjunction with the CRISPR/Cas9 system produced large deletions in wheat. In addition, a genotyping protocol to identify editing events in homoeologs of
Sections du résumé
BACKGROUND
BACKGROUND
Targeted genome editing using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been applied in a large number of plant species. Using a gene-specific single guide RNA (sgRNA) and the CRISPR/Cas9 system, small editing events such as deletions of few bases can be obtained. However larger deletions are required for some applications. In addition, identification and characterization of edited events can be challenging in plants with complex genomes, such as wheat.
RESULTS
RESULTS
In this study, we used the CRISPR/Cas9 system and developed a protocol that yielded high number of large deletions employing a pair of co-expressed sgRNA to target the same gene. The protocol was validated by targeting three genes,
CONCLUSIONS
CONCLUSIONS
Co-expressed pairs of sgRNA targeting single genes in conjunction with the CRISPR/Cas9 system produced large deletions in wheat. In addition, a genotyping protocol to identify editing events in homoeologs of
Identifiants
pubmed: 31673276
doi: 10.1186/s13007-019-0500-2
pii: 500
pmc: PMC6814032
doi:
Types de publication
Journal Article
Langues
eng
Pagination
119Commentaires et corrections
Type : ErratumIn
Informations de copyright
© The Author(s) 2019.
Déclaration de conflit d'intérêts
Competing interestsThe authors declare that they have no competing interests.
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