Cas12a-mediated gene targeting by sequential transformation strategy in Arabidopsis thaliana.
Arabidopsis thaliana
Cas12a
Enhancer
Gene targeting
Genome engineering
Sequential transformation
Journal
BMC plant biology
ISSN: 1471-2229
Titre abrégé: BMC Plant Biol
Pays: England
ID NLM: 100967807
Informations de publication
Date de publication:
12 Jul 2024
12 Jul 2024
Historique:
received:
03
04
2024
accepted:
04
07
2024
medline:
13
7
2024
pubmed:
13
7
2024
entrez:
12
7
2024
Statut:
epublish
Résumé
Gene targeting (GT) allows precise manipulation of genome sequences, such as knock-ins and sequence substitutions, but GT in seed plants remains a challenging task. Engineered sequence-specific nucleases (SSNs) are known to facilitate GT via homology-directed repair (HDR) in organisms. Here, we demonstrate that Cas12a and a temperature-tolerant Cas12a variant (ttCas12a) can efficiently establish precise and heritable GT at two loci in Arabidopsis thaliana (Arabidopsis) through a sequential transformation strategy. As a result, ttCas12a showed higher GT efficiency than unmodified Cas12a. In addition, the efficiency of transcriptional and translational enhancers for GT via sequential transformation strategy was also investigated. These enhancers and their combinations were expected to show an increase in GT efficiency in the sequential transformation strategy, similar to previous reports of all-in-one strategies, but only a maximum twofold increase was observed. These results indicate that the frequency of double strand breaks (DSBs) at the target site is one of the most important factors determining the efficiency of genetic GT in plants. On the other hand, a higher frequency of DSBs does not always lead to higher efficiency of GT, suggesting that some additional factors are required for GT via HDR. Therefore, the increase in DSB can no longer be expected to improve GT efficiency, and a new strategy needs to be established in the future. This research opens up a wide range of applications for precise and heritable GT technology in plants.
Identifiants
pubmed: 38997669
doi: 10.1186/s12870-024-05375-z
pii: 10.1186/s12870-024-05375-z
doi:
Substances chimiques
CRISPR-Associated Proteins
0
Cas12a protein
EC 3.1.-
Bacterial Proteins
0
Endodeoxyribonucleases
EC 3.1.-
Types de publication
Journal Article
Editorial
Langues
eng
Sous-ensembles de citation
IM
Pagination
665Informations de copyright
© 2024. The Author(s).
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