Deep sequencing analysis of CRISPR/Cas9 induced mutations by two delivery methods in target model genes and the CENH3 region of red cabbage (Brassica oleracea var. capitata f. rubra)
Ester Stajič , Agnieszka Kiełkowska , Jana Murovec , Borut Bohanec
AbstractCRISPR/Cas9 is a versatile and highly efficient genome editing tool used in many different plant species. In the present study, we compared the two most commonly used transient expression methods for genome editing, protoplast transfection and infiltration of Agrobacterium tumefaciens, to develop a rapid and efficient validation protocol. Vectors designed to target four different sites in the cabbage genome (two of which were model target genes and two related to the centromere-specific histone H3 (CENH3) gene) were delivered to two red cabbage cultivars, ‘Huzaro F1’ and ‘Rebecca F1’. Targeted deep sequencing analysis showed that CRISPR/Cas9 vectors induced mutations in both cultivars at all target sites and revealed mutation rates of 1.27–11.95% for protoplast transfection and 0.07–14.42% for agroinfiltration. Our results demonstrate successful genome editing in cabbages with CRISPR/Cas9 by two different approaches for the rapid evaluation of genome editing efficiency.
|Journal series||Plant Cell Tissue and Organ Culture, [Plant Cell, Tissue and Organ Culture], ISSN 0167-6857, e-ISSN 1573-5044, (N/A 100 pkt)|
|Publication size in sheets||0.5|
|Keywords in English||CRISPR/Cas9, Brassica oleracea, Agroinfiltration, Protoplast transfection, Genome editing|
|Publication indicators||= 1; : 2017 = 1.122; : 2018 = 2.2 (2) - 2018=2.232 (5)|
|Finansowanie||This work was supported by the Slovenian Research Agency (research programme P4-0077 and PhD student grant 1000-14-0510).|
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