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)
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CRISPR/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.
Comparison of two different transient transformation methods for the validation of sgRNA in red cabbage (B. oleracea var. capitata f. rubra).
KeywordsCRISPR/Cas9 Brassica oleracea Agroinfiltration Protoplast transfection Genome editing
This work was supported by the Slovenian Research Agency (research programme P4-0077 and PhD student grant 1000-14-0510).
BB and JM conceived and designed the research, ES performed the experiments. ES and JM analysed the results, AK contributed to development of protoplast isolation protocol. ES wrote the manuscript. All authors read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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