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

Abstract

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.
Author Ester Stajič
Ester Stajič,,
-
, Agnieszka Kiełkowska (FoBaH / IoPBaB / DoGPBaSP)
Agnieszka Kiełkowska,,
- Department of Genetics, Plant Breeding and Seed Production
, Jana Murovec
Jana Murovec,,
-
, Borut Bohanec
Borut Bohanec,,
-
Journal seriesPlant Cell Tissue and Organ Culture, [Plant Cell, Tissue and Organ Culture], ISSN 0167-6857, e-ISSN 1573-5044, (N/A 100 pkt)
Issue year2019
Vol139
No2
Pages227-235
Publication size in sheets0.5
Keywords in EnglishCRISPR/Cas9, Brassica oleracea, Agroinfiltration, Protoplast transfection, Genome editing
ASJC Classification1108 Horticulture
DOIDOI:10.1007/s11240-019-01665-9
URL https://link.springer.com/content/pdf/10.1007%2Fs11240-019-01665-9.pdf
Languageen angielski
Score (nominal)100
Score sourcejournalList
Publication indicators WoS Citations = 1; Scopus SNIP (Source Normalised Impact per Paper): 2017 = 1.122; WoS Impact Factor: 2018 = 2.2 (2) - 2018=2.232 (5)
Citation count*
Additional fields
FinansowanieThis work was supported by the Slovenian Research Agency (research programme P4-0077 and PhD student grant 1000-14-0510).
Cite
Share Share

Get link to the record


* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.
Back
Confirmation
Are you sure?