PMID- 30610638
OWN - NLM
STAT- MEDLINE
DCOM- 20190617
LR  - 20240104
IS  - 1940-6029 (Electronic)
IS  - 1064-3745 (Linking)
VI  - 1917
DP  - 2019
TI  - Visual Assay for Gene Editing Using a CRISPR/Cas9 System in Carrot Cells.
PG  - 203-215
LID - 10.1007/978-1-4939-8991-1_15 [doi]
AB  - The development of the Clustered Regularly Interspersed Short Palindromic Repeats 
      (CRISPR)/CRISPR-associated (Cas9) system has advanced genome editing and has 
      become widely adopted for this purpose in many species. Its efficient use 
      requires the method adjustment and optimization. Here, we show the use of a model 
      carrot callus system for demonstrating gene editing via CRISPR/Cas9 targeted 
      mutagenesis. The system relies on the utilization of carrot tissue accumulating 
      anthocyanin pigments responsible for a deep purple cell color and generation of 
      knockout mutations in the flavanone-3-hydroxylase (F3H) gene in the anthocyanin 
      biosynthesis pathway. F3H mutant cells targeted by Cas9/gRNA complexes are not 
      able to synthesize anthocyanins and remain white, easily visually distinguished 
      from purple wild-type cells. Mutations are either small indels or larger 
      chromosomal deletions that can be identified by restriction fragment analysis and 
      sequencing. This feasible system can also be applied for validating efficiency of 
      CRISPR/Cas9 vectors.
FAU - Klimek-Chodacka, Magdalena
AU  - Klimek-Chodacka M
AD  - Faculty of Biotechnology and Horticulture, Institute of Plant Biology and 
      Biotechnology, University of Agriculture in Krakow, Krakow, Poland. 
      m.chodacka@ogr.ur.krakow.pl.
FAU - Oleszkiewicz, Tomasz
AU  - Oleszkiewicz T
AD  - Faculty of Biotechnology and Horticulture, Institute of Plant Biology and 
      Biotechnology, University of Agriculture in Krakow, Krakow, Poland.
FAU - Baranski, Rafal
AU  - Baranski R
AD  - Faculty of Biotechnology and Horticulture, Institute of Plant Biology and 
      Biotechnology, University of Agriculture in Krakow, Krakow, Poland.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Methods Mol Biol
JT  - Methods in molecular biology (Clifton, N.J.)
JID - 9214969
RN  - 0 (Anthocyanins)
RN  - 0 (RNA, Guide, CRISPR-Cas Systems)
SB  - IM
MH  - Anthocyanins/metabolism
MH  - Bony Callus/metabolism
MH  - CRISPR-Cas Systems/*genetics
MH  - Daucus carota/*genetics
MH  - Gene Editing/*methods
MH  - RNA, Guide, CRISPR-Cas Systems/genetics
OTO - NOTNLM
OT  - Agrobacterium-mediated transformation
OT  - Anthocyanins
OT  - Callus
OT  - Daucus carota
OT  - F3H
OT  - Flavanone-3-hydroxylase
EDAT- 2019/01/06 06:00
MHDA- 2019/06/18 06:00
CRDT- 2019/01/06 06:00
PHST- 2019/01/06 06:00 [entrez]
PHST- 2019/01/06 06:00 [pubmed]
PHST- 2019/06/18 06:00 [medline]
AID - 10.1007/978-1-4939-8991-1_15 [doi]
PST - ppublish
SO  - Methods Mol Biol. 2019;1917:203-215. doi: 10.1007/978-1-4939-8991-1_15.