Integration of a FT expression cassette into CRISPR/Cas9 construct enables fast generation and easy identification of transgene-free mutants in Arabidopsis
Autoři:
Yuxin Cheng aff001; Na Zhang aff001; Saddam Hussain aff001; Sajjad Ahmed aff001; Wenting Yang aff001; Shucai Wang aff001
Působiště autorů:
Key Laboratory of Molecular Epigenetics of MOE, Northeast Normal University, Changchun, Jilin, China
aff001; College of Life Science, Linyi University, Linyi, Shandong, China
aff002
Vyšlo v časopise:
PLoS ONE 14(9)
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pone.0218583
Souhrn
The CRISPR/Cas9 genome editing technique has been widely used to generate transgene-free mutants in different plant species. Several different methods including fluorescence marker-assisted visual screen of transgene-free mutants and programmed self-elimination of CRISPR/Cas9 construct have been used to increase the efficiency of genome edited transgene-free mutant isolation, but the overall time length required to obtain transgene-free mutants has remained unchanged in these methods. We report here a method for fast generation and easy identification of transgene-free mutants in Arabidopsis. By generating and using a single FT expression cassette-containing CRISPR/Cas9 construct, we targeted two sites of the AITR1 gene. We obtained many early bolting plants in T1 generation, and found that about two thirds of these plants have detectable mutations. We then analyzed T2 generations of two representative lines of genome edited early bolting T1 plants, and identified plants without early bolting phenotype, i.e., transgene-free plants, for both lines. Further more, aitr1 homozygous mutants were successful obtained for both lines from these transgene-free plants. Taken together, these results suggest that the method described here enables fast generation, and at the mean time, easy identification of transgene-free mutants in plants.
Klíčová slova:
Plant genomics – Phenotypes – Polymerase chain reaction – Genetically modified plants – Seeds – Flowering plants – CRISPR – Arabidopsis thaliana
Zdroje
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