Green fluorescent protein gene as a tool to examine the efficacy of Agrobacterium-delivered CRISPR/Cas9 reagents to generate targeted mutations in the potato genome

CRISPR/Cas9 has emerged as a simple, yet efficient gene editing tool to generate targeted mutations in desired genes in crops plants. Agrobacterium tumefaciens , a reliable and inexpensive DNA-delivery mechanism into plant cells, has been used for the generation of CRISPR/Cas9-mediated mutations in...

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Published in:Plant cell, tissue and organ culture tissue and organ culture, 2022-09, Vol.150 (3), p.587-598
Main Authors: Toinga-Villafuerte, Stephany, Janga, Madhusudhana R., Isabel Vales, M., Rathore, Keerti S.
Format: Article
Language:English
Subjects:
Biomedical and Life Sciences
Cell culture
CRISPR
Crops
Deoxyribonucleic acid
DNA
Fluorescence
Genetic modification
Genome editing
Genomes
Genotypes
Green fluorescent protein
Life Sciences
Mutation
Original Article
Plant bacterial diseases
Plant cells
Plant Genetics and Genomics
Plant Pathology
Plant Physiology
Plant Sciences
Potatoes
Predictions
Proteins
Reagents
Vegetables
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Summary:CRISPR/Cas9 has emerged as a simple, yet efficient gene editing tool to generate targeted mutations in desired genes in crops plants. Agrobacterium tumefaciens , a reliable and inexpensive DNA-delivery mechanism into plant cells, has been used for the generation of CRISPR/Cas9-mediated mutations in crop plants, including potato. However, little information is available as to the progression of gene knockout during various stages of culture following the introduction of CRISPR components in this species. In the current study, the green fluorescent protein ( gfp ) transgene was first introduced in the genome of a potato variety, Yukon Gold. Two GFP-expressing lines, one with a single gfp copy integrated and another with four gfp copies integrated, were subjected to CRISPR/Cas9-mediated mutations in the transgene(s) using three different gRNAs. Disappearance of GFP fluorescence was monitored during the entire culture/regeneration process. Although all three gRNAs successfully knocked out the transgene(s), their efficiencies differed greatly and did not completely match the predicted scores by some guide RNA prediction tools. The nature of mutations in various knockout events was analyzed. Several lines containing four gfp -copies showed four different types of mutations. These findings suggest that it is possible to target all four alleles of a desired native gene in the tetraploid potato. Key message Potato lines, with a single or four copies of the gfp transgene integrated, were subjected to CRISPR/Cas9-mediated mutations. The results indicated that it should be possible to target and knock out multiple copies of a native gene in the tetraploid genotypes of this crop.
ISSN:0167-6857
1573-5044
DOI:10.1007/s11240-022-02310-8