CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations.
The CRISPR/Cas9 system has successfully been used in various organisms for precise targeted gene editing. Although it has been demonstrated that CRISPR/Cas9 system can induce mutation in tomato plants, the stability of heredity in later generations and mutant specificity induced by the CRISPR/Cas9 system in tomato plants have not yet been elucidated in detail. In this study, two genes, SlPDS and SlPIF4, were used for testing targeted mutagenesis in tomato plants through an Agrobacterium tumefaciens-mediated transformation method. A high mutation frequency was observed in all tested targets in the T0 transgenic tomato plants, with an average frequency of 83.56%. Clear albino phenotypes were observed for the psd mutants. High frequencies of homozygous and biallelic mutants were detected even in T0 plants. The majority of the detected mutations were 1- to 3-nucleotide deletions, followed by 1-bp insertions. The target mutations in the T0 lines were stably transmitted to the T1 and T2 generations, without new modifications or revision. Off-target activities associated with SlPDS and SlPIF4 were also evaluated by sequencing the putative off-target sites, and no clear off-target events were detected. Our results demonstrate that the CRISPR/Cas9 system is an efficient tool for generating stable and heritable modifications in tomato plants.
Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310058, China.|Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University, Hangzhou 310058, China.
Changtian
C
Pan
Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310058, China.|Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University, Hangzhou 310058, China.
Lei
L
Ye
Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University, Hangzhou 310058, China.
Li
L
Qin
Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University, Hangzhou 310058, China.
Xue
X
Liu
Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University, Hangzhou 310058, China.
Yanjun
Y
He
Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University, Hangzhou 310058, China.
Jie
J
Wang
Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University, Hangzhou 310058, China.
Lifei
L
Chen
Key Laboratory of Horticultural Plant Growth, Development and Quality Improvement, Ministry of Agriculture, Hangzhou 310058, China.|Zhejiang Provincial Key Laboratory of Horticultural Plant Integrative Biology, Department of Horticulture, Zhejiang University, Hangzhou 310058, China.
Gang
G
Lu
D064113:CRISPR-Cas Systems; D018390:Gene Targeting; D017343:Genes, Plant; D018745:Genome, Plant; D005838:Genotype; D040582:Inheritance Patterns; D018551:Solanum lycopersicum / Q000235:genetics*; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D030821:Plants, Genetically Modified; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
D000094704:RNA, Guide, CRISPR-Cas Systems