CRISPR Genome Editing of Transcription Factors Related to Aluminum Toxicity in Tomato (Solanum lycopersicum)
The objective of this study is to set up the CRISPR/Cas9 system for tomatoes in the Plant Biotech Lab at Tennessee State University. Two transcription factors were selected as these genes were shown changes at transcript and protein abundance levels in response to treatments of Al toxicity stress in tomatoes (Solanum lycoperisicum). The selected genes were found specifically expressed in roots in Tomato eFP browser. The online-based tool CRISPR P v2.0 was used for guide RNA (gRNA) design. For gRNA design, two gRNAs were selected based on the number of on & off target sites, location (gene exon, intron, UTR, or intergenic), preferred restriction endonuclease site, and GC content (30-80%). The p201N vector with 2X35S promoter and kanamycin resistance genes was used to prepare the CRISPR/Cas9 construct. The two gRNAs were assembled onto the p201N Cas9 vector using NEBuilder DNA assembly. The CRISPR/Cas9 constructs were first transformed into Top 10 E. coli competent cells provided with the NEBuilder DNA assembly kit. Positive colonies grown on an agar plate supplemented with 50mg/L kanamycin were selected and plasmids were extracted and sequenced. Plasmids containing the gRNA sequences were used to transform Agrobacterium tumefaciens AGL1 strain using the electroporation method. Tomato ‘Money Maker’ seeds were germinated, and cotyledons were used for inoculation of the A. tumefaciens AGL1 harboring the p201N Cas9-gRNAs constructs. Positive T0 plants with genome integrated with the Cas9 & gRNA constructs were screened using PCR with primers specific for these sequences. Rooted plants were transplanted into the potting mix and self-pollinated to produce seeds. Seedlings in the T1 generation. were genotyped for segregation of Cas9 and mutations on targeted and non-targeted regions.
"CRISPR Genome Editing of Transcription Factors Related to Aluminum Toxicity in Tomato (Solanum lycopersicum)"
ETD Collection for Tennessee State University.