Mining natural sequence and carotenoid variation in tomato to design sgRNA towards CRISPR/Cas9 gene-editing
2019
Guevarra, P.R. | Gardoce, R.R. | Soytong, M. | Mateo, J.M.C. | Aglibot, C.C. | Galvez, H.F.
Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas9) technology is rapidly gaining adoption in crop breeding primarily due to its speed and accuracy. The editing process eliminates the randomness, unpredictability and long screening process of finding useful mutants by mutation breeding as well as generations of backcrossing required to introgress a gene from wild type to elite varieties. Using cultivar VB-15, the study characterized SNPs for tomato lycopene genes based on alignment against the SolGenomics data sequence of Solanum lycopersicum cultivar Heinz 1706 (https://solgenomics.net/). A number of natural SNPs were found in the lycopene beta-cyclase gene (CrtL-b), beta-carotene gene (CrtR-b), lycopene epsilon-cyclase gene (CrtL-e), and lycopene beta-cyclase gene 2 (CtrL-b2). Subsequently, four sgRNAs were designed using sequences flanking the natural SNPs specific for CRISPR/Cas9 genome editing application. The 20bp sgRNA primers were designed 5' upstream of the NGG PAM sequence with optimum percent GC content of 40-50%. Two sgRNAs were designed within the single exon of CrtL-b gene. Genetic populations segregating for the target phenotype alteration were also developed and analyzed for genetic linkage to any of these tomato lycopene SNPs. Other genomic regions of tomato that potentially confer or regulate the phenotype were also analyzed for genetic linkage particularly the loci in the vicinity of NGG PAM sequences. To demonstrate the range of phenotypic variation, lycopene was extracted using hexane:acetone and quantified using differential wavelength scanning in a UV-Vis spectrophotometer. Results will validate design of sgRNA for successful gene-edit to induce the desired mutation. This study presents the pioneer characterization of natural SNPs in caretonoid genes between tomato cultivars VB-15 and Heinz 1706.
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