CRISPR/Cas9 editing of carotenoid genes in tomato

Abstract

CRISPR/Cas9 technology is rapidly spreading as genome editing system in crop breeding. The efficacy of CRISPR/Cas9 in tomato was tested on Psy1 and CrtR-b2, two key genes of carotenoid biosynthesis. Carotenoids are plant secondary metabolites that must be present in the diet of higher animals because they exert irreplaceable functions in important physiological processes. Psy1 and CrtR-b2 were chosen because their impairment is easily detectable as a change of fruit or flower color. Two CRISPR/Cas9 constructs were designed to target neighboring sequences on the first exon of each gene. Thirty-four out of forty-nine (69%) transformed plants showed the expected loss-of-function phenotypes due to the editing of both alleles of a locus. However, by including the seven plants edited only at one of the two homologs and showing a normal phenotype, the editing rate reaches the 84%. Although none chimeric phenotype was observed, the cloning of target region amplified fragments revealed that in the 40% of analyzed DNA samples were present more than two alleles. As concerning the type of mutation, it was possible to identify 34 new different alleles across the four transformation experiments. The sequence characterization of the CRISPR/Cas9-induced mutations showed that the most frequent repair errors were the insertion and the deletion of one base. The results of this study prove that the CRISPRCas9 system can be an efficient and quick method for the generation of useful mutations in tomato to be implemented in breeding programs.

Efficient Targeted Mutagenesis in Apple and First Time Edition of Pear Using the CRISPR-Cas9 System

“…Furthermore, bi-allelic mutants were the most frequent outcome and only one putatively mono-allelic homozygous line was observed among the 51 independent edited lines analyzed. Multiple mutated sequences and frequent biallelic mutations were also observed in grape (Nakajima et al, 2017; Wang X. et al, 2018), orange (Peng et al, 2017) or tomato (D’Ambrosio et al, 2018) T0 plants.…”

Tomato Fruit Development and Metabolism

“…Tomato fruits typically provide the principal dietary source of carotenoids in many Western diets (Carrari and Fernie, 2006). The characteristic red tomato color is a result of the accumulation of the carotenoid lycopene in both the fruit skin and pulp (Seymour et al, 2013; Borghesi et al, 2016; D’Ambrosio et al, 2018). During tomato ripening, the concentrations of carotenoids increase by between 10- and 14-fold, mainly due to the accumulation of lycopene (Fraser et al, 1994), which increases as the fruit matures (Tamasi et al, 2019).…”

“…Light signaling and plant hormones, particularly ethylene and auxins, have been identified as important regulators of carotenoid biosynthesis during tomato fruit ripening (Cruz et al, 2018). Almost all the enzymes acting in the carotenoid biosynthesis pathway have been cloned, and metabolic engineering approaches have been developed to enhance pigment quantity and quality (Carrari and Fernie, 2006; Alseekh et al, 2015; D’Ambrosio et al, 2018). The first committed step of carotenoid biosynthesis is the formation of phytoene, which is dependent on the catalytic activity of phytoene synthase.…”

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“…Например, при редактировании гена Gmdrb2ab люцерны «transgene-clean»-поколения удалось получить только в случае единичной вставки; в случае множественных вставок, когда наследование признаков не соответствует простым моделям наследования, этого достичь не удалось [32]. Так же не увенчались успехом попытки получить нетрансгенные поколения, наследующие целевые изменения в генах SlPsy1 томата [55], GmHen1 люцерны [32] и GhCLA1 хлопка [56].…”