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.
Similar content being viewed by others
References
Bae S, Park J, Kim JS (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30:1473–1475
Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V (2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9:39
Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52
Brooks C, Nekrasov V, Lippman ZB, Van Eck J (2014) Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol 166:1292–1297
Cai Y, Chen L, Liu X et al (2015) CRISPR/Cas9-mediated genome editing in soybean hairy roots. PLoS ONE 10:1–13
Cardi T, D’Agostino N, Tripodi P (2017) Genetic transformation and genomic resources for next-generation precise genome engineering in vegetable crops. Front Plant Sci 8:241
Cermak T, Curtin SJ, Gil-Humanes J et al (2017) A multi-purpose toolkit to enable advanced genome engineering in plants. Plant Cell 29:1196–1217
Cong L, Ran FA, Cox D et al (2013) Multiplex genome engineering using CRISPR/VCas systems. Science 339:819–823
D’Ambrosio C, Giorio G, Marino I et al (2004) Virtually complete conversion of lycopene into β-carotene in fruits of tomato plants transformed with the tomato lycopene β-cyclase (tlcy-b) cDNA. Plant Sci 166:207–214
D’Ambrosio C, Stigliani AL, Giorio G (2011) Overexpression of CrtR-b2 (carotene beta hydroxylase 2) from S. lycopersicum L. differentially affects xanthophyll synthesis and accumulation in transgenic tomato plants. Transgenic Res 20:47–60
Deltcheva E, Chylinski K, Sharma CM, Gonzales K (2011) CRISPR RNA maturation by trans -encoded small RNA and host factor RNase III. Nature 471:602–607
Ding Y, Li H, Chen LL, Xie K (2016) Recent advances in genome editing using CRISPR/Cas9. Front Plant Sci 7:1–12
Endo M, Mikami M, Toki S (2016) Biallelic gene targeting in rice. Plant Physiol 170:667–677
Feng Z, Mao Y, Xu N et al (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci 111:4632–4637
Fray RG, Grierson D (1993) Identification and genetic analysis of normal and mutant phytoene synthase genes of tomato by sequencing, complementation and co-suppression. Plant Mol Biol 22:589–602
Galpaz N, Ronen G, Khalfa Z, Zamir D, Hirschberg J (2006) A chromoplast-specific carotenoid biosynthesis pathway is revealed by cloning of the tomato white-flower locus. Plant Cell 18:1–14
Giorio G, Stigliani AL, D’Ambrosio C (2008) Phytoene synthase genes in tomato (Solanum lycopersicum L.)—new data on the structures, the deduced amino acid sequences and the expression patterns. FEBS J 275:527–535
Hirschberg J (2001) Carotenoid biosynthesis in flowering plants. Curr Opin Plant Biol 4:210–218
Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR–Cas9 for genome engineering. Cell 157:1262–1278
Ito Y, Nishizawa-Yokoi A, Endo M, Mikami M, Toki S (2015) CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening. Biochem Biophys Res Commun 467:76–82
Jiang F, Doudna JA (2017) CRISPR–Cas9 structures and mechanisms. Annu Rev Biophys 46:505–529
Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP (2013) Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Res 41:1–12
Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S (2015) Multigene editing in the Escherichia coli genome via the CRISPR–Cas9 system. Appl Environ Microbiol 81:2506–2514
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821
Kachanovsky DE, Filler S, Isaacson T, Hirschberg J (2012) Epistasis in tomato color mutations involves regulation of phytoene synthase 1 expression by cis-carotenoids. Proc Natl Acad Sci 109:19021–19026
Lawrenson T, Shorinola O, Stacey N et al (2015) Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol 16:258
Lei Y, Lu L, Liu HY, Li S, Xing F, Chen LL (2014) CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. Mol Plant 7:1494–1496
Li J, Aach J, Norville JE, Mccormack M, Bush J, Church GM, Sheen J (2013) Multiplex and homologous recombination-mediated plant genome editing via guide RNA/Cas9. Nat Biotechnol 31:688–691
Li R, Li R, Li X et al (2018) Multiplexed CRISPR/Cas9-mediated metabolic engineering of γ-aminobutyric acid levels in Solanum lycopersicum. Plant Biotechnol J 16:415–427
Liang Z, Chen K, Li T, Zhang Y et al (2017) Efficient DNA-free genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nat Commun 8:1–5
Lin Y, Cradick TJ, Brown MT et al (2014) CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences. Nucleic Acids Res 42:7473–7485
Liu YS, Gur A, Ronen G, Causse M et al (2003) There is more to tomato fruit colour than candidate carotenoid genes. Plant Biotechnol J 1:195–207
Liu W, Xie X, Ma X, Li J, Chen J, Liu YG (2015) DSDecode: a web-based tool for decoding of sequencing chromatograms for genotyping of targeted mutations. Mol Plant 8:1431–1433
Liu X, Wu S, Xu J, Sui C, Wei J (2017) Application of CRISPR/Cas9 in plant biology. Acta Pharm Sin B 7:292–302
Ma X, Zhang Q, Zhu Q et al (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284
Ma X, Zhu Q, Chen Y, Liu YG (2016) CRISPR/Cas9 platforms for genome editing in plants: developments and applications. Mol Plant 9:961–974
Mali P, Yang L, Esvelt KM et al (2013) RNA-guided human genome engineering via Cas9. Science 339:823–826
Moise AR, Al-Babili S, Wurtzel ET (2014) Mechanistic aspects of carotenoid biosynthesis. Chem Rev 114:164–193
Nisar N, Li L, Lu S, Khin NC, Pogson BJ (2015) Carotenoid metabolism in plants. Mol Plant 8:68–82
Nonaka S, Arai C, Takayama M, Matsukura C, Ezura H (2017) Efficient increase of Γ-aminobutyric acid (GABA) content in tomato fruits by targeted mutagenesis. Sci Rep 7:1–14
Pan C, Ye L, Qin L et al (2016) CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Sci Rep 6:2–10
Pattanayak V, Lin S, Guilinger JP, Ma E, Doudna JA, Liu DR (2013) High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31:839–843
Podevin N, Davies HV, Hartung F, Nogué F, Casacuberta JM (2013) Site-directed nucleases: a paradigm shift in predictable, knowledge-based plant breeding. Trends Biotechnol 31:375–383
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR–Cas9 system. Nat Protoc 8:2281–2308
Ricroch A, Clairand P, Harwood W (2017) Use of CRISPR systems in plant genome editing : toward new opportunities in agriculture. Emerg Top Life Sci 1:169–182
Roldan MVG, Périlleux C, Morin H, Huerga-Fernandez S, Latrasse D, Benhamed M, Bendahmane A (2017) Natural and induced loss of function mutations in SlMBP21 MADS-box gene led to jointless-2 phenotype in tomato. Sci Rep 7:1–10
Stigliani AL, Giorio G, D’Ambrosio C (2011) Characterization of P450 carotenoid β- and ε-hydroxylases of tomato and transcriptional regulation of xanthophyll biosynthesis in root, leaf, petal and fruit. Plant Cell Physiol 52:851–865
Sun T, Yuan H, Cao H, Yazdani M, Tadmor Y, Li L (2018) Carotenoid metabolism in plants: the role of plastids. Mol Plant 11:58–74
Svitashev S, Young JK, Schwartz C, Gao H, Falco SC, Cigan AM (2015) Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiol 169:931–945
Tsai SQ, Zheng Z, Nguyen NT et al (2015) GUIDE-Seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat Biotechnol 33:187–197
Ueta R, Abe C, Watanabe T et al (2017) Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9. Sci Rep 7:1–8
Waltz E (2016) CRISPR-edited crops free to enter market, skip regulation. Nat Biotechnol 34:582
Waltz E (2018) With a free pass, CRISPR-edited plants reach market in record time. Nat Biotechnol 36:6
Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R (2013) One-step generation of mice carrying mutations in multiple genes by CRISPR/cas-mediated genome engineering. Cell 153:910–918
Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu JL (2014) Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32:947–951
Wright AV, Nuñez JK, Doudna JA (2016) Biology and applications of CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell 164:29–44
Xing HL, Dong L, Wang ZP et al (2014) A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14:327
Yuan H, Zhang J, Nageswaran D, Li L (2015) Carotenoid metabolism and regulation in horticultural crops. Hortic Res 2(July):15036. https://doi.org/10.1038/hortres.2015.36
Zhang H, Zhang J, Wei P et al (2014) The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation. Plant Biotechnol J 12:797–807
Zhou H, Liu B, Weeks DP, Spalding MH, Yang B (2014) Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Res 42:10903–10914
Zsögön A, Cermak T, Voytas D, Peres LEP (2017) Genome editing as a tool to achieve the crop ideotype and de novo domestication of wild relatives: case study in tomato. Plant Sci 256:120–130
Acknowledgements
We thank all the Colleagues of Agronomic Service Unit of the Metapontum Agrobios Research Centre for help with plant maintenance and seed collection. We are also grateful to Laura Giorio for providing language help. This work was funded by ALSIA (Agenzia Lucana per lo Sviluppo e l’Innovazione in Agricoltura) in the framework of the “Piano Annuale di Attività 2016”.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
D’Ambrosio, C., Stigliani, A.L. & Giorio, G. CRISPR/Cas9 editing of carotenoid genes in tomato. Transgenic Res 27, 367–378 (2018). https://doi.org/10.1007/s11248-018-0079-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11248-018-0079-9