Characterization of Gene Function in Tomato Using CRISPR/CAS9 System
Nuclease based genome editing, specifically the CRISPR/CAS9 system, is being hailed as a novel tool to improve quality of crop plants. To test potential of CRISPR/CAS9 system and to characterize gene functions contributing to improve yield and quality of horticultural crops, we have developed knockout mutants in tomato (Solanum lycopersicum), a widely-consumed vegetables crop throughout world. A CRISPR/CAS9 construct was made containing the guide RNA of auxin-responsive protein/ IAA9 (SlAUX/IAA9) gene and MET1 gene. SlAUX/IAA9 encodes a short-lived transcription factor that affects a number of plant growth and developmental processes including leaf architecture. MET1 gene is responsible for maintenance of DNA methylation in plants leading to epigenetic changes effecting plant growth and developmental processes. We show that CRISPR/CAS9 system effectively knocked out SlAUX/IAA9 and generated mutations in MET1 gene sequence in tomato. The resulting AUX/IAA9 knockouts exhibited multiple phenotypes including change of leaf architecture from compound leaves to simple leaves. The knockout plants showed reduced plant height; early fruit set and reduced seed number in fruit. The SlAUX/IAA9 knockout did not altered patterns of ethylene production and lycopene accumulation in ripening fruits but significantly reduced ethylene production during fruit ripening. The changes associated with the knock out mutations were stable and inherited in the successive generation for the SlAUX/IAA9 knockout. However, some other genes contributed to variation in leaf architecture in the simple SlAUX/IAA9 leaf for the second generation of mutants. The MET1 Mutants showed a delayed flowering, fruiting and ripening suggesting a role of MET1 gene in maintaining DNA methylation essential for normal fruiting processes. The MET1 mutants showed that the epigenetic changes associated with MET1 mutants were reversed. Taken together, these results show that CRISPR/CAS9 system provides a method of choice to develop desirable mutants.
Handa, Purdue University.
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