Utility of I-SceI and CCR5-ZFN Nucleases in Excising Selectable Marker Genes from Transgenic Plants.

OBJECTIVES:Removal of selection marker genes from transgenic plants is highly desirable for their regulatory approval and public acceptance. This study evaluated the use of two nucleases, the yeast homing endonuclease, I-SceI, and the designed zinc finger nuclease, CCR5-ZFN, in excising marker genes from plants using rice and Arabidopsis as the models.RESULTS:In an in vitro culture assay, both nucleases were effective in precisely excising the DNA fragments marked by the nuclease target sites. However, rice cultures were found to be refractory to transformation with the I-SceI and CCR5-ZFN overexpressing constructs. The inducible I-SceI expression was also problematic in rice as the progeny of the transgenic lines expressing the heat-inducible I-SceI did not inherit the functional gene. On the other hand, heat-inducible I-SceI expression in Arabidopsis was effective in creating somatic excisions in transgenic plants but ineffective in generating heritable excisions. The inducible expression of CCR5-ZFN in rice, although transmitted stably to the progeny, appeared ineffective in creating detectable excisions. Therefore, toxicity of these nucleases in plant cells poses major bottleneck in their application in plant biotechnology, which could be avoided by expressing them transiently in cultures in vitro.