Improvements of TKC Technology Accelerate Isolation of Transgene-Free CRISPR/Cas9-Edited Rice Plants

(整期优先)网络出版时间:2019-02-12
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EliminationoftheCRISPR/Cas9constructsineditedplantsisaprerequisiteforassessinggeneticstability,conductingphenotypiccharacterization,andapplyingforcommercializationoftheplants.However,removaloftheCRISPR/Cas9transgenesbygeneticsegregationandbybackcrossislaboriousandtimeconsuming.WepreviouslyreportedthedevelopmentofthetransgenekillerCRISPR(TKC)technologythatusesapairofsuicidegenestotriggerself-eliminationofthetransgeneswithoutcompromisinggeneeditingefficiency.TheTKCtechnologyenablesisolationoftransgene-freeCRISPR-editedplantswithinasinglegeneration,greatlyacceleratingcropimprovements.Here,wepresentedtwonewTKCvectorsthatshowgreatefficiencyinbotheditingthetargetgeneandinundergoingself-eliminationofthetransgenes.ThenewvectorsreplacedtheCaMV35SpromoterusedinourpreviousTKCvectorwithtworicepromoterstodriveoneofthesuicidegenes,providingadvantagesoverourpreviousTKCvectorundercertainconditions.Thevectorsreportedhereofferedmoreoptionsandflexibilitytoconductgeneeditingexperimentsinrice.