Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters

The efficacy of the CRISPR/Cas9 system in grapevine(Vitis vinifera L.)has been documented,but the optimization of this system,as well as CRISPR/Cas9-mediated multiplex genome editing,has not been explored in this species.Herein,we identified four VvU3 and VvU6 promoters and two ubiquitin(UBQ)promoters in grapevine and demonstrated that the use of the identified VvU3/U6 and UBQ2 promoters could significantly increase the editing efficiency in grape by improving the expression of sgRNA and Cas9,respectively.Furthermore,we conducted multiplex genome editing using the optimized CRISPR/Cas9 vector that contained the conventional multiple sgRNA expression cassettes or the polycistronic tRNA-sgRNA cassette(PTG)by targeting the sugar-related tonoplastic monosaccharide transporter(TMT)family members TMT1 and TMT2,and the overall editing efficiencies were higher than 10%.The simultaneous editing of TMT1 and TMT2 resulted in reduced sugar levels,which indicated the role of these two genes in sugar accumulation in grapes.Moreover,the activities of the VvU3,VvU6,and UBQ2 promoters in tobacco genome editing were demonstrated by editing the phytoene desaturase(PDS)gene in Nicotiana benthamiana leaves.Our study provides materials for the optimization of the CRISPR/Cas9 system.To our knowledge,our simultaneous editing of the grape TMT family genes TMT1 and TMT2 constitutes the first example of multiplex genome editing in grape.The multiplex editing systems described in this manuscript expand the toolbox of grape genome editing,which would facilitate basic research and molecular breeding in grapevine.