Studies On CRISPR-Cas9 System Optimization And Interactions Analyses Of Whirly Proteins In Arabidopsis

CRISPR-Cas9 system is a novel gene editing tool.It is easy to operate and has high mutation efficiency,which has been extensively applied in various animal and plant species.However,concerering different plant species and different genes,conmercial CRISPR-Cas9 system must be optimized.Whirly transcription factor family is a plant specific single-stranded DNA-binding protein family.The parts of function of Whirly 1 and Whirly2 proteins have been reported,but the relationship among three members of Whirly family still remains unclear.In this study,we intended to use dicotyledonous CRISPR-Cas9 system to make gene editing of the three Whirly family genes in Arabidopsis to obtain the single,double and triple Whirly gene mutations.To further identify the relationship of these three Whirly proteins,we simultaneously constructed the bimolecular fluorescence complementation(BIFC)and yeast two-hybrid(Y2H)vector systems to study the interactions of these Whirly proteins in vitro and in vivo,The main results are listed as follows:1.Two CRISPR-Cas9 systems for gene editing in dicotyledonous and monocotyledonous plants were optimized respectively.To achieve this goal,a yeast CRISPR-Cas9 system was used as a precursor and undergone some modifications.After that,a series of backbone plasmids including pCBIM(or CBUN)-NLS-Cas9-NLS-Flag and pBlunt-AtU6(or-OsU6/-OsU3)-sgRNA were constructed.The plasmid pair pCBIM-NLS-Cas9-NLS-Flag and pBlunt-AtU6-sgRNA are used for gene editing in dicotyledonous plants,this system uses CaMV35S promoter to drive the human Cas9 gene expression.The plasmid pair pCBUN-NLS-Cas9-NLS-Flag and pBlunt-Os U6/-OsU3-sgRNA are used for gene editing in monocotyledonous plants,this system uses maize Ubi promoter to drive the human Cas9 gene expression.2.Gene editing of the Whirly gene family by using dicotyledonous CRISPR-Cas9 system in Arabidopsis.We selected different combination conditions:1.different promoters:CaMV35S promoter、YAO promoter;2.Different Cas9:Human Cas9 or modified plant specific Cas9;3.Different transformation system:floral dip transformation method or root induced callus transformation and regeneration system.At end,a commercial dicotyledonous CRISPR-Cas9 system with plant optimized Cas9 gene driven by the CaMV35S promoter for Whirly gene family editing,an Arabidopsis root induced callus transformation and regeneration system was adopted and used for gene editing the WT or the single or double mutation of the Whirly genes background.The transformed callus were screened by hygromycin and the resistant rosette tissues were regenerated.After the rooting experiment,the regenerated seedlings will be used to identify the target gene editing in these transgenic plants.3.Idenfy the interactions of Whirly family members by bimolecular fluorescence complementation(BIFC)and yeast two-hybrid(Y2H)assay.The resultsThe result showed that all of these three Whirly proteins have low interactions with themselves.The interactions of Whirlyl and Whirly3 were also very low,but the interactions between Whirlyl with Whirly2 were strong if Whirlyl or Whirly3 both linked to the DNA binding domain(BD)and Whirly2 linked to the activation domain(AD)of the GAL4 protein.Howver,the results of BIFC in anion epidermal cells by biolist assay showed that among Whirly proteins there were no inter-or mutual interactions.,furtherl Next step mildly co-transformation to protoplasts by PEG would be detected and further CoIP will be done in future.