Application Of BeYDV Geminivirus And CRISPR-LbCpf1 System In Cotton Genome Editing

Cotton(Gossypium hirsutum)is one of the important economic crops and the main source of raw materials for industrial textile and agricultural oil.It is of great significance to study the molecular mechanism of functional genes though establishing effective technical methods in cotton.In this study,the replication initiation protein(Rep)was been used to recognize the long intergenic region(LIR)structure and initiate rolling-circle replication in the bean dwarf virus,then a highly efficient gene knockout vector was construct combining with CRISPR-Cas9 gene editing system.On this basis,the cotton endogenous homologous templates were constructed into the replication loop and a gene editing knock-in system based on BeYDV Geminivirus was been constructed in cotton.In addition,we optimized the editing efficiency of the CRISPR-Cpf1 system and established a CRISPR-Cpf1 gene editing knock-in system based on the method of combining the CRISPR-Cpf1 editing system with PTC(Polycistronic t RNA-crRNA)and TDT(Target-Donor-Target)structures.The main results of this study are as follows.First,we constructed three knockout vectors based on CRISPR-Cas9 and compared their editing efficiency in cotton.In order to improve the editing efficiency of the CRISPR-Cas9 gene editing system in cotton,two different gene knockout vectors were constructed based on the cotton gene editing system pRGEB32-Gh U6.7.One is the pBeYDV-Cas9-KO gene editing system which the promoter and sgRNA were constructed in the replication loop(LIR-Donor-SIR-Rep-LIR)of the BeYDV geminivirus virus.The second is to replace the UBI promoter driving Cas9 protein with the pRGEB32-35 s gene editing system of constitutive 35 S promoter.The three vectors that the chloroplast synthesis related gene Gh CLA1 were as the target gene were transformed into cotton by Agrobacterium tumefaciens genetic transformation.The results showed that the efficiency of editing At and Dt subgroup simultaneously in cotton by BeYDV-Cas9-KO system was as high as 73.3%;the editing type is mainly fragment deletion,and the length of base deletion mainly ranges from 1 bp to 10 bp.The editing sites were mainly located at positions 11-17 upstream of PAM.We did not find the off-target editing of BeYDV-Cas9-KO by detecting the miss site corresponding to sgRNA.It shows that the pBeYDV-Cas9-KO gene editing system has stronger editing specificity for target genes in cotton,and the editing range is more than larger.Second,we constructed three knock-in vectors based on Cas9 and the BeYDV Geminivirus virus in cotton.In order to establish the knock-in system in cotton,we constructed the homology arms and Cas9 protein into the replication elements of the BeYDV geminivirus on the basis of the BeYDV geminivirus-mediated pBeYDV-Cas9-KO gene editing vector.At the same time,the cotton chloroplast synthesis-related gene Gh CLA1 was selected as the target gene,and 12 BeYDV-mediated knock-in vectors were constructed.Foreign gene RFP was knock in multiple loci of Gh CLA1 gene to verify the knock in system.The results showed that two vectors Be KI-3and Be KI-4 that could knock in cotton were screened,and the knock-in efficiency of the two vectors in cotton was 5.6% and 6.5% respectively.Third,temperature had an effect on the editing efficiency of CRISPR-LbCpf1 in cotton.The T1 generation materials of Gh CLA1 gene and the callus of Gh PGF gene was treated with different temperature,and the expression profile of LbCpf1 protein and editing efficiency were tested under different temperature conditions.The results show that the activity of LbCpf1 nuclease is sensitive to temperature.The activity of LbCpf1 protein is the highest under the condition of 34℃,the CRISPR-LbCpf1 system showed better editing efficiency and reached 91.5%;the editing efficiency that At Dt subfamily was edited simultaneously was as high as 65.0%;the editing efficiency of the second crRNA was better;no off-target effect was found after temperature treatment.In addition,we knocked out Gh PGF gene to obtain a glandfree and nontransgenic cotton material,which can be stably inherited to the next generation.At last,we constructed the gene knock-in vectors based on CRISPR-Cpf1 system in cotton.We utilized the characteristic of Cpf1 protein with double cleavage ability of DNA and RNA,and established the pRGEB3-Cpf I-KI gene knock-in system with PTC(Polycistronic t RNA-crRNA)and TDT(Target-Donor-Target)structure.We designed two targets on Gh PGF gene.Sbf I restriction site or RFP gene was inserted in the middle of the homology arms of pRGEB3-Cpf I-KI vector.We have constructed four knock-in vectors successfully.The results showed that the gene knock-in system based on CRISPR-Cpf1 could accurately insert foreign fragments or genes into the cotton genome.Two vectors KI-Sbf I-1CR and KI-RFP-1CR were screened,and the knock-in efficiency was 6.3% and2.7% respectively.In summary,the vector of gene knockout in cotton was optimized by using CRISPR-Cas9 combined with the replication function of BeYDV Geminivirus virus,the double cleavage characteristics of CRISPR-Cpf1 and the homologous template of TDT structure.At the same time,a gene editing system which can realize gene knock-in in cotton is provided.It provides new technical method for cotton genome gene editing,and provides an experimental basis for the optimization of gene knock-in technology.In the future,multiple gene editing technologies will be applied to analyze the gene functions in cotton.