Study On Editing Diploid Potato Genome By CRISPR/Cpf1

Site-directed editing of plant genomes is of great significance to the genetic improvement of crops.At present,CRISPR/Cas9 system has been widely used in genome editing of many crops including potato because of its simplicity and high efficiency.However,because the CRISPR/Cas9 system can only recognize the leading sequence whose PAM sequence is 5'-NGG-3',it greatly limits the scope of application of its fixed-point editing.Therefore,how to extend the editing scope of CRISPR system is an important research direction in the field of gene editing.CRISPR/Cpf1 is a new type of CRISPR system.CRISPR/Cpf1 can specifically recognize 24 bp,after the PAM sequence is 5'-TTTN-3'.This greatly enriches the selection range of target sites for CRISPR systems,so it has become another research hotspot after CRISPR/Cas9 systems.However,the research of CRISPR/Cpf1 system in Solanaceae crops was not very deep,and the application of CRISPR/Cpf1 system in potato has not been reported.Therefore,in this paper,the application of Solanaceae system in potato is studied systematically.Firstly,on the basis of previous studies,two variants of Cpf1 protein,LbCpf1 and AsCpf1,were optimized based on dicotyledonous plants,and then the expression system of CrRNA was analyzed.Four optimized expression systems were constructed:nuclease-mediated RIBO system,Cys4-based CrRNA excision Csy4 system,polycistron-RNA expression T-RNA system,and Cpf1-mediated CrRNA cleavage DR system.Then the two Cpf1 protein variants and four CrRNA expression systems were combined to generate eight sconstructs which could be transformed into potato.To quickly examine the efficiency of eight sconstructs,a genetic transformation system of Agrobacterium rhizogenes was established.This system has the advantages of simple operation,short transformation period and quick detection of positive transformation root by visual fluorescence screening.In order to verify the feasibility of this system,two targets of potato StCDF1 gene were selected and targeted by CRISPR/Cas9 system.The two selected targets showed high mutationefficiency in the root-growing system.After that,a target with the highest efficiency was selected and verified by transgenic experiments.A heterozygous mutant lacking 5-bp was identified in transgenic progenies,which indicatedt hat the system could be used for rapid verification of gene editing vectors.Finally,eight sets of CRISPR/Cpf1 experimental schemes were tested using the established Agrobacterium tumefaciens transformation system.The results showed that two positive mutant fluorescent roots were identified in the CrRNA expression system of csy4 based on AsCpf1.Later,after further deep sequencing analysis of all the positive fluorescent roots,the genome in the two fluorescent roots of AsCpf1/Csy4 expressing gRNA was mutated.This verifies the feasibility of the application of CRISPR/Cpf1 system in potato.