FAD2 Gene Editing In Peanut Using CRISPR/Cas9

Clustered regularly interspaced short palindromic repeat-associated endonuclease Cas9(CRISPR/Cas9)is a novel genome editing technology that has recently been used extensively.The CRISPR/Cas9 system is derived from the adaptive immune system of Streptococcus thermophilus which is evolved against exogenous viruses and plasmids.It has been applied to various bio-related fields because of simplicity,high efficiency,and specificity.Although CRISPR/Cas9 system has been used in a variety of plants,the application of this technology has not been reported in peanut.The peanut oleic acid content of seeds is an important agronomic trait in peanut,it is a major contributor to the quality of oil.The fatty acid desaturase-2(FAD2)genes is responsible for the conversion of oleic acid to linoleic acid.Peanut natural mutant with mutated EAD2 genes have the character of high oleic acid content,however,the other main agronomic traits of this mutant could not achieve the desired levels.The conventional breeding method has made a great contribution in the development of cultivars having high oleate with the desirable traits.However,it is low-efficiency and time consuming because of the gene linkage disequilibrium during the selection process.Genetic transformation is an effective alternative and rapid way to generate a mutant with a desirable trait.Although ZFNs and TALENs can be used to generate an ideal mutant,but they are cost expensive and labor consuming.A more efficient technology to increase the content of oleic acid is eagerly needed.In this study,we used CRISPR/Cas9 to edit the peanut FAD2 genes in order to create a peanut mutant with an improved oleic acid content.There are two specific objectives:1)to establish an effective CRISPR/Cas9 genome editing system in peanut;2)to figure out the transformation rate of different transformation methods.The main results are as follows:1.A plant expression vector FAD2-p201B-Cas9-sgRNA was successfully constructed,in which a fragment of sgRNA was cloned into the p201B Cas9 vector.2.In protoplast transformation,target mutations were detected by sequencing.The mutation proportion was 61.29%(19/31),and the mutation type was a base substitution of guanine(G)with thymine(T)at 451 bp after the start codon in the FAD2B gene,leading to the production of stop codon in the middle of FAD2B gene and the deduced defective fatty acid desaturase.3.In hairy-root transformation,target mutations with genome editing were detected by PCR-RE and sequencing.The results indicated that the mutation proportion was 11.9%(10/84),and the mutation including two types:1)an adenosine(A)insertion at 442 bp after the start codon in the FAD2B gene;2)a base substitution which has been detected in protoplast transformation.4.We have established an integrated tissue culture system to obtain complete gene editing peanut plant in cotyledonary nodes transformation.The transformation rate was 93.15%(68/73)as detected by PCR.5.We also used half-seed transformation,and it only takes two weeks to generate leaf and root from the beginning of transformation.The transformation rate was 85.85%(91/106)as detected by PCR.Taken together,our results demonstrated that CRISPR/Cas9 system can be applied to peanut as an efficient tool for genome editing.The optimized tissue culture system can make it easier and faster for gene editing application in peanut.