An CRISPR/Cas9-mediated Potato Genome Editing System Without Foreign Gene Insertion

In recent years,the CRISPR / Cas9(Clustered regularly interspaced short palindromic repeats-Cas9)genome editing technology has been widely used in animals and plants.In this research,CRISPR / Cas9 was used to knockout the amylose synthesis-related target genes(GBSS I)to generate new potato lines without foreign DNA insertion.The cultivars including Atlantic,Longshu NO.3 and Favorite were used materials,to transfer the RNP(ribonucleoprotein)and mRNA,which synthesized in vitro,to the explants by gene gun bombardment transformation.A total of 168 transformed plants were btained using RNP as the experimental reagent,and among them 13 transformed plants shown the target gene knockout,while the transformed plants using mRNA as the experimental reagent had not been tested by sequencing yet.The main results of this experiment are as following: 1)The structure of differentiated callus from the explants was too compact and tough to deliver the plasmid into the calli by gene gun bombardment,when using the stem segment of Longshu No.3 as the experimental materials.2)The structure of differentiated callus from the explants is too loose when using the stem segment of Favorite as the experimental materials.As a result,the transformed callus could not differentiate into seedlings.3)The transformants with GBSS I gene knocked out were obtained when using leaf callus of Favorite as the experimental materials,and the transformation efficiency was 0.039-0.36%.The research showed that CRISPR / Cas9 and RNP transformation by gene gun bombardment could be used as a new method for genome editing in potato without foreign DNA insertion.