Applications of CRISPR/Cas9 in a rice protein expression system via an intron-targeted insertion approach.

The sugar starvation-inducible rice αAmy3 promoter and signal peptide are widely used to produce valuable recombinant proteins in rice suspension culture cells. Conventionally, the recombinant gene expression cassette is inserted into the genome at random locations by Agrobacterium- or particle bombardment-mediated transformation. CRISPR/Cas9 gene editing enables gene insertion at a precise target site in the genome. In this study the CRISPR/Cas9 approach was modified for intron-targeted insertion by adding an artificial 3' splicing site upstream of the recombinant gene. Knock-in transgenic rice cell lines containing the recombinant GFP gene inserted in intron 1 of αAmy3 were generated. The endogenous αAmy3 promoter regulated recombinant gene expression and the αAmy3 signal peptide directed secretion of the recombinant GFP protein into the culture medium. In addition, the recombinant GFP protein was localized in amyloplasts, identical to the subcellular localization of endogenous αAmy3 reported previously. This modified CRISPR/Cas9 knock-in approach is simple and highly efficient, and the recombinant gene insertion frequency attained 12.5%. The approach can be applied in the production of pharmaceutical proteins in rice suspension cell cultures. The high efficiency of the GFP reporter gene knock-in method and the maintenance of target gene behavior also make the strategy applicable to endogenous gene functional studies in rice.