Page 18 - S. Plant biology

[S. Plant biology-11]

Editing of glutelin genes in rice using CRISPR-Cas9

techniques

Deepanwita Chandra¹, Kyoungwon Cho¹, Ok Soo Han¹

¹Department of Food, Bioscience and Biotechnology, Chonnam National University, Gwangju 61186, South Korea

Glutelins are major rice storage proteins, accounting for 60%-80% of the total seed protein content. They are
encoded by 15 gene copies in the genome, classified into four sub-families such as GluA, GluB, GluC, and GluD
based on the similarity of their amino acid sequence, and accumulated into Protein body II (PBII) Storage Vacuoles

(PSVs) via Golgi apparatus. It has been known that nutritional-value of rice seed storage proteins (SSPs) is not high

enough and their contents limit the yield of a valuable foreign protein in seeds. To challenge these issues, we have
tried to develop glutelin knockout rice lines using CRISPR-Cas9 technique. CRISPR-Cas9 Technique has recently
emerged as an efficient and easy to handle genome editing tool. Here we have designed four sgRNAs targeting

more than one glutelin genes. We have checked that the sgRNAs activate the cleavage of the targeted genes in
vitro and in vivo (rice protoplast system). We have constructed different sgRNA-Cas9 vectors for Agrobacterium-

mediated rice transformation and carried out transformation. We have 5-20 lines of putative transgenic plants for
each sgRNA-Cas9 constructs (more than 100 putative transgenic plants). Use of wheat dwarf virus (WDV) system

(geminivirus) for the delivery of repair template for HDR mediated knock-in has already been established in rice.
We want to KI a reporter gene (AsRed2) into glutelin genes to check the efficiency of WDV system over convention

T-DNA system for delivering of template DNA in rice protoplasts as well as plants.