DESIGN OF CRISPR/CAS9 SYSTEM FOR EDITING OsNRAMP7 INVOLVED IN METAL TRANSPORT IN TBR225 RICE VARIETY

Abstract

Rice (Oryza sativa L.) is one of the major food crops but is deficient in minerals such as Zn. In rice, there were eight genes belonging to the Natural resistance-associated macrophage protein (NRAMP) family, of which OsNRAMP1, OsNRAMP4, OsNRAMP5, OsNRAMP6, and OsNRAMP8 were reported to transport metals such as iron (Fe), zinc, mangan (Mn). Recently, AtNRAMP5, closely related to OsNRAMP7, has been identified as a function in Zn uptake in Arabidopsis. To identify the function of OsNRAMP7 in a local rice variety -TBR225, the expression of the interested gene was evaluated and a Clustered regular interspaced short palindromic repeats/CRISPR-associated Cas9 (CRISPR/Cas9) system for editing TBR225 OsNRAMP7 was constructed.  The accumulation of OsNRAMP7 mRNA was increased in Zn-treated TBR225 rice plants. Two single guide RNAs for the OsNRAMP7-editing using CRISPR/Cas9 system was designed based on 0.52 kb DNA fragment containing the ExonII region of OsNRAMP7 isolated from the genomic DNA of TBR225. The CRISPR/Cas9 system for directed editing TBR225 OsNRAMP7 containing two CRISPR RNA (crRNA) sequences recognizing two different sites on the ExonII of OsNRAMP57 was developed. The recombinant vector was validated by PCR and sequencing analysis. The results will be used for studying possible function of OsNRAMP7 via knocking out of the gene.