Design of the CRISPR/Cas9-based OsNRAMP2 editing construct in TBR225 rice (Oryza sativa L.) variety

Abstract

Micronutrients are indispensable for plant growth and development. In rice, several protein-coding genes of the NRAMP (Natural resistance-associated macrophage protein) family have been identified as being involved in metal accumulation. In this study, a segment of the coding sequence of OsNRAMP2 was isolated from the total DNA of the rice variety TBR225, one of the most popular varieties in Northern Vietnam. Based on the isolated DNA sequences, two crRNA sequences (CRISPR RNA), capable of binding two specific sites on exon II and exon III of OsNRAMP2, were designed using bioinformatics tools. Mutation sites in the early exons are more likely to cause sense changes in the target protein. The crRNAs were ligated to the BtgZI and BsaI sites on the pENTR4-V2 vector; the DNA fragment encoding both sgRNAs was inserted into the pCas9 vector to create a complete CRISPR/Cas9 (Clustered regularly in- terspaced short palindromic repeats/CRISPR associated protein 9) editing construct, which may cause the OsRAMP2 gene mutation in TBR225 rice. This will be the premise for studying the function of OsNRAMP2 in TBR225.