(CRISPR/Cas9 Cassette Construction for OsARF2 Gene Editing and Development of Transgenic Rice Nipponbare Containing CRISPR/Cas9-OsARF2

Abstract

<p>High Affinity K+ transporter 5 (HAK5) is one of the K + channel that involves in K + uptake. HAK5 gene expression is repressed under K + sufficient condition by the Auxin Response Factor 2 (ARF2) transcription factor. K + absorption can be increased by inactivation of ARF2 gene, e.g. by directed mutation using CRISPR/Cas9 method. The aims of this study were to create CRISPR/Cas9 construct carrying gRNA of OsARF2 gene and obtain transgenic rice Nipponbare containing CRISPR/Cas9- gRNAOsARF2. Two different sites in OsARF2 gene were succesfully designed as gRNA of the gene. Both oligo duplex RNA guides, gRNAOsARF2-A and gRNAOsARF2-B, were ligated into pDIRECT_21A plasmid using Golden Gate method. CRISPR/Cas9-gRNAOsARF2-A and CRISPR/Cas9-gRNAOsARF2-B cassettes were succesfully introduced separately into japonica rice cv. Nipponbare genome via Agrobacterium-mediated transformation, resulting putative transgenic lines. Analysis of Cas9 gene integration by PCR showed that 11 of 28 N-ARF-2-A putative transgenic lines contained Cas9 transgene. The sequencing analysis of the two selected lines showed that the OsARF2 gene of the CRISPR/Cas9-gRNAOsARF2 construct had not been mutated. Further studies are needed to determine the presence of mutations in the OsARF2 gene from other T0 transgenic lines obtained in this study together with that of the T1 transgenic lines.</p>