Genome Editing Platforms in Rice (Oryza sativa L.): Basic methodology and troubleshooting

Abstract

Abstract Nowadays, genome editing approaches are one of the most frequently used for studying the function of a new gene(s) and for the development of elite mutant lines with desired traits. The technology has to boost up the craze among the researchers for editing the crop genome. However, information regarding the constructions of CRISPR/Cas9 gene cassette to develop edited rice plants is scattered. In the present study, we have shown a systematic stepwise protocol for designing gRNA, cloning of gRNA in CRISPR/Cas9 binary vector, Agrobacterium-mediated transformation, screening and confirmation of edited plants along with troubleshooting at each step to accelerate the application of the CRISPR/Cas9 system for rice improvement. The CHOPCHOP web tool was used for designing primers for gRNA. In this study, we are mentioning a specific trait for gene editing because we are giving overall easy and efficient protocols for generating edited plants for any trait. Plants with the presence of CaMV35S promoter, OsU3 promoter, PAT gene, and Cas9 gene were treated as gene-edited plants whereas the absence of the desired band in plants was treated as wild type. The performance of genome editing technology in the laboratory depends upon the systematic steps to finally find the desirable edited plant, and this simplified method of CRISPR/Cas9 mediated gene editing will accelerate functional genomics studies in rice.