Employing template-directed CRISPR-based editing of theOsALSgene to create herbicide tolerance in Basmati rice

Abstract

AbstractRice (Oryza sativa) is one of the primary food crops which contributes major portion of daily calorie intake. It is used as model crop for various genome editing studies. Basmati rice was also explored for establishing non-homologous end joining-based genome editing. But it was not clear whether homology-directed repair (HDR)-based genome editing can be done in Basmati rice. The current study was designed to establish HDR-based genome editing in Basmati rice to develop herbicide tolerance. There is severe weed spread when rice is grown via direct planted rice method in various countries to save labour and water resources. Therefore, the use of herbicides is necessary to control weeds. These herbicides can also affect cultivated rice which creates the need to develop herbicide-tolerant rice. In current study, we introduced a point mutation in Acetolactate Synthase gene to convert tryptophan to leucine at position 548. For this purpose, different constructs for HDR were tested with different RNA scaffold and orientation of repair templates. Out of four different architectures, the one having repair template identical to the target DNA strand precisely edited the target site. We successfully established template-directed CRISPR-Cas9 system in Super Basmati rice by detecting desired substitutions at the target site in Acetolactate Synthase locus. Moreover, this editing of Acetolactate Synthase gene resulted in the production of herbicide tolerance in Super Basmati rice. This study suggests that such type of HDR system can be used to precisely edit other genes for crop improvement.