Kashaf Zafar, Muhammad Zuhaib Khan, Imran Amin, Zahid Mukhtar, Mehak Zafar, Shahid Mansoor

AoB PLANTS, plac059, https://doi.org/10.1093/aobpla/plac059

Published: 13 January 2023

Abstract

Rice (Oryza sativa) is one of the primary food crops which contribute major portion of daily calories 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-based genome editing can be done in Basmati rice. The current study was designed to establish homology directed repair-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 labor 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 the position 548. For this purpose, different constructs for homology-directed repair 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 homology-directed repair system can be used to precisely edit other genes for crop improvement.

See https://academic.oup.com/aobpla/advance-article/doi/10.1093/aobpla/plac059/6986705?login=false

Figure 1: BS sensitivity of Super Basmati rice callus and seedlings. (A) BS sensitivity of callus on BS supplemented medium at 0, 0.1, 0.2, 0.3 and 0.5 μM, respectively. The degree of callus growth was compared after 25 days after. (B) Seedling growth on different BS concentrations from 0 to 5.0 μM.