Zafar Kashaf, Khan Muhammad Zuhaib, Amin Imran, Mukhtar Zahid, Zafar Mehak, Mansoor Shahid
Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Constituent College of Pakistan Institute of Engineering and Applied Sciences, Jhang Road, Faisalabad 37000, Pakistan.
Department of Biotechnology, Balochistan University of Information Technology, Engineering and Management Sciences (BUITEMS), Quetta 87300, Pakistan.
AoB Plants. 2023 Jan 13;15(2):plac059. doi: 10.1093/aobpla/plac059. eCollection 2023 Feb.
Rice () 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 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 locus. Moreover, this editing of 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.
水稻（）是主要的粮食作物之一，提供了日常卡路里摄入的大部分。它被用作各种基因组编辑研究的模式作物。人们还探索了用巴斯马蒂水稻建立基于非同源末端连接的基因组编辑。但尚不清楚基于同源定向修复（HDR）的基因组编辑是否能在巴斯马蒂水稻中实现。当前的研究旨在在巴斯马蒂水稻中建立基于HDR的基因组编辑以培育除草剂耐受性。在各国，当通过直播水稻法种植水稻以节省劳动力和水资源时，杂草蔓延严重。因此，使用除草剂来控制杂草是必要的。这些除草剂也会影响栽培水稻，这就产生了培育耐除草剂水稻的需求。在当前研究中，我们在基因中引入了一个点突变，将第548位的色氨酸转换为亮氨酸。为此，测试了不同的HDR构建体与不同的RNA支架和修复模板方向。在四种不同的结构中，具有与目标DNA链相同的修复模板的结构精确地编辑了目标位点。我们通过检测位点目标位点的期望替换，在超级巴斯马蒂水稻中成功建立了模板导向的CRISPR-Cas9系统。此外，该基因的这种编辑导致超级巴斯马蒂水稻产生了除草剂耐受性。这项研究表明，这种类型的HDR系统可用于精确编辑其他基因以改良作物。
