Intron-targeted gene insertion in rice using CRISPR/Cas9: A case study of the Pi-ta gene
Yang Xu,
Fangquan Wang,
Zhihui Chen,
Jun Wang,
Wen-Qi Li,
Fangjun Fan,
Yajun Tao,
Ling Zhao,
Weigong Zhong,
Qian-Hao Zhu,
Jie Yang
Affiliations
Yang Xu
Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing 210014, Jiangsu, China; Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China; Provincial Key Laboratory of Agrobiology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China
Fangquan Wang
Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing 210014, Jiangsu, China; Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China; Provincial Key Laboratory of Agrobiology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China
Zhihui Chen
Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing 210014, Jiangsu, China; Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China
Jun Wang
Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing 210014, Jiangsu, China; Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China
Wen-Qi Li
Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing 210014, Jiangsu, China; Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China
Fangjun Fan
Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing 210014, Jiangsu, China; Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China
Yajun Tao
Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing 210014, Jiangsu, China
Ling Zhao
Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing 210014, Jiangsu, China
Weigong Zhong
Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing 210014, Jiangsu, China; Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China
Qian-Hao Zhu
CSIRO Agriculture and Food, GPO Box 1700, Canberra ACT 2601, Australia
Jie Yang
Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing 210014, Jiangsu, China; Jiangsu Co-Innovation Center for Modern Production Technology of Grain Crops, Yangzhou University, Yangzhou 225009, Jiangsu, China; Provincial Key Laboratory of Agrobiology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China; Institute of Life Science, Jiangsu University, Zhenjiang 212013, Jiangsu, China; Corresponding author at: Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement, Nanjing 210014, Jiangsu, China.
Intron-targeted gene insertion strategy using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated Cas9) has been shown to be a potential tool for crop genetic improvement by targeted mutagenesis or gene replacement of an elite allele into widely cultivated rice varieties. The rice blast resistant protein Pi-ta, differs from its susceptible counterpart, pi-ta, by a single amino acid in exon 2. To create new materials resistant to the rice blast disease, we inserted a genomic fragment containing the exon 2 and 3′ untranslated region (3′ UTR) of Pi-ta into intron 1 of pi-ta in rice materials susceptible to rice blast using the intron-targeted insertion strategy. The gene insertion frequency was 3.8%. Several novel transgene-free progeny with stably inherited homozygous insert were identified in the T1 generation, which have been crossed to rice germplasm bearing other resistance gene (R gene) for pyramiding of R genes. This work verified the feasibility of using the genome editing technology in improvement of qualitative agronomic trait in crops.