Intron-targeted gene insertion in rice using CRISPR/Cas9:A case study of the Pi-ta gene

【作者】 许扬； Fangquan Wang； Zhihui Chen； Weigong Zhong； Jun Wang； Wenqi Li； Fangjun Fan； Yajun Tao； Ling Zhao； Qian-Hao Zhu； 杨杰；

【Author】 Yang Xu;Fangquan Wang;Zhihui Chen;Weigong Zhong;Jun Wang;Wenqi Li;Fangjun Fan;Yajun Tao;Ling Zhao;Qian-Hao Zhu;Jie Yang;Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement;CSIRO Agriculture, GPO Box 1600;

【机构】 Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Nanjing Branch of Chinese National Center for Rice Improvement； CSIRO Agriculture, GPO Box 1600；

【摘要】 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 resistance 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.更多还原

【Abstract】 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 resistance 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.更多还原