Sequence-specific nucleases have been exploited to create targeted gene knockouts in various plants1, but replacing a fragment and even obtaining gene insertions at specific loci in plant genomes remain a serious challenge. Here, we report efficient intron-mediated site-specific gene replacement and insertion approaches that generate mutations using the non-homologous end joining (NHEJ) pathway using the clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) system. Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a donor DNA template including the same pair of sgRNA sites, we achieved gene replacements in the rice endogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) at a frequency of 2.0%. We also obtained targeted gene insertions at a frequency of 2.2% using a sgRNA targeting one intron and a donor DNA template including the same sgRNA site. Rice plants harbouring the OsEPSPS gene with the intended substitutions were glyphosate-resistant. Furthermore, the site-specific gene replacements and insertions were faithfully transmitted to the next generation. These newly developed approaches can be generally used to replace targeted gene fragments and to insert exogenous DNA sequences into specific genomic sites in rice and other plants.
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The authors thank J.-L. Qiu from Institute of Microbiology, Chinese Academy of Sciences and D.F. Voytas from University of Minnesota for helpful discussions and insightful comments on this manuscript. This work was supported by grants from the Ministry of Science and Technology (2016YFD0101804), the Ministry of Agriculture of China (2016ZX08010002 and 2014ZX0801003B) and the National Natural Science Foundation of China (31420103912, 31271795, 31570369 and 31501376).
The authors have filed a patent application based on the results reported in this paper.
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Li, J., Meng, X., Zong, Y. et al. Gene replacements and insertions in rice by intron targeting using CRISPR–Cas9. Nature Plants 2, 16139 (2016) doi:10.1038/nplants.2016.139
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