Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9
Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9
复制标题
使用 CRISPR/Cas9 通过内含子靶向进行水稻中的基因替换和插入
DOI：
10.1038/nplants.2016.139
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发表时间：
2016-10-01
期刊：
影响因子：
18
通讯作者：
Gao, Caixia
中科院分区：
文献类型：
--
作者：
Li, Jun;Meng, Xiangbing;Gao, Caixia
序列特异性核酸酶已被用于在多种植物中创建靶向基因敲除(1)，但在植物基因组的特定位点上替换片段甚至获得基因插入仍然是一个严峻的挑战。在这里，我们报道了有效的内含子介导的位点特异性基因替换和插入方法，该方法使用非同源末端连接（NHEJ）途径，使用集群规则间隔短回文重复序列(CRISPR)-CRISPR相关蛋白9 （Cas9）系统产生突变。利用一对靶向相邻内含子的单引导rna （sgRNAs）和一个包含相同sgRNA位点的供体DNA模板，我们以2.0%的频率实现了水稻内源基因5-烯醇丙酮酰石草酸-3-磷酸合成酶（EPSPS）的基因替换。我们还获得了频率为2.2%的靶向基因插入，使用sgRNA靶向一个内含子和一个包含相同sgRNA位点的供体DNA模板。含有OsEPSPS基因的水稻植株具有草甘膦抗性。此外，位点特异性基因的替换和插入被忠实地传递给下一代。这些新开发的方法通常可用于替换目标基因片段，并将外源DNA序列插入水稻和其他植物的特定基因组位点。
Sequence-specific nucleases have been exploited to create targeted gene knockouts in various plants(1), 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.