Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants.
Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants.
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DOI：
10.1038/srep26685
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发表时间：
2016-05-26
影响因子：
4.6
通讯作者：
Osakabe K
中科院分区：
文献类型：
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作者：
Osakabe Y;Watanabe T;Sugano SS;Ueta R;Ishihara R;Shinozaki K;Osakabe K
使用CRISPR/Cas9系统的基因组编辑可用于修饰植物基因组，然而，为了使编辑技术在各种植物物种中有用，仍然需要改进特异性和适用性。在这里，使用由截短的gRNA（tru-gRNA）/Cas9组合介导的基因组编辑，我们为OST 2（拟南芥中的质子泵）产生了新的等位基因，没有脱靶效应。通过跟踪Cas9和tru-gRNA的表达，使用组成型启动子检测到CRIPSR/Cas9转基因植物中新产生的突变，具有高达32.8%的高平均突变率，并且没有脱靶效应。减少Cas9中的核定位信号降低了突变率。相比之下，由分生组织和生殖组织特异性启动子驱动的tru-gRNA Cas9盒增加了拟南芥中的可遗传突变率，表明生殖系中的高表达可以产生双等位基因突变。最后，新的突变等位基因获得OST 2表现出改变气孔关闭响应环境条件。这些结果表明了在分子育种中的进一步应用，以使用优化的植物CRISPR/Cas9系统改善植物功能。
Genome editing using the CRISPR/Cas9 system can be used to modify plant genomes, however, improvements in specificity and applicability are still needed in order for the editing technique to be useful in various plant species. Here, using genome editing mediated by a truncated gRNA (tru-gRNA)/Cas9 combination, we generated new alleles for OST2, a proton pump in Arabidopsis, with no off-target effects. By following expression of Cas9 and the tru-gRNAs, newly generated mutations in CRIPSR/Cas9 transgenic plants were detected with high average mutation rates of up to 32.8% and no off-target effects using constitutive promoter. Reducing nuclear localization signals in Cas9 decreased the mutation rate. In contrast, tru-gRNA Cas9 cassettes driven by meristematic- and reproductive-tissue-specific promoters increased the heritable mutation rate in Arabidopsis, showing that high expression in the germ line can produce bi-allelic mutations. Finally, the new mutant alleles obtained for OST2 exhibited altered stomatal closing in response to environmental conditions. These results suggest further applications in molecular breeding to improve plant function using optimized plant CRISPR/Cas9 systems.