Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice.
Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice.
复制标题
DOI：
10.1007/s11103-015-0342-x
复制
发表时间：
2015-08
影响因子：
5.1
通讯作者：
Endo M
中科院分区：
文献类型：
--
作者：
Mikami M;Toki S;Endo M
CRISPR/Cas9系统是一种用于多种生物体基因组编辑的有效工具。尽管最近有几个报道在植物中使用CRISPR/Cas9系统成功地进行了靶向诱变，但在每种情况下，感兴趣的靶基因、所使用的Cas9表达系统和向导RNA（gRNA）以及用于转化和随后诱变的组织都不同，因此不能直接比较所报道的靶向诱变的频率。在这里，我们在标准化实验条件下使用不同的Cas9和/或gRNA表达盒评估了水稻的突变频率。我们将Cas9和gRNA表达盒分别或依次引入水稻愈伤组织中，并评估在相同内源靶向序列处的诱变频率。根据所使用的Cas9表达盒，突变频率显著不同。此外，由OsU 6启动子驱动的gRNA比由OsU 3启动子驱动的gRNA具有上级优势。使用携带最佳组合的Cas9/gRNA表达盒的一体化表达载体导致水稻愈伤组织中靶向诱变的频率大大提高，并且在T0代中产生双等位基因突变体植物。本文提出的方法可以适用于优化用于多种植物中基因组编辑的Cas9/gRNA盒的构建。本文的在线版本（doi：10.1007/s11103-015-0342-x）包含补充材料，可供授权用户使用。
The CRISPR/Cas9 system is an efficient tool used for genome editing in a variety of organisms. Despite several recent reports of successful targeted mutagenesis using the CRISPR/Cas9 system in plants, in each case the target gene of interest, the Cas9 expression system and guide-RNA (gRNA) used, and the tissues used for transformation and subsequent mutagenesis differed, hence the reported frequencies of targeted mutagenesis cannot be compared directly. Here, we evaluated mutation frequency in rice using different Cas9 and/or gRNA expression cassettes under standardized experimental conditions. We introduced Cas9 and gRNA expression cassettes separately or sequentially into rice calli, and assessed the frequency of mutagenesis at the same endogenous targeted sequences. Mutation frequencies differed significantly depending on the Cas9 expression cassette used. In addition, a gRNA driven by the OsU6 promoter was superior to one driven by the OsU3 promoter. Using an all-in-one expression vector harboring the best combined Cas9/gRNA expression cassette resulted in a much improved frequency of targeted mutagenesis in rice calli, and bi-allelic mutant plants were produced in the T0 generation. The approach presented here could be adapted to optimize the construction of Cas9/gRNA cassettes for genome editing in a variety of plants. The online version of this article (doi:10.1007/s11103-015-0342-x) contains supplementary material, which is available to authorized users.