A rapid method for detection of mutations induced by CRISPR/Cas9-based genome editing in common wheat
A rapid method for detection of mutations induced by CRISPR/Cas9-based genome editing in common wheat
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DOI：
10.5511/plantbiotechnology.20.0404b
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发表时间：
2020-06-01
影响因子：
1.6
通讯作者：
Kawaura, Kanako
中科院分区：
文献类型：
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作者：
Kamiya, Yoko;Abe, Fumitaka;Kawaura, Kanako
利用CRISPR/Cas9对普通小麦进行基因组编辑是有用的，因为普通小麦具有异源六倍体的性质，并且可以同时诱导三个同源基因的突变。虽然农杆菌介导的转化在基因组编辑中具有优势，但在小麦中效率较低，耗时较长。因此，在体内使用具有高效诱变作用的引导rna （gRNAs）是在短时间内产生基因组编辑突变系的关键因素之一。本研究以普通小麦中的三个基因为研究对象，建立了一种快速检测瞬时表达系统诱导突变的方法。在未成熟小麦胚中实现了grna和Cas9的生物学瞬时表达。一周后使用PCR-RFLP检测突变，并通过基因组克隆测序进行验证。我们确认了几种类型的突变，根据目标序列以不同的速率发生。此外，在农杆菌转化的植物中，在编辑率较高的目标上，突变频率往往更高。这些结果表明，这种快速检测编辑突变的方法可用于多种应用，例如筛选靶序列或修饰载体，以便在小麦中进行高效的CRISPR/Cas9基因组编辑。
Genome editing using CRISPR/Cas9 is useful for common wheat because common wheat has allohexaploid nature and it can induce mutations simultaneously in three homoeologous genes. Although Agrobacterium-mediated transformation has advantages in genome editing, it still has low efficiency and requires relatively long time in wheat. Therefore, the use of guide RNAs (gRNAs) with efficient mutagenesis in vivo is one of the critical factors for producing genome-edited mutant lines in a short time. In this study, we targeted three genes in common wheat and established a rapid method for detection of mutations induced by the biolistic transient expression system. Biolistic transient expression of the gRNAs and Cas9 was achieved in immature wheat embryos. Mutations were detected a week later using PCR-RFLP and verified by the sequencing of genomic clones. We confirmed several types of mutations that occurred at different rates depending on the target sequences. Furthermore, frequencies of mutations tended to be higher at the targets that were edited at higher rates in the plants transformed by Agrobacterium. These results show that this method of rapid detection of edited mutations could be used for variety of applications, such as screening of target sequences or modified vectors for efficient CRISPR/Cas9 genome editing in wheat.