A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum
A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum
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DOI：
10.3389/fpls.2020.00614
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发表时间：
2020-05-20
影响因子：
5.6
通讯作者：
Hasterok, Robert
中科院分区：
文献类型：
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作者：
Hus, Karolina;Betekhtin, Alexander;Hasterok, Robert
CRISPR/Cas9系统能够进行精确的基因组编辑，是功能基因组研究的有用工具。在这里，我们报告了在模式草二穗短柄草及其异源四倍体亲属B中进行靶向基因组编辑的详细方案。本发明描述了gRNA设计、测试gRNA效率的瞬时原生质体测定、农杆菌介导的转化以及再生植物的选择和分析。在B。二穗草中，我们靶向编码八氢番茄红素去饱和酶（PDS）的基因，PDS是叶绿素生物合成途径中的关键酶。获得的突变体的白化病表型证实了该方案用于功能基因分析的有效性。此外，我们还靶向了两个与细胞壁维持相关的基因，它们编码一种成束蛋白样阿拉伯半乳聚糖蛋白（弗拉）和一种果胶甲基酯酶（PME），也在B中。二穗子在两个B中靶向编码可能参与DNA重组的细胞周期蛋白依赖性激酶（CDKG 1和CDKG 2）的两个基因。二穗型和B型。杂种Cas9活性主要诱导插入或缺失，导致移码突变，其可能导致过早的终止密码子。由于短柄草属物种和关键温带谷物和牧草之间的密切系统发育关系，该协议应易于适应的目标基因支撑农艺学上的重要性状。
The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.