Steinert Jeannette, Schmidt Carla, Puchta Holger
Botanical Institute, Molecular Biology and Biochemistry, Karlsruhe Institute of Technology, 76133, Karlsruhe, Germany.
Methods Mol Biol. 2017;1669:365-376. doi: 10.1007/978-1-4939-7286-9_27.
Since the discovery of the CRISPR/Cas system and its in vivo application for site-specific targeted mutagenesis, this technique is wildly used in a great variety of organisms, such as many plant species. Commonly used for this application is the Cas9 enzyme from Streptococcus pyogenes. Here, we describe the application of two Cas9 orthologs from Streptococcus thermophilus and Staphylococcus aureus for targeted non-homologous end-joining mediated mutagenesis in Arabidopsis thaliana. With both orthologs, we could show efficient inheritance of the induced mutations. As both Cas9 orthologs are smaller in size than the enzyme of S. pyogenes and as the Protospacer adjacent motifs (PAMs) differ between all orthologs, they are attractive alternative tools for genome engineering in plants.
自从CRISPR/Cas系统被发现及其在体内用于位点特异性靶向诱变以来，这项技术已广泛应用于多种生物体，比如许多植物物种。化脓性链球菌的Cas9酶常用于此应用。在此，我们描述了嗜热链球菌和金黄色葡萄球菌的两种Cas9直系同源物在拟南芥中用于靶向非同源末端连接介导的诱变的应用。使用这两种直系同源物，我们能够证明诱导突变的有效遗传。由于这两种Cas9直系同源物的大小均小于化脓性链球菌的酶，并且由于所有直系同源物之间的原间隔相邻基序（PAM）不同，它们是植物基因组工程中有吸引力的替代工具。
