利用 xCas9 和 Cas9-NG 杂合体扩展 CRISPR/Cas9 介导的植物基因组编辑范围。
Expanding the scope of CRISPR/Cas9-mediated genome editing in plants using an xCas9 and Cas9-NG hybrid.
机构信息
Shanghai Center for Plant Stress Biology, and National Key Laboratory of Plant Molecular Genetics, Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, 200032, China.
Shandong Shunfeng Biotechnology Co., Ltd, Jinan, 250000, China.
出版信息
J Integr Plant Biol. 2020 Apr;62(4):398-402. doi: 10.1111/jipb.12886. Epub 2020 Jan 9.
The widely used Streptococcus pyogenes Cas9 (SpCas9) requires NGG as a protospacer adjacent motif (PAM) for genome editing. Although SpCas9 is a powerful genome-editing tool, its use has been limited on the targetable genomic locus lacking NGG PAM. The SpCas9 variants xCas9 and Cas9-NG have been developed to recognize NG, GAA, and GAT PAMs in human cells. Here, we show that xCas9 cannot recognize NG PAMs in tomato, and Cas9-NG can recognize some of our tested NG PAMs in the tomato and Arabidopsis genomes. In addition, we engineered SpCas9 (XNG-Cas9) based on mutations from both xCas9 and Cas9-NG, and found that XNG-Cas9 can efficiently mutagenize endogenous target sites with NG, GAG, GAA, and GAT PAMs in the tomato or Arabidopsis genomes. The PAM compatibility of XNG-Cas9 is the broadest reported to date among Cas9s (SpCas9 and Cas9-NG) active in plant.
广泛使用的化脓性链球菌 Cas9（SpCas9）需要 NGG 作为基因组编辑的前导相邻基序（PAM）。尽管 SpCas9 是一种强大的基因组编辑工具，但由于缺乏 NGG PAM 的靶向基因组位点，其应用受到限制。已经开发出 SpCas9 的变体 xCas9 和 Cas9-NG 以识别人细胞中的 NG、GAA 和 GAT PAMs。在这里，我们表明 xCas9 不能在番茄中识别 NG PAMs，而 Cas9-NG 可以识别我们在番茄和拟南芥基因组中测试的一些 NG PAMs。此外，我们基于 xCas9 和 Cas9-NG 的突变工程化了 SpCas9（XNG-Cas9），并发现 XNG-Cas9 可以有效地使具有 NG、GAG、GAA 和 GAT PAMs 的内源靶位点在番茄或拟南芥基因组中发生突变。XNG-Cas9 的 PAM 兼容性是迄今为止报道的 Cas9s（SpCas9 和 Cas9-NG）在植物中活性最广泛的。
Zotero 插件