Expanding the scope of CRISPR/Cas9-mediated genome editing in plants using an xCas9 and Cas9-NG hybrid.,Journal of Integrative Plant Biology

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Expanding the scope of CRISPR/Cas9-mediated genome editing in plants using an xCas9 and Cas9-NG hybrid.
Journal of Integrative Plant Biology ( IF 9.3 ) Pub Date : 2020-01-09 , DOI: 10.1111/jipb.12886
Qingfeng Niu ₁ , Siqun Wu ₁ , Yansha Li ₂ , Xiaoxuan Yang ₁ , Ping Liu ₁ , Yaping Xu ₁ , Zhaobo Lang ₁

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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.

中文翻译：

使用xCas9和Cas9-NG杂种扩大植物中CRISPR / Cas9介导的基因组编辑的范围。

广泛使用的化脓性链球菌Cas9（SpCas9）需要NGG作为基因组编辑的原间隔邻近基序（PAM）。尽管SpCas9是一种功能强大的基因组编辑工具，但其使用仅限于缺少NGG PAM的可靶向基因组基因座。SpCas9变种xCas9和Cas9-NG已被开发用于识别人细胞中的NG，GAA和GAT PAM。在这里，我们证明xCas9无法识别番茄中的NG PAM，而Cas9‐NG可以识别我们在番茄和拟南芥基因组中测试的某些NG PAM 。此外，我们基于xCas9和Cas9-NG的突变设计了SpCas9（XNG-Cas9），发现XNG-Cas9可以利用番茄，拟南芥中的NG，GAG，GAA和GAT PAM有效诱变内源性靶位点。基因组。XNG-Cas9的PAM兼容性是迄今为止在植物中活跃的Cas9（SpCas9和Cas9-NG）中最广泛的报道。

更新日期：2020-01-09

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