The 30th International Conference on Arabidopsis Research (ICAR 2019)

495 / 2019-03-13 15:54:12

Cas9-NG greatly expands the targeting scope of genome-editing toolkit by recognizing NG and other atypical PAMs in rice

CRISPR, Cas9-NG, PAM, gene editing, base editing, Oryza sativa L.

From models to crops > From models to crops

The CRISPR technologies enabling precise genome manipulation are valuable for the gene function study and molecular crop breeding. However, the requirement of a protospacer adjacent motif (PAM), such as NGG, TTN, etc., for Cas protein recognition restricts the targetable genomic loci in applications. Very lately, Cas9-NG which recognizes a minimal NG PAM was reported to expand the targeting space in genome editing in human cells, but little is known about its applications in plants. Here, we evaluated the nuclease activity of Cas9-NG toward various NGN PAMs by targeting endogenous genes in transgenic rice, and revealed that Cas9-NG edits all NGG, NGA, NGT and NGC sites with impaired activities, and gene-edited plants were dominated by mono-allelic mutations. The Cas9-NG-engineered base editors were further developed and used to generate OsBZR1 gain-of-function plants, which other available Cas9-engineered base editors are not able to. Moreover, Cas9-NG-based transcriptional activator efficiently up-regulated the expression of endogenous target genes in rice. In addition, we discovered that Cas9-NG recognizes NAC, NTG, NTT, NCG apart from NG PAM. All these findings show that Cas9-NG greatly expands the targeting scope of genome-editing tools and imply great potentials for targeted genome editing, base editing and genome regulation in plants.