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New CRISPR/Cas9 toolkits for efficient genome editing in rice


Recently, an article “Cas9-NG greatly expands the targeting scope of genome-editing toolkit by recognizing NG and other atypical PAMs in rice”, which was about the op-timization and expansion of the PAM recognition sequence of rice genome editing technology, was reported in the Molecular Plant (IF = 9.326) by Prof. ZHOU Huanbin's group from the Institute of Plant Protection of the Chinese Academy of Agricultural Sciences (IPP-CAAS).

CRISPR/Cas9 system derived from the microbial adaptable immune systems in Streptococcus pyogenes have been successfully and widely applied in rice genome editing, showing a powerful potential to advance rice functional genomics research and crop breeding. However, the requirement of the protospacer adjacent motif (PAM) NGG for Cas9 recognition restricts the targetable genomic loci in applica-tions. Especially for the base editing, it is difficult to obtain the suitable PAM for the designated target base.

To address this problem, two engineered Cas9 variants, Cas9-NG and xCas9, were adopted to optimize and extend the genome targeting scope of CRISPR-Cas9 technology. The results showed that Cas9-NG, outperforming xCas9, efficiently induced mu-tations of target genes at the relaxed NG PAM and the diverse set of atypical PAMs (NAC, NTG, NTT and NCG). Moreover, efficient rice genome-editing toolkits were constructed for single and multiplex genes knockout, base editing (conversion be-tween C•G and T•A) and transcriptional activation by using Cas9-NG, respectively. To some extent, the successful application of Cas9-NG in rice genome editing overcame the restriction of PAM recognition, and made the editable locus in rice genome increase eight times. The targeted sites resistant to Cas9 before can be modified through Cas9-NG now, and the targeting scope of genome editing tools was greatly expanded. All of this will be of great value in deciphering rice functional genomics, speeding up the molecular rice breeding program, and extending the commercial life of the current rice cultivars.This work was accomplished by extensive collaborations among the research groups of Profs. ZHOU Huanbin and ZHOU Xueping from IPP-CAAS, and Prof. LIN Honghui from College of Life Sciences of Sichuan University, and supported by grants from the National Natural Science Foundation of China (31871948), the National Key Research and Development Program of China (2017YFD0200900) and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.



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Copyright:Institute of Plant Protection,Chinese Academy of Agricultural Sciences 

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