Systematic identification of endogenous RNA polymeraseⅢpromoters for efficient RNA guidebased genome editing technologies in maize  

作　　者：Xiantao Qi Le Dong Changlin Liu Long Mao Fang Liu Xin Zhang Beijiu Cheng Chuanxiao Xie 

机构地区：[1]Institute of Crop Science,Chinese Academy of Agricultural Sciences,Beijing 100081,China [2]Anhui Agricultural University,Anhui Province,Hefei 230036,Anhui,China

出　　处：《The Crop Journal》2018年第3期314-320,共7页作物学报（英文版）

基　　金：supported by the National Science Foundation of China(31771808);Ministry of Science and Technology(2015BAD02B0203);National Engineering Laboratory of Crop Molecular Breeding;the Chinese Academy of Agricultural Sciences(Y2017XM03)

摘　　要：Single-guide RNA(sg RNA) is one of the two core components of the CRISPR(clustered regularly interspaced short palindromic repeat)/Cas(CRISPR-associated) genome-editing technology. We established an in vitro Traffic Light Reporter(TLR) system, which is designated as the same colors as traffic lights such as green, red and yellow were produced in cells. The TLR can be readily used in maize mesophyll protoplast for a quick test of promoter activity. The TLR assay indicates the variation in transcription activities of the seven Pol III promoters, from 3.4%(U6-1) to over 21.0%(U6-6). The U6-2 promoter, which was constructed to drive sg RNA expression targeting the Zm Wx1 gene, yielded mutation efficiencies ranging from 48.5% to 97.1%. Based on the reported and unpublished data, the in vitro TLR assay results were confirmed to be a readily system and may be extended to other plant species amenable to efficient genome editing via CRISPR/Cas. Our efforts provide an efficient method of identifying native Pol III-recognized promoters for RNA guide-based genome-editing systems in maize.Single-guide RNA（sg RNA） is one of the two core components of the CRISPR（clustered regularly interspaced short palindromic repeat）/Cas（CRISPR-associated） genome-editing technology. We established an in vitro Traffic Light Reporter（TLR） system, which is designated as the same colors as traffic lights such as green, red and yellow were produced in cells. The TLR can be readily used in maize mesophyll protoplast for a quick test of promoter activity. The TLR assay indicates the variation in transcription activities of the seven Pol III promoters, from 3.4%（U6-1） to over 21.0%（U6-6）. The U6-2 promoter, which was constructed to drive sg RNA expression targeting the Zm Wx1 gene, yielded mutation efficiencies ranging from 48.5% to 97.1%. Based on the reported and unpublished data, the in vitro TLR assay results were confirmed to be a readily system and may be extended to other plant species amenable to efficient genome editing via CRISPR/Cas. Our efforts provide an efficient method of identifying native Pol III-recognized promoters for RNA guide-based genome-editing systems in maize.

关 键 词：CRISPR/Cas Genome editing RNA polymerase III promoters MAIZE 

分 类 号：S[农业科学]