AtGCS promoter-driven clustered regularly interspaced short palindromic repeats/Cas9 highly efficiently generates homozygous/biallelic mutations in the transformed roots by Agrobacterium rhizogenes–mediated transformation
Shuang Liu, Xiuyuan Wang, Qianqian Li, Wentao Peng, Zunmian Zhang, Pengfei Chu, Shangjing Guo, Yinglun Fan, Shanhua Lyu
Abstract
Agrobacterium rhizogenes –mediated (ARM) transformation is an efficient and powerful tool to generate transgenic roots to study root-related biology. For loss-of-function studies, transgenic-root-induced indel mutations by CRISPR/Cas9 only with homozygous/biallelic mutagenesis can exhibit mutant phenotype(s) (excluding recessive traits). However, a low frequency of homozygous mutants was produced by a constitutive promoter to drive Cas9 expression. Here, we identified a highly efficient Arabidopsis thaliana gamma- g lutamyl c ysteine s ynthetase promoter, termed AtGCSpro , with strong activity in the region where the root meristem will initiate and in the whole roots in broad eudicots species. AtGCSpro achieved higher homozygous/biallelic mutation efficiency than the most widely used CaMV 35S promoter in driving Cas9 expression in soybean, Lotus japonicus , and tomato roots. Using the p AtGCSpro -Cas9 system, the average homozygous/biallelic mutation frequency is 1.7-fold and 8.3-fold higher than the p 2 × 35Spro -Cas9 system for single and two target site(s) in the genome, respectively. Our results demonstrate the advantage of the p AtGCSpro -Cas9 system used in ARM transformation, especially its great potential in diploids with multiple-copy genes targeted mutations and polyploid plants with multiplex genome editing. AtGCSpro is conservatively active in various eudicots species, suggesting that AtGCSpro might be applied in a wide range of dicots species.