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

doi:10.3389/fpls.2022.952428

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

Front Plant Sci. 2022 Oct 18:13:952428. doi: 10.3389/fpls.2022.952428. eCollection 2022.

Authors

Shuang Liu¹, Xiuyuan Wang¹, Qianqian Li¹, Wentao Peng¹, Zunmian Zhang¹, Pengfei Chu¹, Shangjing Guo¹, Yinglun Fan¹, Shanhua Lyu¹

Affiliation

¹ College of Agriculture, Liaocheng University, Liaocheng, China.

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-glutamylcysteine synthetase 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 pAtGCSpro-Cas9 system, the average homozygous/biallelic mutation frequency is 1.7-fold and 8.3-fold higher than the p2 × 35Spro-Cas9 system for single and two target site(s) in the genome, respectively. Our results demonstrate the advantage of the pAtGCSpro-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.

Keywords: CRISPR/Cas9; agrobacterium rhizogenes–mediated transformation (ARM); gamma-glutamylcysteine synthetase gene; genome editing; hairy root; homozygous/biallelic mutation.