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

• Published in: Frontiers in plant science 2022-10, Vol.13, p.952428-952428
• Main Authors: Liu, Shuang, Wang, Xiuyuan, Li, Qianqian, Peng, Wentao, Zhang, Zunmian, Chu, Pengfei, Guo, Shangjing, Fan, Yinglun, Lyu, Shanhua
• Format: Article
• Language: English
• Subjects: agrobacterium rhizogenes–mediated transformation (ARM); CRISPR/Cas9; gamma-glutamylcysteine synthetase gene; genome editing; hairy root; homozygous/biallelic mutation; Plant Science
• ISSN: 1664-462X; 1664-462X
• DOI: 10.3389/fpls.2022.952428

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.