AUTHOR=Liu Shuang , Wang Xiuyuan , Li Qianqian , Peng Wentao , Zhang Zunmian , Chu Pengfei , Guo Shangjing , Fan Yinglun , Lyu Shanhua 

TITLE=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

JOURNAL=Frontiers in Plant Science

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2022.952428

DOI=10.3389/fpls.2022.952428

ISSN=1664-462X

ABSTRACT=<p><italic>Agrobacterium rhizogenes</italic>–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 <italic>Cas9</italic> expression. Here, we identified a highly efficient <italic>Arabidopsis thaliana</italic> gamma-<italic><underline>g</underline>lutamyl<underline>c</underline>ysteine <underline>s</underline>ynthetase</italic> promoter, termed <italic>AtGCSpro</italic>, with strong activity in the region where the root meristem will initiate and in the whole roots in broad eudicots species. <italic>AtGCSpro</italic> achieved higher homozygous/biallelic mutation efficiency than the most widely used <italic>CaMV 35S</italic> promoter in driving <italic>Cas9</italic> expression in soybean, <italic>Lotus japonicus</italic>, and tomato roots. Using the p<italic>AtGCSpro</italic>-Cas9 system, the average homozygous/biallelic mutation frequency is 1.7-fold and 8.3-fold higher than the p<italic>2 × 35Spro</italic>-Cas9 system for single and two target site(s) in the genome, respectively. Our results demonstrate the advantage of the p<italic>AtGCSpro</italic>-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. <italic>AtGCSpro</italic> is conservatively active in various eudicots species, suggesting that <italic>AtGCSpro</italic> might be applied in a wide range of dicots species.</p>