Gene Editing Profiles in 94 CRISPR-Cas9 Expressing T0 Transgenic Tobacco Lines Reveal High Frequencies of Chimeric Editing of the Target Gene

Authors: SONG, GUOQING - Michigan State University; URBADN, GRACE - Michigan State University; RYNER, JOHN - Michigan State University; Yang, Yingzhen; Zhong, Gan-Yuan

Submitted to: Plants
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/12/2022
Publication Date: 12/13/2022
Citation: Song, G., Urbadn, G., Ryner, J.T., Yang, Y., Zhong, G. 2022. Gene editing profiles in 94 CRISPR-Cas9 expressing T0 transgenic tobacco lines reveal high frequencies of chimeric editing of the target gene. Plants. https://doi.org/10.3390/plants11243494.
DOI: https://doi.org/10.3390/plants11243494

Interpretive Summary: Gene editing is a new biotechnology to modify a gene in plants. However, successful editing rates are often very low. Further, some cells may escape from the editing and the resulted plants may be chimeric with some cells containing edited changes while other cells not. We designed and conducted an experiment to examine this issue further in transgenic tobacco. This transgenic tobacco carried a gene, called gusA, whose function can be detected through a color assay. We introduced into the transgenic gusA tabacco an editing vector that can edit and make the gusA gene lose of function, offering an opportunity to study editing efficiencies and chimeric editing. Through color assay and DNA detection, we found that the editing efficiencies were very low in the transgenic tobacco shoots investigated and almost all edited shoots were chimeric. We could enrich edited cells and reduce chimeric editing in a shoot by carrying out additional shoot induction and propagation.

Technical Abstract: Chimeric editing is often reported in gene editing. To assess how general chimeric editing is, we created a transgenic tobacco line carrying a marker gene, beta-glucuronidase gene (gusA), introduced a CRISPR-Cas9 editing vector into the transgenic tobacco line for knocking out gusA, and then investigated gusA editing efficiencies in T0 and subsequent generations. The editing vector carried a Cas9 gene, which was driven by the Cauliflower mosaic virus 35S promoter, and two guide RNAs gRNA1 and gRNA2, which were driven by Arabidopsis U6 (AtU6) and U3 (AtU3) promoter, respectively. The two gRNAs were designed to knock out a 60-nucleotide fragment of the coding region of gusA. The editing vector was transformed into gusA-containing tobacco leaves using Agrobacterium tumefaciens-mediated transformation and hygromycin selection. Hygromycin-resistant, independent T0 transgenic lines were used to evaluate gusA-editing efficiencies through histochemical GUS assays, polymerase chain reactions (PCR), and Next-Generation Sequencing of PCR amplicons. Of 94 transgenic lines sequenced, none of them had the target fragment of 60 bp of nucleotides removed. Detail analysis showed mutation or editing frequencies ranged from 0 to 48.8% at the AuU6-sgRNA1 site and 0 to 99.7% at the AtU3-gRNA2 site. We found one line (1.1%) which was likely regenerated from a single edited cell containing a deletion near the AtU3-gRNA2 site. All other lines were regenerated from non-edited cells and subsequent editing during or after regeneration created chimeric editing cells in these lines. To overcome the issue of extremely low editing efficiencies in T0 lines, we conducted a second-round of shoot induction from chimeric line(s) to enhance the success of obtaining lines with all or most cells edited.