Find in Library

Chimeric editing is often reported in gene editing. To assess how the general chimeric editing is, we created a transgenic tobacco line carrying a marker, <i>beta-glucuronidase</i> gene (<i>gusA</i>), introduced a CRISPR-Cas9 editing vector into the transgenic tobacco line for knocking out <i>gusA</i>, and then investigated the <i>gusA</i> 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 <i>Arabidopsis</i> U6 (AtU6) and U3 (AtU3) promoter, respectively. The two gRNAs were designed to knock out a 42-nucleotide fragment of the coding region of <i>gusA</i>. The editing vector was transformed into <i>gusA</i>-containing tobacco leaves using <i>Agrobacterium tumefaciens</i>-mediated transformation and hygromycin selection. Hygromycin-resistant, independent T₀ transgenic lines were used to evaluate <i>gusA</i>-editing efficiencies through histochemical GUS assays, polymerase chain reactions (PCR), and next-generation sequencing of PCR amplicons. Profiles of targeted sequences of 94 T₀ transgenic lines revealed that these lines were regenerated from non-edited cells where subsequent editing occurred and created chimeric-edited cells in these lines during or after regeneration. Two of them had the target fragment of 42 bp pairs of nucleotides removed. Detail analysis showed that on-target mutations at the AtU6-gRNA1 site and the AtU3-gRNA2 site were found in 4.3% and 77.7% of T₀ transgenic lines, respectively. To overcome the issue of extremely low editing efficiencies in T₀ lines, we conducted a second round of shoot induction from the chimeric line(s) to enhance the success of obtaining lines with all or most cells edited. The mutation profiles in T₀ transgenic lines provide valuable information to understand gene editing in plant cells with constitutively expressed CRISPR-Cas9 and gRNAs.