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

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, gene (), introduced a CRISPR-Cas9 editing vector into the transgenic tobacco line for knocking out , and then investigated the 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 U6 (AtU6) and U3 (AtU3) promoter, respectively. The two gRNAs were designed to knock out a 42-nucleotide fragment of the coding region of . The editing vector was transformed into -containing tobacco leaves using -mediated transformation and hygromycin selection. Hygromycin-resistant, independent T transgenic lines were used to evaluate -editing efficiencies through histochemical GUS assays, polymerase chain reactions (PCR), and next-generation sequencing of PCR amplicons. Profiles of targeted sequences of 94 T 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 T transgenic lines, respectively. To overcome the issue of extremely low editing efficiencies in T 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 T transgenic lines provide valuable information to understand gene editing in plant cells with constitutively expressed CRISPR-Cas9 and gRNAs.