ABSTRACT
KEY MESSAGE: Co-transformation of multiple T-DNA in a binary vector enabled CRISPR/Cas9-mediated HR in tobacco. HR occurred in a limited region around the gRNA target site. In this study, CRISPR/Cas9-mediated homologous recombination (HR) in tobacco (Nicotiana tabacum L. 'SR-1') was achieved using binary vectors comprising two (T1-T2) or three (T1-T2-T3) independent T-DNA regions. For HR donor with the tobacco acetolactate synthase gene, SuRB, T-DNA1 contained ΔSuRBW568L, which lacked the N-terminus region of SuRB and was created by three nucleotide substitutions (ATG to GCT; W568L), leading to herbicide chlorsulfuron (Cs) resistance, flanked by the hygromycin (Hm)-resistant gene. T-DNA2 consisted of the hSpCas9 gene and two gRNA inserts targeting SuRB and An2. For the 2nd HR donor with the tobacco An2 gene encoding a MYB transcription factor involved in anthocyanin biosynthesis, T-DNA3 had a 35S promoter-driven An2 gene lacking the 3rd exon resulting in anthocyanin accumulation after successful HR. After selecting for Hm and Cs resistance from among the 7462 Agrobacterium-inoculated explants, 77 independent lines were obtained. Among them, the ATG to GCT substitution of endogenous SuRB was detected in eight T1-T2-derived lines and two T1-T2-T3-derived lines. Of these mutations, four T1-T2-derived lines were bi-allelic. All the HR events occurred across the endogenous SuRB and 5' homology arm of the randomly integrated T-DNA1. HR of the SuRB paralog, SuRA, was also found in one of the T1-T2-derived lines. Sequence analysis of its SuRA-targeted region indicated that the HR occurred in a limited (< 153 bp) region around the gRNA target site. Even though some T1-T2-T3-derived lines introduced three different T-DNAs and modified the An2 gRNA target site, no signs of HR in the endogenous An2 could be observed.
