Knockout of Tobacco Homologs of Gene Confers a Limited Resistance to Aminoglycoside Antibiotics.

To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the () gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient mediated gene transfer. A gene was introduced into and together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of homologs (s). All three sgRNAs directed the introduction of InDels into s, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in s. The s-knockout seedlings of the T generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like , s have a role in plants' sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs.