CRISPR/Cas9-mediated site-directed mutagenesis of the PDS gene of Nicotiana tabacum L.

CRISPR/Cas9 system is one of the effective tools for editing plant genomes. Using the CRISPR/Cas9 system, we have obtained different-type Nicotiana tabacum transformants carrying in their genome the mutational events of insertiondeletion type in the PDS gene, encoding15-cis-phytoene desaturase enzyme. The endogenous tobacco NtPDS gene was chosen as a target, since introducing mutations into its nucleotide sequence leads to appearing easily visualized characters. The tetraallelic knockout of the NtPDS gene led to complete albinism in plants. Albino transformants were characterized by a slower growth, dwarfism, and were not viable when cultivated in vitro. Mutants with variegated leaves in the in vitro culture formed roots and did not differ from the control in leaf size. A nucleotide sequence of the protospacer of the target NtPDSt gene was analyzed on a random sample, including 21 transgenic plants of the T0 generation with different phenotypes – from completely albino to wild type. Sequencing results showed that all transformants with visible phenotypic manifestations carried mutant sequences in the NtPDS gene with a frequency of 51.0–80.0 %, while the efficiency of site-directed mutagenesis was 33.33 %. In transformants of a wild-type phenotype, no mutated sequences were detected in the target gene.