DEVELOPMENT OF THE CRISPR/CAS9 SYSTEM FOR THE GENOME EDITING OF THE NtPDS GENE OF TOBACCO (NICOTIANA TABACUM)

Abstract

The CRISPR/Cas9 system is one of the most powerful tools for the editing of plant genomes. In the presented study, the vector constructs developed on the basis of the CRISPR/Cas9 system were used to edit the Nicotiana tabacum genome. The NtPDS gene encoding the 15-cis-phytoene desaturase enzyme was chosen as the target. A knockout of this gene in plants results in the albinism phenotype and dwarfism. Using the in silico bioinformatics platforms, three vector constructs based on the binary pRGEB31 vector were modeled: pRGEB31 + gRNA4-pds, pRGEB31 + gRNAJp2-pds, and pRGEB31 + gRNADeT186-pds, carrying in its composition the CRISRP/Cas9 system with spacers to different parts of the structural domains of the NtPDS gene. Vector constructs were assembled using molecular cloning techniques. The accuracy and correctness of their assembly was confirmed by Sanger sequencing. Вy means of Agrobacteriummediated transformation of leaf discs, the genetic constructs were introduced into the genome of the N. tabacum cv. Petit Havana SR1 model object. During the cultivation of tobacco leaf discs, it was possible to initiate callusogenesis and morphogenesis processes using all three constructs, however, the maximum frequency of these processes was observed when using the pRGEB31 + gRNA4-pds construct.