METHOD FOR DETERMINING THE GENOME-WIDE LOCALISATION AND NUMBER OF COPIES OF T-DNA IN THE GENOME OF TRANSFORMED PLANTS USING Cas9-TARGETED NANOPORE SEQUENCING Russian patent published in 2022 - IPC A01H5/00 B82Y30/00 

FIELD: biotechnology.

SUBSTANCE: presented is a method for determining the genome-wide localisation and number of copies of T-DNA in the genome of transformed plants using Cas9-targeted nanopore sequencing. The method includes the following stages: isolating the high molecular weight fraction of the DNA of individual transgenic plants (HMW DNA), pooling the DNA of individual transgenic plants; selecting gRNAs specific to the T-DNA region and synthesising gRNAs in vitro on a double-stranded DNA matrix molecule; assembling Cas9/gRNA ribonucleoprotein complexes; performing Cas9-targeted nanopore sequencing; identifying the T-DNA insertion sites in the plant pool using the NanoCasTE approach; selecting specific primers for each T-DNA insertion into regions flanking the insertion site; and genotyping individual plants.

EFFECT: new method for finding the location and number of copies of T-DNA in the genome of plants and creating markers for genotyping transformed plants using Cas9-targeted nanopore sequencing.

1 cl, 5 dwg, 1 ex