METHODS AND COMPOSITIONS FOR TARGETED GENETIC MODIFICATION BY SINGLE-STEP MULTIPLE TARGETING Russian patent published in 2019 - IPC A01K67/27 C12N15/85 C12N15/90 C12N9/22 

FIELD: biotechnology.

SUBSTANCE: invention relates to the biotechnology. Described is a method of modifying a target genome locus in a mammalian cell, comprising: introducing into a cell a nuclease agent which performs a single- or double-stranded rupture within the target genomic locus; introduction into the cell of the first large targeting vector (LTVEC), which has a length of at least 10 kbp and comprises a first polynucleotide insert flanked by first 5' homologous arm and first 3' homologous arm, and a second LTVEC, having a length of at least 10 kbp and comprises a second polynucleotide insert flanked by second 5' homologous arm and second 3' homologous arm, wherein the total size of the first polynucleotide insert and the second polynucleotide insert is at least 100 kbp, wherein first 3' homologous arm of the first LTVEC has a first overlapping sequence, homologous to second 5' homologous to second LTVEC arm, and first 5' homologous arm of first LTVEC and second 3' homologous arm of the second LTVEC are homologous to the corresponding genomic segments within the target genomic locus, wherein the target genomic locus is modified by integrating the first polynucleotide insert and the second polynucleotide insert between the corresponding genome segments; and selecting the target-oriented cell containing the first polynucleotide insert and the second polynucleotide insert integrated into the target genome locus.

EFFECT: invention enables to modify the target genomic locus.

25 cl, 4 dwg, 9 tbl, 5 ex