Nucleic acid molecule and method to make biallelic modifications in a target gene or locus which is part of the genetic material of a cell

摘要

The aim of this invention is to provide a nucleic acid molecule and a method to modify at the same time both alleles of a target gene or region of the genome of a cell. This nucleic acid molecule has the ability to edit and modify both alleles of a gene or locus, currently present in the genetic material of a cell. The nucleic acid molecule encodes for certain nuclease proteins which once expressed will cleave the target gene or locus in the cell DNA. Afterwards the nucleic acid molecule will integrate itself in the cleavage site in at least one of the two alleles at first, by means of the innate homologous recombination repair mechanism of the cell. That is possible due to the homology regions that the introduced molecule is carrying, homologous to the target gene or locus. Once the nucleic acid molecule is integrated in the first allele, the nucleases will eventually produce another cut in the remaining allele and, by using again the homologous recombination repair pathway of the cell, the cleaved allele will be repaired using as a template the previously modified allele, producing the integration of the molecule in the second allele of the target gene or locus. After the nucleic acid molecule has been integrated in both alleles, the activation of the transposable element encoded in the molecule will remove all the undesired sequences leaving only the desired modifications in the target gene or locus. Such modifications will be present and will be identical in both alleles making possible by this method the generation of mutations in wild type genes, the insertion of complete genes in genomes, the insertion and removal of specific sequences and the repair of genetic mutations present in the genome, among other uses.