Use of terminal deoxynucleotidyl transferase for mutagenic DNA repair to generate variability, at a determined position in DNA

摘要

The invention relates to a method of generating junctional variability in the nucleotide sequence of a polynucleotide of interest present in an intrachromosomal substrate/context in a eukaryotic cell which is competent for canonical Non Homologous End Joining pathway (NHEJ) repair, involving the generation of double-strand break (DSB) in the DNA sequence of said polynucleotide, and involving the use of polymerase Terminal Deoxynucleotidyl Transferase (TdT) in conditions enabling said TdT to add Non-templated nucleotides (N nucleotides) before ligation through the canonical Non Homologous End Joining pathway (NHEJ) thereby allowing a mutagenic repair to take place at the DSB site. The invention also relates to a library of eukaryotic cells and a collection of recombinant clones obtained by implementing the method of the invention on a population of eukaryotic cells, as well as a method for determining occurrence(s) of generation of double strand break(s) in a cell, or in a population of cells, after evaluation of the generated junctional variability. The invention further relates to the use of TdT as a marker of DSB events.