A method of in vivo mutagenesis of a preselected target region (PTR) of an intracellular DNA within a cell culture, which PTR comprises at least one sequence of interest (SOI) which is at least 60 nucleobases long, the method comprising: a) providing a pool of partially overlapping single stranded DNA (ssDNA) oligonucleotides which upon alignment form a continuous sequence that is complementary to the SOI, wherein the pool contains a diversity of mutagenizing oligonucleotides covering nucleobase mismatches at every position of said SOI and combination of said nucleobase mismatches, wherein each mutagenizing oligonucleotide is hybridizing with the PTR and comprises at least one mismatching nucleobase up to 20% mismatching nucleobases, compared to the SOI； b) integrating the pool into said intracellular DNA by homologous recombination and inducing in vivo mutagenesis of the intracellular DNA through hybridizing the oligonucleotides to the PTR, thereby producing a cell library comprising a repertoire of variant cells covering point mutations at every position and combinations of said point mutations within said PTR.
