The disclosure provides methods and compositions for generating conditional knock-out alleles using donor constructs together with sequence-specific nucleases to generate conditional knock-out alleles. Specifically, the donor construct comprises a 5&prime homology region, a 5&prime recombinase recognition site, a donor sequence, a 3&prime recombinase recognition site, and a 3&prime homology region. Further disclosed are the donor sequences each comprises a target sequence having at least one neutral mutation. Different sequence-specific nucleases can be used with the donor constructs are further disclosed.
