A new I-CreI derived single-chain meganuclease comprising two domains, each domain comprising a portion of a parent I-CreI monomer which extends at least from the beginning of the first alpha helix to the end of the C-terminal loop and said two domains being joined by a peptidic linker which allows them to fold as a I-CreI dimer that is able to bind and cleave a chimeric DNA target comprising one different half of each parent homodimeric I-CreI meganuclease target sequence. Use of said I-CreI derived single-chain meganuclease for genetic engineering, genome therapy and antiviral therapy.