Determination of in vivo DNA double-strand break localization and application thereof

A method for determining the in vivo specificity of an endonuclease, comprising:

• a) incubating a host cell comprising the endonuclease and a linear polynucleotide comprising a known sequence, wherein the linear polynucleotide comprising the known sequence is integrated into double-strand breaks in the genome in a sequence independent manner by non-homologous end joining;
• b) detecting at least 50 in vivo insertion sites of the linear polynucleotide in the genome of the host cell by (i) amplifying polynucleotides from the genome using primers priming in the linear poiynucieotide comprising the known sequence and (ii) determining at least one nucleic acid sequence which is in the genome and adjacent to the linear polynucleotide comprising the known sequence;
• c) determining the in vivo recognition sites of the endonuclease based on the sequence information obtained in step b);
• and
• d) assessing the in vivo specificity of the endonuclease.

A method for obtaining an endonuclease with an altered in vivo specificity, comprising:

• a) providing at least one mutant of an endonuclease with a known recognition sequence;
• b) determining the in vivo specificity of the mutant of an endonuclease by a method comprising:
• i) incubating a host cell comprising the endonuclease and a linear polynucleotide comprising a known sequence, wherein the linear polynucleotide comprising the known sequence is integrated into double-strand breaks in the genome in a sequence independent manner by non-homologous end joining;
• ii) detecting at least 50 in vivo insertion sites of the linear polynucleotide in the genome of the host cell by (aa) amplifying polynucleotides from the genome using primers priming in the linear polynucleotide comprising the known sequence and (bb) determining at least one nucleic acid sequence which is in the genome and adjacent to the linear polynucleotide comprising the known sequence;
• iii) determining the in vivo recognition sites of the endonuclease based on the sequence information obtained in step ii);
• and
• iv) assessing the in vivo specificity of the endonuclease;
• c) comparing the recognition sites recognized by the at least one mutant endonuclease with the recognition sites recognized by the unmodified endonuclease;
• and
• d) obtaining an endonuclease with an altered in vivo specificity.