Determination of in vivo DNA double-strand break localization and application thereof
The present invention relates to a method for determining the in vivo localization of double-strand breaks in a host cell, comprising incubating a host cell suspected to comprise DNA double-strand breaks and a linear polynucleotide comprising a known sequence, detecting the in vivo insertion sites of said polynucleotide in the genome of said host cell, and assessing the in vivo localization of double-strand breaks. Further envisaged by the present invention is a method for obtaining an endonuclease with altered in vivo specificity. Finally, the present invention is directed to a kit for determining in vivo specificity of an endonuclease.
1. A method for determining the in vivo distribution of double-strand breaks induced by an endonuclease in a host cell, comprising:
a) incubating the host cell and a linear polynucleotide comprising a known sequence and encoding the endonuclease, wherein the linear polynucleotide comprising the known sequence and encoding the endonuclease 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 polynucleotide 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 positions of double-strand breaks based on the sequence information obtained in step b); and
d) determining the in vivo distribution of double-strand breaks induced by the endonuclease in the host cell.
2. The method of claim 1 , wherein the linear polynucleotide is an integrase-deficient lentivirus.
3. The method of claim 1 , wherein double-strand breaks are induced by a zinc-finger nuclease.
4. The method of claim 1 , wherein the linear polynucleotide comprises an integrase-deficient lentivirus and encodes the zinc-finger nuclease.
5. The method of claim 1 , wherein the insertion sites are detected by a method comprising PCR amplification followed by DNA sequencing.
6. The method of claim 5 , wherein the PCR is LAM-PCR.
7. 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.
8. The method of claim 7 , wherein the endonuclease is a Zinc-finger endonuclease.
9. The method of claim 7 , wherein the linear polynucleotide is an integrase-deficient lentivirus.
10. The method of claim 7 , wherein the insertion sites are detected by a method comprising PCR amplification followed by DNA sequencing.
11. The method of claim 10 , wherein the PCR is LAM-PCR.
12. 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.
13. The method of claim 12 , wherein the altered in vivo specificity is an increased in vivo specificity for a specific recognition sequence.
14. The method of claim 12 , wherein the endonuclease is a Zinc-finger endonuclease.
15. The method of claim 1 , wherein the determined position of the in vivo localization of double-strand breaks is within 5000 nucleotides from the position of double-strand breaks.