Methods for detecting site-specific and spurious genomic deamination induced by base editing technologies
Methodologies to detect off-target mutations induced by the deaminase activity of Base Editing technology.
1. A method of detecting deaminated sites in substrate DNA, the method comprising:
providing a sample comprising substrate DNA;
deaminating the substrate DNA using a base editing fusion protein comprising a deaminase domain and a DNA binding domain;
contacting the deaminated substrate with Endonuclease MS from Thermococcus kodakarensis (TkoEndoMS) to induce double strand breaks (DSBs) at deamination sites in the substrate DNA to produce DNA fragments with single-stranded, five-base pair overhanging ends;
treating the DNA fragments with uracil DNA glycosylase and endonuclease VIII;
end-repairing and/or A-tailing the ends of the DNA fragments;
ligating an adapter oligonucleotide to the ends; and
sequencing the DNA fragments.
2. The method of claim 1 , wherein the substrate DNA is genomic DNA (gDNA).
3. The method of claim 1 , wherein the adapter oligonucleotide comprises a single deoxyuridine.
4. The method of claim 1 , wherein sequencing the DNA fragments comprises determining a sequence of at least 10 nucleotides at the ends of the DNA fragments.
5. The method of claim 1 , wherein the base editing fusion protein is selected from the group consisting of a zinc-finger domain; a transcription-activator-like effector domain; or a catalytically-inactive Cas9 or Cpf1, with a selected guide RNA.
6. The method of claim 1 , wherein the adapter oligonucleotide comprises sequences for use in high throughput sequencing.
7. The method of claim 1 , wherein the substrate DNA is synthesized DNA.
8. The method of claim 1 , wherein the base editing fusion protein is selected from the group consisting of a zinc-finger domain; a transcription-activator-like effector domain; or a catalytically-inactive Cas9 or Cpf1, with a selected guide RNA, and wherein the substrate DNA is genomic DNA.
9. The method of claim 8 , wherein the base editing fusion protein is a catalytically-inactive Cas9 or Cpf1, with a selected guide RNA.
10. The method of claim 1 , wherein the base editing fusion protein is selected from the group consisting of a zinc-finger domain; a transcription-activator-like effector domain; or a catalytically-inactive Cas9 or Cpf1, with a selected guide RNA, and wherein the substrate DNA is synthesized DNA.
11. The method of claim 10 , wherein the base editing fusion protein is a catalytically-inactive Cas9 or Cpf1, with a selected guide RNA.