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US 2020/0239930 A1Published application
Methods for Detecting Site-Specific and Spurious Genomic Deamination Induced by Base Editing Technologies
AssigneeThe General Hospital Corporation
InventorsJ. Keith Joung, James Angstman, Jason Michael Gehrke
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Abstract
Methodologies to detect off-target mutations induced by the deaminase activity of Base Editing technology.
Claims10
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, preferably a zinc-finger domain;
- a transcription-activator-like effector domain;
- or a catalytically-inactive Cas9 or Cpf1, with a selected guide RNA;
- 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, 5 base pair overhanging ends centered at the deamination site;
- treating the DNA fragments with uracil DNA glycosylase and endonuclease VIII to remove the deoxyuracil base from the ends of the DNA fragments;
- end-repairing and/or A-tailing the ends of the DNA fragments;
- and
- ligating an adapter oligonucleotide (preferably comprising sequences for use in high throughput sequencing) to the end;
- and
- sequencing the DNA fragments.
8 more elements
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 nicking Cas9 protein (nCas9);
- contacting the deaminated substrate DNA with uracil DNA glycosylase and endonuclease VIII, to induce DSBs;
- end-repairing and/or A-tailing the ends of the DNA fragments;
- ligating adapter oligonucleotide (comprising sequences for use in high throughput sequencing) to the end;
- and
- sequencing the DNA fragments.
4 more elements
A method for detecting and quantifying base editor-induced cytosine to thymine mutation events in living cells, the method comprising:
- providing a sample comprising substrate genomic DNA from cells exposed to a base editor protein comprising a deaminase domain fused to DNA binding domain, preferably a zinc-finger domain;
- a transcription-activator-like effector domain;
- or a Cas9 or Cpf1 nickase or catalytically-inactive Cas9 or Cpf1, with a selected guide RNA;
- using 3D PCR to selectively amplify alleles that have undergone deamination events, to create a population of amplicons that is enriched for deaminated alleles;
- and
- sequencing the enriched population of amplicons, preferably using next generation sequencing or TOPO cloning, to determine the identity of the amplified molecules.
3 more elements
Dependencies are inferred from the claim text.