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US 10,501,794 B2Granted
Genomewide unbiased identification of DSBs evaluated by sequencing (GUIDE-seq)
AssigneeThe General Hospital Corporation
InventorsJ. Keith Joung, Shengdar Tsai
Life of this patent
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Abstract
Unbiased, genomewide and highly sensitive methods for detecting mutations, e.g., off-target mutations, induced by engineered nucleases.
Claims16
A method for detecting double stranded breaks (DSBs) in genomic DNA (gDNA) of a cell, the method comprising:
- contacting the cell with a blunt-ended double-stranded oligodeoxynucleotide (dsODN), wherein both strands of the dsODN are orthogonal to the genome of the cell, and further wherein (a) the 5′ ends of the dsODN are phosphorylated, and (b) phosphorothioate linkages are present on both 3′ ends, or phosphorothioate linkages are present on both 3′ ends and both 5′ ends;
- maintaining the cell under conditions sufficient for the cell to repair the DSBs, integrating a dsODN at one or more DSBs;
- amplifying a portion of gDNA comprising an integrated dsODN;
- and
- sequencing the amplified portion of the gDNA,
- thereby detecting a DSB in the gDNA of the cell.
3 more elements
A method for detecting double stranded breaks (DSBs) in gDNA of a cell, the method comprising:
- contacting the cell with a blunt-ended double-stranded oligodeoxynucleotide (dsODN), wherein both strands of the dsODN are orthogonal to the genome of the cell, and further wherein (a) the 5′ ends of the dsODN are phosphorylated, and (b) phosphorothioate linkages are present on both 3′ ends, or phosphorothioate linkages are present on both 3′ ends and both 5′ ends;
- maintaining the cell under conditions sufficient for the DSBs to occur in the genomic DNA of the cell, and for the cell to repair the DSBs, integrating a dsODN at one or more DSBs;
- amplifying a portion of gDNA comprising an integrated dsODN;
- and
- sequencing the amplified portion of the genomic DNA,
- thereby detecting a DSB in the gDNA of the cell.
3 more elements
Dependencies are inferred from the claim text.