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US 12,612,619 B2Granted
Compositions and methods for improved gene editing
AssigneeUniversity of Massachusetts
InventorsCraig Cameron Mello, Krishna Sumanth Ghanta, Gregoriy Dokshin, Aamir Mir, Hassan Gneid, Jonathan Kenneth Watts, Erik Joseph Sontheimer
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Abstract
The disclosure provides novel methods and compositions for gene editing. In particular, the disclosure relates to compositions and methods of making modified nucleic acid donor templates for highly efficient and precise gene editing.
Claims49
A method of introducing a DNA donor sequence into a target sequence of a genome in a cell, the method comprising:
- i) contacting the cell with a double-stranded nucleic acid donor molecule, wherein both strands comprise a DNA donor sequence comprising a 3′ end, a 5′ end, and one or more terminal adaptors covalently attached to the DNA donor sequence;
- and
- ii) contacting the cell with an agent that creates a double-stranded break at or near the target sequence,
- wherein each DNA donor sequence comprises a first region located at the 3′ end having homology to a first portion of the target sequence and a second region located at the 5′ end having homology to a second portion of the target sequence, and wherein the first region and the second region are independently about 20 bases to about 1,000 bases in length, and
- wherein one of the one or more terminal adaptors is attached at the 5′ end of each of the DNA donor sequences and comprises ethylene glycol, polyethylene glycol (PEG), a polyamine having at least two amino groups, an alkanediol, a single-stranded RNA (ssRNA), or a combination thereof.
2 more elements
A method of reducing off-target integration of a DNA donor sequence into a target sequence of a genome in a cell, the method comprising:
- i) contacting the cell with a double-stranded nucleic acid donor molecule, wherein both strands comprise a DNA donor sequence comprising a 3′ end, a 5′ end, one or more terminal adaptors covalently attached to the DNA donor sequence, and one or more terminal adaptor ligand moieties;
- and
- ii) contacting the cell with an agent that creates a double-stranded break at or near the target sequence,
- wherein each DNA donor sequence comprises a first region located at the 3′ end having homology to a first portion of the target sequence and a second region located at the 5′ end having homology to a second portion of the target sequence, and wherein the first region and the second region are independently about 20 bases to about 1,000 bases in length, and
- wherein one of the one or more terminal adaptors is attached at the 5′ end of each of the DNA donor sequences and comprises ethylene glycol, polyethylene glycol (PEG), a polyamine having at least two amino groups, an alkanediol, and a single-stranded RNA (ssRNA), or a combination thereof.
2 more elements
A method of introducing a nucleic acid donor sequence into a target sequence of a genome in a cell, the method comprising:
- i) contacting the cell with a double-stranded nucleic acid donor molecule, wherein both strands comprise:
- (a) a nucleic acid donor sequence comprising:
- a 3′ end comprising a first region having homology to a first portion of the target sequence;
- and
- a 5′ end comprising a second region having homology to a second portion of the target sequence;
- and
- (b) one or more terminal adaptors at the 3′ end of each strand and/or at the 5′ end of each strand;
- ii) contacting the cell with an agent that creates a double-stranded break at or near the target sequence;
- wherein the one or more terminal adaptors comprises one or more of ethylene glycol, polyethylene glycol (PEG), a polyamine having at least two amino groups, an alkanediol, and a single-stranded RNA (ssRNA),
- wherein if the ethylene glycol, the PEG, the polyamine having at least two amino groups, or the alkanediol is present, the one or more terminal adaptors further comprises the ssRNA.
7 more elements
A method of introducing a DNA donor sequence into a target sequence of a genome in a cell, the method comprising:
- i) contacting the cell with a double-stranded nucleic acid donor molecule comprising one or more terminal adaptors, wherein both strands comprise a DNA donor sequence comprising a 3′ end and a 5′ end, wherein both strands comprise at least one terminal adaptor covalently attached to the 3′ end and/or the 5′ end of the DNA donor sequence, wherein the at least one terminal adaptor comprises a single stranded RNA (ssRNA) of 1 to about 50 bases in length comprising a 2′-O-alkyl modified nucleotide;
- and
- ii) contacting the cell with an agent that creates a double-stranded break at or near the target sequence,
- wherein each DNA donor sequence comprises a first region located at the 3′ end having homology to a first portion of the target sequence and a second region located at the 5′ end having homology to a second portion of the target sequence, and wherein the first region and the second region are independently about 20 bases to about 1,000 bases in length.
1 more element
Dependencies are inferred from the claim text.