Nucleobase editor systems and methods of use thereof
AssigneePeking University
InventorsWensheng WEI, Zongyi YI, Xiaoxue ZHANG
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Abstract
Provided are systems for strand-specific editing of DNA, including mitochondrial DNA in humans. The systems provided herein comprise a single stand nickase and a deaminase each, or together, associated with a double-stranded DNA binding polypeptide such that DNA editing occurs in an editing region. Also provided are components of the systems for editing DNA, and methods of use thereof.
Claims63
A nucleobase editor system comprising:
- a first unit comprising a first double-stranded (ds) DNA binding polypeptide associated with a single-stranded (ss-) nickase;
- and
- a second unit comprising a second dsDNA binding polypeptide associated with a deaminase,
- wherein the nucleobase editor system is configured such that the first dsDNA binding polypeptide of the first unit and the second dsDNA binding polypeptide of the second unit, when associated with a dsDNA, position the ss-nickase and the deaminase such that a site action for the ss-nickase and a site of action for the deaminase are within an editing region on the dsDNA.
A nucleobase editor system comprising:
- a single-stranded (ss-) nickase;
- a deaminase;
- and
- a double-stranded (ds) DNA binding polypeptide,
- wherein the ss-nickase, the deaminase, and dsDNA binding polypeptide form a complex configured such that the dsDNA binding polypeptide, when associated with a dsDNA, positions the ss-nickase and the deaminase such that a site action for the ss-nickase and a site of action for the deaminase are within an editing region on the dsDNA.
A non-naturally occurring polynucleotide encoding:
- a first unit comprising a first double-stranded (ds) DNA binding polypeptide associated with a single-stranded (ss-) nickase;
- and/or
- a second unit comprising a second dsDNA binding polypeptide associated with a deaminase.
A non-naturally occurring polynucleotide encoding:
- a first unit comprising a first double-stranded (ds) DNA binding polypeptide associated with a single-stranded (ss-) nickase;
- and
- a second unit comprising a second dsDNA binding polypeptide associated with a deaminase,
- wherein the first unit and second unit, when expressed, are configured such that the first dsDNA binding polypeptide of the first unit and the second dsDNA binding polypeptide of the second unit, when associated with a dsDNA, position the ss-nickase and the deaminase with a site of action for the ss-nickase and a site of action for the deaminase within an editing region on the dsDNA.
A non-naturally occurring polynucleotide encoding:
- a single-stranded (ss-) nickase;
- a deaminase;
- and
- a double-stranded (ds) DNA binding polypeptide,
- wherein the ss-nickase, the deaminase, and dsDNA binding polypeptide, when expressed, form a complex configured such that the dsDNA binding polypeptide, when associated with a dsDNA, positions the ss-nickase and the deaminase with a site action for the ss-nickase and a site of action for the deaminase within an editing region on the dsDNA.
A method of treating an individual having a disease associated with a DNA mutation, the method comprising administering one or more polynucleotides encoding:
- a first unit comprising a first double-stranded (ds) DNA binding polypeptide associated with a single-stranded (ss-) nickase;
- and
- a second unit comprising a second dsDNA binding polypeptide associated with a deaminase,
- wherein the first unit and second unit, when expressed in the individual, are configured such that the first dsDNA binding polypeptide of the first unit and the second dsDNA binding polypeptide of the second unit, when associated with a dsDNA, position the ss-nickase and the deaminase with a site of action for the ss-nickase and a site of action for the deaminase within an editing region on the dsDNA.
A kit for a nucleobase editing system, the kit comprising:
- a first unit comprising a first double-stranded (ds) DNA binding polypeptide associated with a single-stranded (ss-) nickase;
- and
- a second unit comprising a second dsDNA binding polypeptide associated with a deaminase,
- wherein the nucleobase editor system is configured such that the first dsDNA binding polypeptide of the first unit and the second dsDNA binding polypeptide of the second unit, when associated with a dsDNA, position the ss-nickase and the deaminase with a site of action for the ss-nickase and a site of action for the deaminase within an editing region on the dsDNA.
A kit for a nucleobase editor system, the kit comprising one or more polynucleotides encoding:
- a first unit comprising a first double-stranded (ds) DNA binding polypeptide associated with a single-stranded (ss-) nickase;
- and
- a second unit comprising a second dsDNA binding polypeptide associated with a deaminase,
- wherein the nucleobase editor system, when expressed, is configured such that the first dsDNA binding polypeptide of the first unit and the second dsDNA binding polypeptide of the second unit, when associated with a dsDNA, position the ss-nickase and the deaminase with a site of action for the ss-nickase and a site of action for the deaminase within an editing region on the dsDNA.
A non-naturally occurring polypeptide having nickase activity, the non-naturally occurring polypeptide comprising the amino acid sequence of SEQ ID NO:1 with the following mutations of E91A and F94A (SEQ ID NO:2).
Dependencies are inferred from the claim text.