Programmable gene editing using guide rna pair
AssigneeMassachusetts Institute of Technology
InventorsOmar Abudayyeh, Jonathan Gootenberg
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Abstract
Provided herein are compositions, methods, and systems comprising a DNA binding nickase, a reverse transcriptase, an integration enzyme, and a guide RNA pair. Also described herein are method of use of the guide RNA pair in methods of editing and integrating polynucleotide sequences.
Claims32
A composition comprising:
- a DNA binding nickase or a functional fragment or variant thereof;
- a reverse transcriptase (RT) or a functional fragment or variant thereof;
- an integration enzyme or a functional fragment or variant thereof, wherein the integration enzyme is selected from the group consisting of an integrase, a recombinase, and a reverse transcriptase;
- and
- a guide RNA (gRNA) pair comprising:
- a first heterologous gRNA or functional fragment or variant thereof, comprising:
- a first spacer sequence,
- a first scaffold sequence,
- a first reverse transcription template sequence that comprises at least a first portion of an at least first integration recognition sequence;
- a first primer binding sequence, and
- a second heterologous gRNA or functional fragment or variant thereof, comprising:
- a second spacer sequence,
- a second scaffold sequence,
- a second reverse transcription template sequence that comprises at least a second portion of the first integration recognition sequence,
- a second primer binding sequence,
- wherein the first heterologous RNA and the second heterologous RNA collectively encode all of the first integration recognition sequence.
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8-9. (canceled)
13-18. (canceled)
The composition of claim 13, wherein the first and second heterologous gRNAs form a double stranded nucleic acid.
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25-26. (canceled)
The composition of claim 25, wherein the M-MLV reverse transcriptase domain comprises one or more of the mutations selected from the group consisting of D200N, T306K, W313F, T330P, and L603W.
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33-35. (canceled)
A method of site-specifically integrating an exogenous nucleic acid into a cell genome, the method comprising:
- (a) incorporating an integration sequence at a target location in the cell genome by introducing into a cell:
- i. a DNA binding nickase or a functional fragment or variant thereof;
- ii. a reverse transcriptase (RT) or a functional fragment or variant thereof;
- and
- iii. a guide RNA (gRNA) pair comprising:
- a first heterologous gRNA or functional fragments or variants thereof, comprising:
- a first spacer sequence,
- a first scaffold sequence,
- a first reverse transcription template sequence that comprises at least a first portion of an at least first integration recognition sequence;
- a first primer binding sequence
- and
- a second heterologous gRNA or functional fragments or variants thereof, comprising:
- a second spacer sequence,
- a second scaffold sequence,
- a second reverse transcription template sequence that comprises at least a second portion of the first integration recognition sequence,
- a second primer binding sequence
- wherein:
- the first and second heterologous gRNAs interact with the DNA binding nickase and target the target location in the cell genome,
- the DNA binding nickase nicks a strand of the cell genome, and
- the reverse transcriptase reverse transcribes (i) the first reverse transcription template sequence into a first extended sequence that encodes the at least first portion of the first integration recognition sequence and (ii) the second reverse transcription template sequence into a second extended sequence that encodes the at least second portion of the first integration recognition sequence,
- the first and second extended sequences comprise at least about 5 complementary nucleotides with respect to each other, wherein annealing of the complementary nucleotides forms a duplex which results in the insertion of the at least first integration recognition sequence into the target location;
- and
- (b) integrating the nucleic acid into the cell genome by introducing into the cell:
- i. a DNA or RNA strand comprising the nucleic acid linked to a sequence that is complementary or associated to the integration sequence;
- and
- ii. an integration enzyme or a functional fragment or variant thereof, wherein the integration enzyme is selected from the group consisting of an integrase, a recombinase, and a reverse transcriptase,
- wherein the integration enzyme incorporates the nucleic acid into the cell genome at the at least first integration recognition sequence by integration, recombination, or reverse transcription of the sequence that is complementary or associated to the integration sequence, thereby introducing the nucleic acid into the target location of the cell genome of the cell.
38-77. (canceled)
A gRNA pair that specifically binds to a DNA binding nickase, wherein the gRNA pair comprises a first heterologous gRNA or functional fragments or variants thereof, and a second heterologous gRNA or functional fragments or variants thereof, and wherein the first and second heterologous gRNAs separately comprise a scaffold sequence, a primer binding sequence, an integration sequence, a spacer sequence, and optionally a reverse transcription template sequence.
A polypeptide comprising a DNA binding nickase linked to a reverse transcriptase, an integration enzyme, and a gRNA pair.
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Dependencies are inferred from the claim text.