Crispr oligonucleotides and gene editing
AssigneesLife Technologies Corporation, Thermo Fisher Scientific Geneart GmbH
InventorsNamritha Ravinder, Korbinian Heil, Yizhu Guo, Xiquan Liang, Robert Potter
Life of this patent
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Abstract
The present disclosure generally relates to compositions and methods for the genetic modification of cells. In particular, the disclosure relates to CRISPR reagents and the use of such reagents.
Claims21
A method for producing a nucleic acid molecule, the method comprising performing polymerase chain reaction (PCR) in a reaction mixture containing
- (i) a double stranded nucleic acid segment, and
- (ii) at least one oligonucleotide capable of hybridizing to nucleic acid at one terminus of the double stranded nucleic acid segment, wherein the nucleic acid molecule is produced by the PCR reaction, and wherein the product nucleic acid molecule contains at or near one terminus a promoter suitable for in vitro transcription.
4-6. (canceled)
A method for producing a nucleic acid molecule, the method comprising performing polymerase chain reaction (PCR) in a reaction mixture comprising
- (i) a double stranded nucleic acid segment comprising a first terminus and a second terminus,
- (ii) a first oligonucleotide comprising a first terminus and a second terminus, wherein the second terminus of the first oligonucleotide is capable of hybridizing to the first terminus of the double stranded nucleic acid segment, and
- (iii) a second oligonucleotide comprising a first terminus and a second terminus,
- wherein the second terminus of the second oligonucleotide is capable of hybridizing to the first terminus of the first oligonucleotide, to produce the nucleic acid molecule, wherein the product nucleic acid molecule contains a promoter suitable for in vitro transcription at or near one terminus and encodes a CRISPR RNA.
10-14. (canceled)
A method for producing a guide RNA molecule, the method comprising:
- (a) separately producing a crRNA molecule and a tracrRNA molecule, and
- (b) contacting the crRNA molecule and the tracrRNA molecule with each other under conditions that allow for the covalently linking of the 3′ terminus of the crRNA to the 5′ terminus of the tracrRNA to produce the guide RNA molecule.
(canceled)
A method for gene editing at a target locus within a cell, the method comprising introducing into the cell at least one CRISPR protein and at least one CRISPR RNA, wherein the at least one CRISPR RNA has a region of sequence complementarity of at least 10 base pairs to the target locus.
(canceled)
(canceled)
Dependencies are inferred from the claim text.