Nucleic acid sequence replacement by nhej
AssigneeMax-DelbrüCk-Centrum FüR Molekulare Medizin In Der Helmholtz-Gemeinschaft
InventorsRalf KÜHN, Eric DANNER
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Abstract
A method for modifying double stranded DNA (dsDNA) employing an RNA guided DNA endonuclease to generate two double strand breaks in the dsDNA molecule to be modified, and replacement of the sequence positioned between the double strand breaks with a substitute DNA sequence using the non-homologous end joining (NHEJ) pathway, and corresponding kits and compositions for modifying double stranded DNA molecules.
Claims17
An in vitro method for modifying a double stranded DNA (dsDNA) molecule in a cell, the method comprising
- introducing into the cell
- i. an RNA guided DNA endonuclease or a nucleic acid encoding an RNA guided DNA endonuclease,
- ii. at least one guide RNA, and
- iii. an exogenous nucleic acid molecule comprising or encoding a DNA substitute sequence,
- generating at least two double strand breaks of the dsDNA molecule to be modified, wherein
- i. the dsDNA molecule comprises at least two target sequences (target sequences 1 and 2) which are targeted by the at least one guide RNA, and
- i. the at least two double strand breaks occur within or adjacent to the at least two target sequences,
- replacing a DNA sequence of the dsDNA molecule to be modified, wherein the replaced sequence is located between the double strand breaks (between target sequences 1 and 2) and is replaced by the DNA substitute sequence of the exogenous nucleic acid molecule by the non-homologous end joining (NHEJ) pathway.
A kit for modifying a double stranded DNA (dsDNA) molecule in a cell, comprising:
- an RNA guided DNA endonuclease or a nucleic acid encoding an RNA guided DNA endonuclease,
- at least one guide RNA, and
- a nucleic acid molecule comprising or encoding a DNA substitute sequence,
- wherein:
- the at least one guide RNA is configured for generating at least two double strand breaks of a dsDNA molecule to be modified within or adjacent to a target sequence 1 and a target sequence 2 within the dsDNA molecule, and
- the nucleic acid molecule comprising or encoding a DNA substitute sequence is configured for replacement of the DNA sequence of the dsDNA molecule located between the two double strand breaks with the DNA substitute sequence by the NHEJ pathway.
A composition for use as a medicament, for modifying a double stranded DNA (dsDNA) molecule in a cell, comprising:
- an RNA guided DNA endonuclease or a nucleic acid encoding an RNA guided DNA endonuclease,
- at least one guide RNA, and
- a nucleic acid molecule comprising or encoding a DNA substitute sequence,
- wherein:
- the at least one guide RNA is configured for generating at least two double strand breaks of a genomic dsDNA molecule within or adjacent to a target sequence 1 and a target sequence 2 within the genomic dsDNA molecule, and
- the nucleic acid molecule comprising or encoding a DNA substitute sequence is configured for replacement of the DNA sequence of the genomic dsDNA molecule located between the two double strand breaks with the DNA substitute sequence by the NHEJ pathway.
Dependencies are inferred from the claim text.