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US 10,947,559 B2Granted
Inducible modification of a cell genome
AssigneeAstraZeneca AB
InventorsMarcello Maresca, Mohammad Bohlooly-Yeganeh, Himjyot Jaiswal, Lorenz M. Mayr, Xiufeng Xu
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Abstract
The present disclosure is directed, in some embodiments, to compositions and methods for inducible modification of a cell genome.
Claims23
A circular engineered nucleic acid construct comprising:
- (a) a promoter operably linked to a nucleic acid encoding a regulatory protein;
- (b) an inducible promoter operably linked to a nucleic acid encoding an enzyme that cleaves nucleic acid, a nucleic acid encoding an enzyme that nicks nucleic acid, or a nucleic acid encoding an enzyme that catalyzes exchange of nucleic acid, wherein activity of the inducible promoter is modulated by the regulatory protein;
- (c) at least two insulators located downstream from (a) and upstream from (b);
- (d) at least one insulator located downstream from (b) and upstream from (a);
- and
- (e) at least one deoxyribonucleic acid (DNA)-binding domain recognition sequence located downstream from (b) and upstream from (a).
3 more elements
A method of modifying a cell genome, comprising:
- introducing into a cell a circular engineered nucleic acid construct comprising
- (a) a promoter operably linked to a nucleic acid encoding a regulatory protein;
- (b) an inducible promoter operably linked to a nucleic acid encoding an enzyme that cleaves nucleic acid, a nucleic acid encoding an enzyme that nicks nucleic acid, or a nucleic acid encoding an enzyme that catalyzes exchange of nucleic acid, wherein activity of the inducible promoter is modulated by the regulatory protein;
- (c) at least two insulators located downstream from (a) and upstream from (b);
- (d) at least one insulator located downstream from (b) and upstream from (a);
- and
- (e) at least one deoxyribonucleic acid (DNA)-binding domain recognition sequence located downstream from (b) and upstream from (a);
- introducing into the cell an engineered nucleic acid comprising a promoter operably linked to a nucleic acid encoding a guide RNA (gRNA) that targets a genomic region of the cell;
- introducing into the cell an engineered nucleic acid comprising a promoter operably linked to a nucleic acid encoding a hybrid nuclease that binds to the at least one DNA-binding domain recognition sequence;
- and
- incubating the cell in the presence of an effector substance that modulates activity of the regulatory protein under conditions that result in modification of the cell genome.
8 more elements
A method of modifying a cell genome, comprising:
- introducing into a cell a circular engineered nucleic acid construct comprising
- (a) a promoter operably linked to a nucleic acid encoding a regulatory protein;
- (b) an inducible promoter operably linked to a nucleic acid encoding an enzyme that cleaves nucleic acid, nicks nucleic acid, or catalyzes exchange of nucleic acid, wherein activity of the inducible promoter is modulated by the regulatory protein;
- (c) at least two insulators located downstream from (a) and upstream from (b);
- (d) at least one insulator located downstream from (b) and upstream from (a);
- (e) a promoter operably linked to a nucleic acid encoding a guide RNA (gRNA) that targets a genomic region of the cell;
- and
- (f) at least one deoxyribonucleic acid (DNA)-binding domain recognition sequence located downstream from (b) and upstream from (a);
- introducing into the cell an engineered nucleic acid comprising a promoter operably linked to a nucleic acid encoding a hybrid nuclease that binds to the at least one DNA-binding domain recognition sequence;
- and
- incubating the cell in the presence of an effector substance that modulates activity of the regulatory protein under conditions that result in modification of the cell genome.
8 more elements
An engineered nucleic acid comprising the sequence of any one of SEQ ID NO:
- 8 or 12-20.
Dependencies are inferred from the claim text.