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US 2021/0230630 A1Published application
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.
Claims28
A 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).
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20-26. (canceled)
28-38. (canceled)
An engineered nucleic acid comprising:
- (a) a promoter operably linked to a nucleic acid encoding a Cas9 nuclease that does not comprise a nuclear localization signal, wherein the nucleic acid encoding the enzyme is flanked by estrogen receptor (ERT2) sequences;
- and
- (b) a deoxyribonucleic acid (DNA)-binding nuclease recognition sequence.
A method of modifying a cell genome, comprising:
- introducing into a cell an engineered nucleic acid comprising
- (a) a promoter operably linked to a nucleic acid encoding a Cas9 nuclease that does not comprise a nuclear localization signal, wherein the nucleic acid encoding the enzyme is flanked by estrogen receptor (ERT2) sequences;
- and
- (b) a deoxyribonucleic acid (DNA)-binding nuclease recognition sequence;
- 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;
- and
- incubating the cell in the presence of tamoxifen under conditions that result in modification of the cell genome.
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A transgenic mouse comprising a genome comprising an engineered nucleic acid construct comprising the sequence of any one of SEQ ID NO:
- 8 or 12-20.
47-49. (canceled)
Dependencies are inferred from the claim text.