DIET CONTROLLED EXPRESSION OF A NUCLEIC ACID ENCODING CAS9 NUCLEASE AND USES THEREOF
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United States of America Patent
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Jun 20, 2019
app pub date -
Jun 2, 2017
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Jun 3, 2016
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Abstract
The present invention relates to genome editing by the mean of Cas nucleases. The inventors found that the expression of Cas nucleases may be finely controlled by the use of regulatory elements comprising a minimal promoter and at least one amino acid response element (AARE) nucleic acid, which are responsive to a diet deficient in at least one essential amino acid, or tunicamycin. For example, a FLAG-Cas9-GFP fusion and a Cas9-FLAG-RFP fusion could be expressed in 293 T cells. In addition, in the presence of a donor plasmid bearing a puromycin resistant gene, integration of the said puromycin resistant gene may be performed at the site of the safe harbour locus AASV1 on the genome of 293 T cells. Therefore, the invention relates to a nucleic acid for the controlled expression of a nucleic acid encoding a Cas nuclease in an individual, comprising (i) a regulatory polynucleotide comprising a minimal promoter and from one to twenty AARE nucleic acids, and (ii) a nucleic acid encoding a Cas nuclease, which is placed under the control of the said regulatory polynucleotide.
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