Drug-inducible CRISPR/Cas9 system for genome editing and transcription regulation
A genome editing and inducible technology, applied in the field of molecular biology, can solve problems such as the inapplicability of regulatory drugs or methods
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Embodiment 1
[0067] Example 1 Drug-Inducible CRISPR / Cas9 System for Genome Editing
[0068] This example is used to illustrate the construction of the drug-inducible CRISPR / Cas9 system for genome editing described in the present invention.
[0069] Build method:
[0070] The drug-induced genome editing system constructed in the present invention includes the following two parts:
[0071] (1) Cas9 and two ERs in series with its C-terminus T2 Composition, namely Cas9-2ER T2 .
[0072] (2) in Cas9-2ER T2 Insert one / two tandem NES into the plasmid, i.e. Cas9-NES-2ER T2 / Cas9-2NES-2ER T2 .
[0073] The source of each part of the plasmid is as follows:
[0074] The Cas9 element was amplified from the plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (from Zhang Feng's laboratory, Addgene plasmid #42230 40). ER T2 Elements were amplified from plasmid pAd-CreER (a gift from T.C. He's laboratory, University of Chicago). NES sequence according to the report of Ding et al. (Ding, Y., Ai, H.W., Hoi,...
Embodiment 2
[0078] This embodiment is used to illustrate the application of the system described in Embodiment 1.
[0079] 1. Experimental materials
[0080] Cas9-2ER T2 , Cas9-NES-2ER T2 and Cas9-2NES-2ER T2 Plasmids, sgRNAs targeting different sites, donor plasmids for TLR systems and single-stranded DNA donors (ssDNA donors) for FCR experiments.
[0081] 2. Experimental method
[0082] HEK293T cells (ATCC) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 2mM GlutaMAX (Thermo Fisher), 100U / ml penicillin and 100μg / ml streptomycin, and placed at 37°C, 5% CO 2 cultured in an incubator. Monoclonal TLR and FCR stably transfected cell lines were obtained by lentiviral packaging and infection. The cell transfection reagent was the DNA transfection reagent of Biotool Company, and the transfection method was carried out according to the instructions. The total amount of DNA transfected per well was consistent in each experiment. Cell medium was changed 5 hou...
Embodiment 3
[0097] Example 3 A drug-inducible CRISPR / Cas9 system capable of simultaneous genome editing and transcriptional activation
[0098] In this example, genome editing of the BFP gene and transcriptional activation of the CD43 gene are taken as an example to illustrate the construction of a drug-inducible CRISPR / Cas9 system that simultaneously performs genome editing and transcriptional activation.
[0099] Build method:
[0100] The drug-inducible CRISPR / Cas9 system for simultaneous genome editing and transcriptional activation of the present invention includes Cas9-2NES-2ER T2 -GCN4 and scFv-2ER T2 -VPH, where Cas9-2NES-2ER T2 It is the plasmid constructed in Example 1, GCN4 is a short peptide of 10 copies of yeast transcriptional activator GCN4, V is VP64, P is P65, and H is HSF1. The sources and construction methods of each element of the plasmid are as follows:
[0101] VP64 was amplified using the pLenti-EF1a-SOX2 plasmid (Addgene plasmid #35388) from Feng Zhang’s labora...
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