There is current lack of detailed protocols, or yet I couldn't find one!
The nuclease-inactive Cas9 (dCas9) enzyme is widely used for this purpose.
I read a publication in which mouse embryonic fibroblasts were reprogrammed into skeletal myocytes by a fusion of dCas9 with two transactivators VP64 at both termini.
Further efficacy of this over-expressing machine was achieved by adding a blue fluorescent protein and two nuclear localization signals to the complex.
Guide RNA is also needed to activate the complex in the correct gene.
The delivery of the transgene for the dCas9 was achieved by lentiviral transduction.
Original paper: A CRISPR/Cas9-Based System for Reprogramming Cell Lineage Specification.
The TALE-TF system seems useful for increasing the expression of an endogenous gene: http://www.nature.com/nmeth/journal/v10/n3/full/nmeth.2373.html
Many days ago I studied an article about light indiucble crispr-cas9 system.
The nuclease-inactive Cas9 (dCas9) enzyme is widely used for this purpose.
I read a publication in which mouse embryonic fibroblasts were reprogrammed into skeletal myocytes by a fusion of dCas9 with two transactivators VP64 at both termini.
Further efficacy of this over-expressing machine was achieved by adding a blue fluorescent protein and two nuclear localization signals to the complex.
Guide RNA is also needed to activate the complex in the correct gene.
The delivery of the transgene for the dCas9 was achieved by lentiviral transduction.
Original paper: A CRISPR/Cas9-Based System for Reprogramming Cell Lineage Specification.
I agree with Lorenzo. I would also recommend a paper by Konermann et al. Nature 517:583-588, 2015. All the plasmids are accessible free from Addgene.
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Gene Expression