Highly efficient Cas9-mediated transcriptional programming.

Chavez A1, Scheiman J2, Vora S3, Pruitt BW4, Tuttle M4, P R Iyer E2, Lin S5, Kiani S6, Guzman CD4, Wiegand DJ4, Ter-Ovanesyan D2, Braff JL4, Davidsohn N2, Housden BE7, Perrimon N7, Weiss R8, Aach J9, Collins JJ10, Church GM2.
Source: Nat Methods
Publication Date: (2015)
Issue: 12(4): 326-8
Research Area:
Stem Cells
Gene Expression
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
4D-Nucleofector® X-Unit
approximately 5x10^5 cells were nucleofected with 1.5µg of dCas9-AD piggy-bac expression vector and 340ng of transposase vector (System Biosciences) using the Amaxa P3 Primary Cell 4D-Nucleofector X Kit (Lonza), program CB-150. Following electroporation, cells were seeded onto 24-well matrigel-coated plates in the presence of 10uM ROCK inhibitor (R&D systems) and allowed to recover for two days before expanding to 6-well plates in the presence of 20ug/ml hygromycin to select for a mixed population of dCas9-AD integrant containing cells.
The RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).