CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs

Mohammad A. Mandegar, Nathaniel Huebsch, Ekaterina B. Frolov, Edward Shin, Annie Truong, Michael P. Olvera, Amanda H. Chan, Yuichiro Miyaoka, Kristin Holmes, C. Ian Spencer, Luke M. Judge, David E. Gordon, Tilde V. Eskildsen, Jacqueline E. Villalta, Max A. Horlbeck, Luke A. Gilbert, Nevan J. Krogan, Søren P. Sheikh, Jonathan S. Weissman, Lei S. Qi, Po-Lin So, and Bruce R. Conklin
Source: Cell Stem Cell
Publication Date: (2016)
Issue: 18 (4): 541-53
Research Area:
Stem Cells
Gene Expression
Basic Research
Cells used in publication:
Species: human
Tissue Origin: ovarian
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Nucleofectorâ„¢ I/II/2b
A very precise and tunable system fot silencing specific alleles in iPSCs is described. the system relies on a doxycycline-inducible deactivated Cas9 which is fused to a KRAB repression domain. For plasmid transfections, the human stem cell nucleofector kit 1 solution was used on the Amaxa nucleofector 2b device (program A-23; Lonza). To generate the CRISPRi and CRISPRn iPSC lines, two million WTC or WTB iPSCs were nucleofected with the appropriate knockin vector (5 mg) and each AAVS1TALEN pair (2 mg). CEM CRISPRi cells were generated by electroporation of 0.5 mg of each AAVS1 TALEN pair and 1 mg of the Gen1 CRISPRi vector with an Amaxa nucleofector 2b device and Amaxa cell line nucleofector kit C (Lonza). Cells were selected in 1 mg/ml G418, and clonal lines were generated by dilution in 96-well plates.
Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs).CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.