This cell line has been engineered for use with the CRISPR Synergistic Activation Mediator (SAM) system to induce transcriptional activation and expression of any gene of interest. Cells stably express a mutated dCas9 (Streptococcus pyogenes CRISPR-associated protein 9), lacking any endonuclease activity, fused to VP64, a transcriptional activator. Stable dCas9-VP64 expression is maintained with Blasticidin resistance. Cells also stably express a construct consisting of P65 (Transcription Factor p65, or Nuclear Factor NF-κB p65) and HSF1 (Heat Shock Factor 1) fused with an MS2 tag (from the MS2 coat protein), which is maintained with Hygromycin resistance. When these cells are transfected with an MS2 aptamer fused to a single guide RNA (sgRNA)targeting the promoter region of the gene of interest, dCas9-VP64 and MS2-P65-HSF1 are recruited to the promoter in the genomic DNA by the MS2 aptamer and begin transcription, inducing high level expression of the desired protein.

Cells were functionally validated by transfecting the cells with an sgRNA-MS2 targeting PD-1, resulting in high expression of PD-1.

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