Multi-gene targeting by CRISPRa and CRISPRi in primary human T cells using synthetic gRNAs 9040

Abstract Description Dysregulation of cytokine production in stimulated T cells has been linked to autoimmunity, immunodeficiencies, and cancer. However, the signaling events and regulators that lead to these outcomes remain poorly understood. The CRISPR system, which has been widely used to knock out genes, has also been adapted to modulate transcription. CRISPR activation (CRISPRa) relies on a nuclease deficient Cas9 (dCas9) fused to transcriptional activator domains including VP64, p65, and RTA (VPR), while CRISPR interference (CRISPRi) effectors fuse dCas9 to transcriptional repressor domains including KRAB or SALL1-SDS3. Transcriptional regulation relies on precise targeting of the dCas9 effector to the transcriptional start site of a gene by a guide RNA. Guide RNAs can be chemically synthesized with stabilizing modifications, allowing for co-delivery alongside an mRNA transcript encoding the dCas9 effector. Further, multiple synthetic single guide RNAs (sgRNAs) can be delivered simultaneously, allowing for the targeting of multiple genes in a single experiment. Thus, we assessed mRNA and protein levels following electroporation of mRNAs encoding dCas9-VPR or dCas9-SALL1-SDS3 alongside DharmaconTM synthetic sgRNAs targeting CD28, IL2RB, GATA3, LAT, or all four genes simultaneously in T cells. Together, these data highlight the feasibility and utility in performing complex genetic experiments with CRISPRa and CRISPRi, enabling more precise interrogation of immune signaling networks. Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)