CD8 + T cell differentiation orchestrated by transcription regulators is critical for balancing pathogen eradication and long-term immunity by effector and memory CTLs, respectively. The transcription factor Nuclear Factor of Activated T cells (NFAT) family members are known for their roles in T cell development and activation but still largely undetermined in CD8 + T cell differentiation in vivo . Here, we interrogated the role of two NFAT family members, NFAT1 and NFAT2, in the effector and memory phase of CD8 + T cell differentiation using LCMV Arm acute infection model. We found that NFAT1 is critical for effector population generation whereas NFAT2 is required for promoting memory CTLs in a cell intrinsic manner. Moreover, we found that mice lacking both NFAT1 and NFAT2 in T cells display a significant increase in KLRG1 hi CD127 hi population and are unable to clear an acute viral infection. NFAT-deficient CTLs showed different degrees of impaired IFN-γ and TNF-α expression with NFAT1 being mainly responsible for IFN-γ production upon ex-vivo stimulation as well as for antigen-specific cytotoxicity. Our results suggest that NFAT1 and NFAT2 have distinct roles in mediating CD8 + T cell differentiation and function.

T.X. and G.J.M. designed the experiments and wrote the article. T.X. performed experiments, analyzed, and plotted the data. A.K. and A.S. maintained the mouse colony and helped with experiments. H.I.N., A.N.A.G., and L.J. performed the assay for transposase-accessible chromatin sequencing bioinformatics analysis. R.M.P. and M.E.P. provided valuable reagents and guidance in the project. G.J.M. supervised the project.The sequences presented in this article have been submitted to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE161842.

While CRISPR interference (CRISPRi) systems have been widely implemented in pooled lentiviral screening, there has been limited use with synthetic guide RNAs for the complex phenotypic readouts enabled by experiments in arrayed format. Here we describe a novel deactivated Cas9 fusion protein, dCas9-SALL1-SDS3, which produces greater target gene repression than first or second generation CRISPRi systems when used with chemically modified synthetic single guide RNAs (sgRNAs), while exhibiting high target specificity. We show that dCas9-SALL1-SDS3 interacts with key members of the histone deacetylase and Swi-independent three complexes, which are the endogenous functional effectors of SALL1 and SDS3. Synthetic sgRNAs can also be used with in vitro -transcribed dCas9-SALL1-SDS3 mRNA for short-term delivery into primary cells, including human induced pluripotent stem cells and primary T cells. Finally, we used dCas9-SALL1-SDS3 for functional gene characterization of DNA damage host factors, orthogonally to small interfering RNA, demonstrating the ability of the system to be used in arrayed-format screening.

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