CRISPR Regulation of a Novel Gene, BLST, to Modulate Cellular Activity and Senescence

Introduction:: Cell engineering has provided the opportunity to tune cell function to address therapeutic needs and discover novel biology. Recently, in a set of CRISPRa genome wide screens, our lab identified a novel zinc finger protein previously unpublished on, which we are calling BLST, that produces robust cell-engineering phenotypes. Here we investigate BLSTs phenotype when upregulated (CRISPRa) and downregulated (CRISPRi), perform RNAseq in multiple cell types, and perform follow-up experiments to understand how BLST is regulating the observed phenotypes.


Materials and Methods:: : Human embryonic kidney (HEK293) and Adipose derived stem cells (ASCs) were transduced with lentiviral vectors targeting BLST for upregulation (CRISPRa) or downregulation (CRISPRi) and non-target control vectors for both systems. Cell proliferation was evaluated for 5 days following successful transduction and RNA was isolated from BLST and NTC cells for qRT-PCR of BLST expression (n=4). Cell cycle analysis was performed using Vibrant Dyecycle Violet staining. RNAseq was performed for both ASC and HEK cells (data not shown). Primary human nucleus pulposus cells (NPCs) were transduced with CRISPRi -BLST or -NTC expression cassettes and evaluated for proliferation and senescence using SA-β-gal staining 1 and 3 weeks post-transduction (n=4). Additionally, BLST was upregulated in Jurkat cells with CRISPRa, and their proliferation and cytokine production (IL-2) were measured via ELISA. One-way (qRT-PCR, ELISA) and two-way ANOVA (proliferation, SA-β-gal) were used to determine significance (a = 0.05)


Results, Conclusions, and Discussions:: Results and Discussion: Fold change gene expression showed significant activation by VPR and inhibition by KRAB. (1A). Downregulation of BLST in HEK 293 cells and ASCs showed a significant decrease in cell viability within 3 days of expression (1B). CRISPRa of BLST in HEK 293 cells and ASCs significantly decreased doubling time (1C). RNA-seq analysis shows the top molecular processes (Cell Cycle, DNA replication, cellular senescence, and protein processing) affected by BLST upregulation (1D). CRISPRi of BLST in hNPCs prevents proliferation compared to NTC (1E). SA-β-gal staining of BLST downregulated hNPCs shows significant increases in senescence across 1-week and 3-week timepoints (1F-H), indicating BLST may be a key regulator of senescence. CRISPRa of BLST in Jurkat cells increases proliferation and cytokine production when stimulated (1I,1J).

Conclusions: This research examines the effects of CRISPR-guided gene modulation of BLST. Overall, our results suggest that BLST regulates key processes in cell activity and senescence across a broad range of human cell types. BLST appears to be necessary for the normal function of cells in all the cell types we tested it in, indicating a previously unknown and significant cell biology discovery. Furthermore, the apparent regulation of senescence provides the potential for a novel gene that can interact with senescence in disease and aging. Overall, BLST has the potential to be a novel cell-engineering tool for regulating senescence in disease and boosting cell therapy potential across a broad range of cell therapies



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