Multiplex CRISPR/Cas9-based gene activation in BV2 cells
Patrick Miller-Rhodes and Harris A Gelbard University of Rochester Medical Center, Rochester, NY
Next-generation sequencing technologies have vastly expanded our knowledge of factors that regulate the development and homeostasis of microglia. However, the generation of floxed mouse strains capable of achieving cell-specific gene knockout remains a significant bottle-neck in the investigation of such factors in vivo. Moreover, current in vitro approaches for studying gene function in cultured hematopoietic cells are limited by difficulties associated with expressing full-length cDNAs and/or generating knockout or knockdown cell lines. To circumvent these limitations, we developed a straightforward in vitro approach for studying gene function in cultured microglia that relies on recent advances in CRISPR/Cas9 technology. We stably expressed the CRISPR/Cas9 synergistic activation mediator (SAM) complex in the BV2 microglial cell line. When combined with a single guide RNA that targets a desired genomic promoter element, the Cas9-SAM complex activates gene transcription by recruiting endogenous transcriptional machinery to the targeted locus. To establish the utility of this reagent, we used this system to overexpress the transcription factor Mafb from the endogenous locus. Expression of Mafb-targeting sgRNAs resulted in an upregulation of Mafb mRNA and protein. Upregulation of Mafb via this method also resulted in increased expression of the putative Mafb target genes. We also describe a method for multiplex gene activation in BV2-SAM cells. Altogether, the reagents described here can be used broadly to study the function of newly identified microglia-specific genes in the microglia-like BV2 cell line. This work was supported by NIH grants T32 AI049815 (P.M-R.), F31 MH113504 (P.M-R.), RO1 MH104147 (H.A.G.), & PO1 MH64570 (H.A.G.).
Credits: None available.
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