• dCas9-VPR mRNA for quick transcriptional activation and enrichment in any cell line

    dCas9-VPR mRNA for quick transcriptional activation in any cell line

    While genome engineering is widely ascribed with the capacity to knock out genes of interest, new adaptations of these same molecular building-blocks are reshaping the way researchers think about the opposite: overexpression experiments. This technique allows recapitulation of the native gene expression patterns in a manner previously unobtainable using traditional overexpression-plasmid methods. For CRISPR activation (CRISPRa), the guide RNA forms a complex with a nuclease-deactivated Cas9 (dCas9), which is in turn fused to three transcriptional activators (comprising the VPR system). The machinery then acts upstream of the transcription start site to up-regulate expression of a target gene (Figure 1). CRISPRa provides a new set of tools to identify gene function that might otherwise go undetected using loss-of-function studies through gene knockdown or knockout.

    Previously, we have demonstrated that synthetic crRNAs can be successfully used to perform experiments in cells stably expressing dCas9-VPR. However, the constitutive expression or extended timeline needed to create a stable cell line may not be desired for every application. Here we examined the ability to use the synthetic crRNA approach with dCas9-VPR mRNA - alleviating the need to use lentivirus altogether.

    We demonstrate that:

    • Transient delivery of dCas9-VPR mRNA and synthetic guide in multiple cell lines results in robust activation – CRISPRa is possible in any cell line that is amenable to lipid-mediated or electroporation delivery methods.
    • Using EGFP-tagged or puromycin-tagged dCas9-VPR mRNA enables antibiotic or fluorescent selection – CRISPRa can be improved by enrichment methods when delivery is less than optimal.

    Transcriptional activation by transient transfection of dCas9-VPR mRNA and synthetic guide RNA

    To demonstrate that we can get transcriptional activation without the need to generate a stable-expressing dCas9-VPR cell line, we used DharmaFECT Duo to co-transfect Edit-R dCas9-VPR mRNA, Edit-R synthetic tracrRNA, and Edit-R CRISPRa crRNA pools targeting the promotor region of either POU5F1 or IL1R2. We observed robust activation of approximately 1,000-fold (Figure 2).

    To test whether this same activation could be demonstrated in cells where electroporation is necessary for delivery, we co-electroporated K-562 and THP-1 cells with dCas9-VPR mRNA, synthetic tracrRNA and crRNA pools targeting the promotor region of TTN and see strong activation (Figure 3). We also observe robust activation of TTN for K-562 and THP-1 cells co-electroporated with EGFP or puro tagged mRNA, which enables enrichment and a wider array of applications.

    dCas9-VPR mRNA allows for enrichment using antibiotics or fluorescence

    EGFP- or puro-tagged dCas9-VPR mRNA can be used to select for enrichment in gene activation experiments where the cell line may have low delivery efficiency, or in cases where optimization of delivery conditions is not possible.

    For EGFP dCas9-VPR mRNA, we co-transfected EGFP dCas9-VPR mRNA, synthetic tracrRNA, and crRNA pools targeting the promotor region of IL1R2. Following FACS sorting we observed significant enrichment for the top 10% sorted cells when compared to dim and unsorted cell populations (Figure 4).

    We further examined if selection and enrichment could be achieved using our Edit-R Puro dCas9-VPR mRNA. Similarly, we co-transfected cells with Edit-R Puro dCas9-VPR mRNA, Edit-R synthetic tracrRNA, and Edit-R crRNA pools targeting the promotor region of either IL1R2, TFAP2C, or TTN. Following a 24-hour selection with puromycin supplemented media, we observed significant enrichment or gene activation in our puromycin selected population (Figure 5).

    Conclusions

    dCas9-VPR mRNA can be used in both lipid-mediated transfection and electroporation delivery to achieve transcriptional activation in a CRISPRa system. Furthermore, EGFP dCas9-VPR mRNA and Puromycin dCas9-VPR mRNA can be used to select and enrich for gene activation.

    These methods presented are broadly applicable as a strategy to up-regulate the expression for any gene in difficult-to-transfect cell lines, or in laboratories where generating stably expressing dCas9-VPR cell lines may be technically difficult or undesirable.

    Finally, these advances in technology open the door for systems biology experiments previously unfeasible, such as large-scale overexpression screening or overexpressing multiple genes in the same cell.

    Additional Resources

  • Webinar: CRISPRa tools for transcriptional activation studies