Edit-R SAM tracrRNA

A synthetic MS2-containing tracrRNA for transcriptional activation with the dCas9-SAM system

A synthetic SAM tracrRNA optimized for functionality

CRISPR activation (CRISPRa) is a repurposed use of the Streptococcus pyogenes CRISPR-Cas9 system. Review our CRISPRa applications page to get an overview of the technology. In the synergistic activation mediator (SAM)1activation system, fusion proteins dCas9-VP64 and MS2-p65-HSF1 are used in combination with an MS2 aptamer-modified guide RNA to facilitate binding of the MS2-p65-HSF1 activating module. Published expressed versions of this SAM guide RNA contain two MS2 aptamers. Synthetic versions of SAM guide RNA are challenging to synthesize, because an analogous SAM sgRNA or SAM tracrRNA would be 158 and 141 nucleotides respectively. Taking advantage of Horizon Discovery’s long RNA synthesis and optimization capabilities, we have developed a synthetic SAM tracrRNA that contains a single MS2 aptamer sequence on stem loop 2 (Figure 1). When used with gene-specific Edit-R CRISPRa crRNA and Edit-R CRISPRa crRNA arrayed libraries, Edit-R SAM tracrRNA abundantly recruits the MS2-p65-HSF1 fusion to the transcriptional start site (TSS) to initiate activation. Edit-R CRISPRa guide RNA designs come from a published CRISPRa v2 algorithm based on machine learning2 and enable robust over-expression of your gene(s) of interest.

sam tracrrna crispr activation

Robust gene activation with synthetic SAM tracrRNA

We have assessed the activation of synthetic crRNA and SAM tracrRNA by measuring the transcriptional fold activation for six genes of interest (Figure 2). U2OS and A375 cells stably expressing the SAM activation system (dCas9-VP64 and MS2-p65-HSF1) were transfected with synthetic SAM tracrRNA and Edit-R CRISPRa pooled crRNA using DharmaFECT transfection reagents. Cells were harvested 72 hours post-transfection and the relative gene expression was measured using RT-qPCR. Robust activation ranging from 17 to almost 10,000-fold was observed for all six genes in both cell lines.

sam tracrrna sam cell lines

Figure 2. U2OS and A375 cells stably expressing dCas9-VP64 and MS2-p65-HSF1 were plated at 10,000 cells/well and transfected with Edit-R CRISPRa pooled crRNA complexed with Edit-R SAM tracrRNA (25nM), using either DharmaFECT 4 (U2OS-dCas9-SAM) or DharmaFECT 1 (A375-dCas9-SAM) Transfection Reagent. Cells were harvested 72 hours post-transfection and the relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using PPIB as the housekeeping gene and normalized to a non-targeting control.

SAM-mediated transcriptional activation with synthetic crRNA: SAM tracrRNA versus expressed SAM sgRNA

All publications to date for SAM-mediated activation have utilized expressed SAM sgRNA. We investigated how levels of activation compare when using synthetic crRNA: SAM tracrRNA, SAM sgRNA plasmid, or SAM sgRNA lentiviral particles. When SAM tracrRNA is combined with a single crRNA, it can be used to effectively induce short-term overexpression at levels comparable to, or better than, SAM sgRNA expression from commonly used plasmid and lentiviral delivery methods (Figure 3). A synthetic crRNA: SAM tracrRNA option also has some key benefits including:

  • Use in arrayed screening
  • Use in short term assays
  • No cloning, sequencing, or in vitro transcription required
  • Ability to combine individual crRNA into a pool for robust activation

sam tracrrna versu expressed sam sgrna

Figure 3. Activation with synthetic crRNA: SAM tracrRNA: Cells were plated at 10,000 cells/well and were transfected using DharmaFECT 4 (U2OS-dCas9-SAM) or DharmaFECT 1 (A375-dCas9-SAM) Transfection Reagent with synthetic crRNA: SAM tracrRNA (25 nM) targeting POU5F1, TTN, and ASCL1 genes. Cells were harvested 72 hours post-transfection and the relative gene expression was measured using RT-qPCR.

Activation with SAM sgRNA plasmid: Cells were plated at 10,000 cells/well and transfected with 200 ng of SAM sgRNA plasmids (containing two MS2 domains in the tracrRNA region1) targeting POU5F1, TTN, and ASCL1 genes using DharmaFECT kb Transfection Reagent. Cells were harvested 72 hours post-transfection (without antibiotic selection) and the relative gene expression was measured using RT-qPCR.

Activation with lentiviral sgRNA lentiviral particles: Cells were plated at 10,000 cells/well and were transduced with SAM sgRNA lentiviral particles (containing two MS2 domains in the tracrRNA region1) targeting POU5F1, TTN, and ASCL1 genes at a MOI of 0.3 to obtain cells with a single integrant. Cells were selected with 250 µg/mL zeocin for 48 hours prior to analysis with RT-qPCR.

The relative expression of each gene was calculated with the ∆∆Cq method using PPIB as the housekeeping gene and normalized to a non-targeting control.

Multiple crRNA per gene improves activation

When Edit-R SAM tracrRNA is complexed with an individual Edit-R CRISPRa crRNA, robust activation is achieved. When complexed with multiple Edit-R CRISPRa crRNAs targeting a single gene, enhanced activation levels can be achieved (Figure 4). Overall, pooling is a good strategy if maximal gene activation is desired, or if it is beneficial to decrease the scale of an experiment, for example, when performing analysis of multiple genes in an arrayed plate format.

sam tracrrna

Figure 4. U2OS and A375 cells stably expressing dCas9-VP64 and MS2-p65-HSF1 were plated at 10,000 cells/well and transfected with either individual Edit-R CRISPRa crRNA or pooled Edit-R CRISPRa crRNAs, complexed with Edit-R SAM tracrRNA (25nM), using DharmaFECT 4 (U2OS-dCas9-SAM) or DharmaFECT 1 (A375-dCas9-SAM) Transfection Reagent. Cells were harvested 72 hours post-transfection and the relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using PPIB as the housekeeping gene and normalized to a non-targeting control.

 

Featured Products

Edit-R SAM tracrRNA

  • Chemically synthesized SAM tracrRNA for use with synthetic crRNA for fast and easy gene activation in stable cells containing the SAM transcriptional activation system.

Edit-R CRISPRa crRNA Libraries

  • Arrayed collections of synthetic CRISPR RNA for overexpression screening of gene families, druggable, and whole human genome.

Edit-R CRISPRa crRNA

  • Predesigned synthetic guide RNA for over-expression of human and mouse genes using CRISPR activation. Just search for your gene! Available as pooled or individual reagents.

Edit-R CRISPRa Positive crRNA Controls

  • Validated synthetic crRNA pool or individual for experimental optimization of transcription activation experiments.

Edit-R CRISPRa crRNA Non-targeting Control

  • Non-targeting controls to evaluate baseline cellular responses to CRISPRa components in the absence of gene target-specific crRNA.

Cherry-pick Libraries

  • Begin here to turn your gene list into a custom library!

 

Featured Resources

References:

  1. S. Konermann et al., Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature. 517, 583–588 (2015).
  2. M. A. Horlbeck et al., Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. eLife. 5, e19760 (2016).