Edit-R SAM tracrRNA

Synthetic trans-activating CRISPR RNA compatible with the SAM transcriptional activation system


Chemically synthesized SAM tracrRNA for use with synthetic crRNA for fast and easy gene activation in stable cells containing the SAM transcriptional activation system
For gene activation with the synergistic activation mediator (SAM) system, Edit-R SAM tracrRNA can be used with Edit-R synthetic CRISPRa crRNA for gene activation studies. The Edit-R SAM tracrRNA is a chemically synthesized and HPLC-purified long RNA molecule based on the optimized published S. pyogenes tracrRNA sequence (Jinek, 2012) that also contains an MS2 aptamer sequence that binds to the MS2-p65-HSF1 component of the SAM system (Konermann et al., 2015).

Highlights

The Edit-R CRISPRa synthetic crRNA platform can be used with the SAM system for gene activation in mammalian cells and requires the following:

  • A stable cell line expressing both components (NLS-dCas9-VP64 and MS2-p65-HSF1) of the SAM activation system
  • A chemically synthesized SAM tracrRNA, and
  • A chemically synthesized CRISPRa crRNA designed to the gene target site of interest

How much crRNA & tracrRNA do I need?

This table provides the approximate number of experiments that can be carried out for lipid transfection methods at the recommended crRNA:tracrRNA working concentration (25 nM:25nM) in various plate/well formats. Calculations do not account for pipetting errors.

For crRNA libraries, use (# wells)*(nmol per well) to determine the approximate amount of tracrRNA required. For instance, a 100 well library at 0.5 nmol per well would require 50 nmol of tracrRNA. The bulk sizes of tracrRNA are recommended for large library projects.
 
crRNA
nmol
tracrRNA
nmol
96-well plate
100 µL reaction volume
24-well plate
500 µL reaction volume
12-well plate
1000 µL reaction volume
6-well plate
2500 µL reaction volume
2 2 800 160 80 32
5 5 2000 400 200 80
10 10 4000 800 400 160
20 20 8000 1600 800 320

 

  
HazardousNo
Shipping ConditionAmbient
Stability at Recommended Storage ConditionsAt least 24 months
Storage Condition-20 C
CRISPRa SAM workflow options and recommendations

CRISPRa SAM workflow options and recommendations

CRISPRa SAM workflow options and recommendations

Horizon's Edit-R SAM tracrRNA contains an MS2 aptamer on stem loop 2 that recruits MCP-p65-HSF1. Combined with a catalytically inactivated Cas9-VP64 and a gene specific Edit-R CRISPRa crRNA, this utilization of the synergistic activation mediator (SAM) system achieves robust transcriptional activation.

Modifed tracrRNA has a MS2 domain on stem loop 2 and contains 3’ 2xMS modifications.


Transcriptional activation of gene targets in SAM cell lines

Transcriptional activation of gene targets in SAM cell lines

Transcriptional activation of gene targets in SAM cell lines

U2OS and A375 cells stably expressing dCas9-VP64 and MS2-p65-HSF1 (dCas9-SAM) were plated at 10,000 cells/well and transfected using DharmaFECT 4 (U2OS) or DharmaFECT 1 (A375) Transfection Reagent with synthetic crRNA:tracrRNA (25nM). Four predesigned crRNAs targeting each gene were pooled (to a total concentration of 25nM). Cells were harvested 72 hours post-transfection and the relative gene expression was calculated using RT-qPCR. The relative expression of each gene was calculated with the ΔΔCq method using PPIB as the housekeeping gene and normalized to a non-targeting control.


Transcriptional activation of gene targets in U2OS-dCas9-SAM cell lines: individual and pooled crRNA

Transcriptional activation of gene targets in U2OS-dCas9-SAM cell lines: individual and pooled crRNA

Transcriptional activation of gene targets in U2OS-dCas9-SAM cell lines: individual and pooled crRNA

U2OS cells stably expressing dCas9-VP64 and MS2-p65-HSF1 (dCas9-SAM) were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with synthetic crRNA:tracrRNA (25nM). Four predesigned crRNAs targeting each gene were transfected individually or pooled (to a total concentration of 25nM). Cells were harvested 72 hours post-transfection and the relative gene expression was calculated using RT-qPCR. The relative expression of each gene was calculated with the Δ Δ Cq method using PPIB as the housekeeping gene and normalized to a non-targeting control.


Transcriptional activation of gene targets in A375-dCas9-SAM cell lines: individual and pooled crRNA

Transcriptional activation of gene targets in A375-dCas9-SAM cell lines: individual and pooled crRNA

Transcriptional activation of gene targets in A375-dCas9-SAM cell lines: individual and pooled crRNA

A375 cells stably expressing dCas9-VP64 and MS2-p65-HSF1 (dCas9-SAM) were plated at 10,000 cells/well and transfected using DharmaFECT 1 Transfection Reagent with synthetic crRNA:tracrRNA (25nM). Four predesigned crRNAs targeting each gene were transfected individually or pooled (to a total concentration of 25nM). Cells were harvested 72 hours post-transfection and the relative gene expression was calculated using RT-qPCR. The relative expression of each gene was calculated with the Δ ΔCq method using PPIB as the housekeeping gene and normalized to a non-targeting control.