Edit-R dCas9-VPR mRNA

A DNA-free option for dCas9-VPR expression


Purified dCas9-VPR mRNA for co-transfection or electroporation with synthetic crRNA for gene activation

The CRISPR activation (CRISPRa) system is a variation of the canonical CRISPR-Cas9 used for creation of double-strand breaks in genomic DNA. It utilizes a nuclease-deactivated S. pyogenes Cas9 (dCas9), often called "dead Cas9", that is fused to one or more transcriptional activators. When paired with a well-designed guide RNA that targets a gene near a promoter region, the gene's native transcription start site is activated.

Edit-R dCas9-VPR mRNA expresses a human codon-optimized version of the nuclease-deactivated S. pyogenes Cas9 gene, the three transcriptional activators (VP64, p65 and Rta), and two nuclear localization signals (NLS).

The dCas9-VPR mRNA is available in a form that co-expresses either EGFP or puromycin for transfection optimization or enrichment using fluorescence-activated cell sorting (FACS) or antibiotic selection.

Highlights

  • Easily co-transfect Edit-R synthetic guide RNA and dCas9-VPR mRNA using DharmaFECT Duo transfection reagent or co-electroporate into cells that are difficult to transfect
  • No need to generate dCas9-VPR-expressing cell line
  • dCas9-VPR mRNA has fluorescent and antibiotic options to enable enrichment of gene activation in the cell population (see supporting data)
  • Since mRNA is ready for translation, no need for optimal promotor selection
  
HazardousNo
Shipping ConditionDry Ice
Stability at Recommended Storage ConditionsAt least 24 months
Storage Condition-80 C
Fold activation by CRISPRa varies by gene and depends on the endogenous gene expression level

Fold activation by CRISPRa varies by gene and depends on the endogenous gene expression level

Fold activation by CRISPRa varies by gene and depends on the endogenous gene expression level

U2OS cells stably expressing integrated dCas9-VPR were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with synthetic crRNA:tracrRNA pools (25 nM) targeting genes with low to high basal transcript expression levels. Cells were harvested 72 hours post-transfection and the relative gene expression was calculated using qRT-PCR. The relative expression of each gene was calculated with the ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control. The fold activation is shown for the genes ranked from low to high basal transcript expression level in samples treated with NTC control and is shown in the lower graph as basal gene expression relative to GAPDH expression in the same samples.


CRISPRa gene activation in U2OS cells is observed at 24 hours and increases at 48-72 hours

CRISPRa gene activation in U2OS cells is observed at 24 hours and increases at 48-72 hours

CRISPRa gene activation in U2OS cells is observed at 24 hours and increases at 48-72 hours

U2OS cells stably expressing integrated dCas9-VPR were plated at 10,000 cells/well and transfected using DharmaFECT 4 Transfection Reagent with synthetic crRNA:tracrRNA targeting EGFR, IL1R2, POU5F1 or TFAP2C. The four pre-designed crRNAs for CRISPRa were used either individually or pooled (to a total concentration of 25 nM). Cells were harvested at 24, 48, and 72 hours post-transfection and the relative gene expression was calculated using qRT-PCR. The relative expression of each gene was calculated with the ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.


CRISPRa gene activation by electroporation of dCas9-VPR mRNA and synthetic guide RNA

CRISPRa gene activation by electroporation of dCas9-VPR mRNA and synthetic guide RNA

CRISPRa gene activation by electroporation of dCas9-VPR mRNA and synthetic guide RNA

THP-1 and K-562 cells were electroporated using the Lonza 2b system with either dCas9-VPR mRNA (5 µg, Cat #CAS12024), Puro dCas9-VPR mRNA (5 ug, Cat #CAS12026) or EGFP dCas9-VPR mRNA (5 ug, Cat #CAS12025), synthetic tracrRNA (25 nM, Cat #U-002005-05), and pooled CRISPRa crRNA targeting TTN (5 uM, Cat #P-005395-01-0005) or non-targeting control (NTC, 25 nM, Cat #U-009500-10-05). Cells were harvested at 48 hours post-transfection and total RNA was isolated. Relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.


EGFP can be used to select for cells with EGFP dCas9-VPR mRNA and enrich for gene activation

EGFP can be used to select for cells with EGFP dCas9-VPR mRNA and enrich for gene activation

EGFP can be used to select for cells with EGFP dCas9-VPR mRNA and enrich for gene activation

U2OS cells were plated at 200,000 cells per well in clear 6-well plates. After 24 hours, cells were co-transfected with EGFP dCas9-VPR mRNA (2 µg, Cat #CAS12025), synthetic tracrRNA (25 nM, Cat #U-002005-05), and pooled CRISPRa crRNA targeting IL1R2 (25 nM, Cat #P-007960-01-0005) or Non-targeting control (NTC, 25 nM, Cat #U-009500-10-05) using DharmaFECT Duo (2 µg/well, Cat #T-2010-01). At 24 hours post-transfection, cells were trypsinized and FACS was performed and cells were sorted into three categories: Negative, Dim, and Top 10%. Cells were replated in 6-well dishes and allowed to recover. After 24 hours, total RNA was isolated and relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control. The negative population of cells sorted shows no activation. The Dim and Unsorted populations both showed ~120-fold activation and our Top 10% population resulted in 450-fold activation.


Puromycin can be used to select for cells with Puro dCas9-VPR mRNA and enrich for gene activation

Puromycin can be used to select for cells with Puro dCas9-VPR mRNA and enrich for gene activation

Puromycin can be used to select for cells with Puro dCas9-VPR mRNA and enrich for gene activation

U2OS cells were plated at 200,000 cells per well in clear 6-well plates. After 24 hours, cells were co-transfected with Puro dCas9-VPR mRNA (2 µg, Cat #CAS12026), synthetic tracrRNA (25 nM, Cat #U-002005-05), and pooled CRISPRa crRNA targeting either POU5F1 (25 nM Cat #P-019591-01-0005), IL1R2 (25 nM, Cat #P-007960-01-0005), TFAP2C (25 nM, Cat #P-005238-01-0005), TTN (25 nM, Cat #P-005395-01-0005) or non-targeting control (NTC, 25 nM, Cat #U-009500-10-05) using DharmaFECT Duo (2 µg/well, Cat #T-2010-01). At 24 hours post-transfection, 2 µg/ml of puromycin growth media was added to the cells and a duplicate plate received normal growth media. At 48 hours post-transfection a crystal violet assay was performed and images captured to assess viability and total RNA was isolated. Relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control. For all genes tested, we observed 3- to 5-fold enrichment of activation when compared to unselected samples.