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
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.