Edit-R CRISPRa Lentiviral sgRNA

Predesigned genome-wide CRISPRa lentiviral sgRNAs for highly efficient gene activation in human and mouse models; available as glycerol stocks and high-titer purified particles.


Genome-wide human and mouse lentiviral sgRNA reagents are specifically designed for CRISPRa. When used in conjunction with dCas9-VPR expression, you can leverage the power of the CRISPR-Cas9 system for activation of your favorite gene.

Enter a common gene identifier and click SEARCH to find products for your gene.

 
Edit-R Human CRISPRa lentiviral sgRNA
GSGH11887 glycerol stock - $450.00select
Edit-R Human CRISPRa Set of 4 lentiviral sgRNA
GSGH11890 glycerol set - $1,500.00select
Edit-R Mouse CRISPRa lentiviral sgRNA
GSGM11893 glycerol stock - $450.00select
Edit-R Mouse CRISPRa Set of 4 lentiviral sgRNA
GSGM11896 glycerol set - $1,500.00select

The CRISPR activation (CRISPRa) system is a unique 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 referred to as 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 (TSS) is activated.

In the Edit-R CRISPRa Lentiviral sgRNA vector backbone, the gene-specific crRNA and tracrRNA are expressed under the control of a human U6 promoter, while expression of the puromycin resistance marker (PuroR) is driven from the mouse CMV promoter and allows for rapid selection of cells with integrated sgRNA vector. Genome-wide Edit-R CRIPSRa human and mouse lentiviral sgRNA reagents are specifically designed for highly efficient gene activation.

Highlights of Edit-R CRISPRa Lentiviral sgRNA

  • Individual constructs and sets of 4 are available as glycerol stocks and high-titer purified lentivirus particles
  • Set-of-4 format ensures broad coverage of a target genes transcription start site, providing robust activation and expression
  • Designed with a published algorithm by Horlbeck, et. al. (2016) that has demonstrated strong levels of gene activation from optimized designs adjacent to one or more transcription start sites
  • Purified, concentrated high-titer lentivirus particles can be directly transduced into cells, including difficult-to-transfect cells and lines

Required components for an Edit-R CRISPRa gene activation experiment using lentiviral sgRNA:

  • A lentiviral expression plasmid or lentiviral particles for dCas9-VPR[2]; a mammalian codon-optimized S. pyogenes deactivated Cas9 fused to VPR activation domains (also compatible with SunTag technology[3])
  • A predesigned CRISPRa lentiviral sgRNA for the gene of interest
  
HazardousNo
Shipping ConditionDry Ice
Stability at Recommended Storage ConditionsAt least 12 months
Storage Condition-80 C
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Schematic diagram of the Edit-R CRISPRa Lentiviral sgRNA vector

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In the Edit-R CRISPRa Lentiviral sgRNA vector backbone, the gene-specific crRNA and the tracrRNA are expressed under the control of a human U6 promoter, while expression of the puromycin resistance marker (PuroR) is driven from the mouse CMV promoter and allows for rapid selection of cells with integrated sgRNA. The plasmid contains the AmpR resistance marker for growth and selection in E. coli.


Edit-R-CRISPRa-workflow-diagram

Edit-R CRISPRa workflow

Edit-R-CRISPRa-workflow-diagram

CRISPR activation workflow with lentiviral dCas9-VPR and synthetic crRNA:tracrRNA (left side) or Lentiviral expressed sgRNA (right side).


CRISPRa-co-transfection-workflow-diagram

Edit-R CRISPRa plasmid co-transfection workflow

CRISPRa-co-transfection-workflow-diagram

CRISPRa workflow for plasmid co-transfection of lentiviral dCas9-VPR and expressed sgRNA.


lenti-sgrna-transduce-crispra-different-cell-types

Transductions with Edit-R CRISPRa lentiviral sgRNA particles in different dCas9-VPR cell lines

lenti-sgrna-transduce-crispra-different-cell-types

U2OS, HEK293T, MCF 10A and K562 stably expressing integrated dCas9-VPR were plated at 10,000 cells/well and transduced with sgRNA lentiviral particles targeting POU5F1 or TTN at a MOI of 0.3 to obtain cells with a single integrant. Cells were selected with 2 µg/mL puromycin for 4 days prior to analysis with 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.


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

crispra-activation-basal-expression-relationship

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.


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CRISPRa in U2OS cells stably expressing integrated dCas9-VPR was compared using different guide RNAs and delivery methods

edit-r-crispra-guide-rna-types-dcas9-vpr-cells

CRISPRa using synthetic crRNA:tracrRNA: Cells were plated at 10,000 cells/well and were transfected using DharmaFECT 4 Transfection Reagent with synthetic crRNA:tracrRNA (25 nM) targeting ASCL1, EGFP, POU5F1 and TTN genes. Cells were harvested 72 hours post-transfection and the relative gene expression was calculated using qRT-PCR. The relative expression of each gene was ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control. CRISPRa with lentiviral sgRNA transduction: Cells were plated at 10,000 cells/well and were transduced with CRISPRa sgRNA lentiviral particles targeting ASCL1, EGFR, POU5F1 or TTN at a MOI of 0.3 to obtain cells with a single integrant. Cells were selected with 2 µg/mL puromycin for 4 days prior to analysis with 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. CRISPRa with lentiviral sgRNA plasmid transfection: cells were plated at 10,000 cells/well and transfected with CRISPRa sgRNA plasmids (100 ng) targeting ASCL1, EGFR, POU5F1 or TTN using DharmaFECT kb Transfection Reagent. Cells were harvested 72 hours post-transfection (without puromycin selection) and the relative gene expression was calculated using qRT-PCR. The relative expression of each gene was ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.


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Edit-R lentiviral sgRNA for CRISPRa in human induced pluripotent stem cells

ips-cells-nucleofection-plasmid-crispra

(A) Human iPSC (ThermoFisher Cat #A18945) were cultured in feeder-free and serum free culture conditions and visualized for proper morphology on an inverted fluorescent microscope. (B). The human iPSCs were nucleofected according to manufactures protocols using 96-well shuttle solution P3. Three days after nucleofections, cells were harvested, and prepped for 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.


References

  1. Horlbeck MA, Gilbert LA, et. al., Compact and highly active next-generation libraries for CRISPR-mediated gene repression and activation. 2016 Sep 23;5. pii: e19760. doi: 10.7554/eLife.19760. PubMed 27661255
  2. Chavez A, Scheiman J et. al., Highly efficient Cas9-mediated transcriptional programming Nat Methods. 2015 Mar 2. doi: 10.1038/nmeth.3312. 10.1038/nmeth.3312 PubMed 25730490
  3. Tanenbaum ME, Gilbert LA, et. al., A protein tagging system for signal amplification in gene expression and fluorescence imaging. Cell. 2014;159(3):635-646. doi:10.1016/j.cell.2014.09.039.