CRISPRa is an adapted and repurposed use of the Streptococcus pyogenes CRISPR-Cas9 system (widely used for gene knockout) to upregulate gene(s) in their endogenous context. The native CRISPR-Cas9 DNA cutting functionality has been obliterated by point mutations in the RuvCI and HNH nuclease domains (D10A and H840A, Figure 1A) changing the protein to a catalytically deactivated, or dead, Cas9 (dCas9). The dCas9 protein was then further engineered to be fused to different transcription activation domains1-7. Dharmacon™ Edit-R™ CRISPRa system is based on the VPR activation system (Figure 1).
Figure 1. A. CRISPRa uses deactivated Cas9 (dCas9), a nuclease-deficient mutant variant of the Cas9 protein. The schematic of dCas9 shows two point mutations (D10A, H840A) that inactivate the DNA cleavage activity. B. The VPR CRISPRa system comprised of dCas9 fused to three transcriptional activators (VP64, p65 and Rta19) (dCas9-VPR) which act upstream of the transcriptional start site (TSS) to up-regulate expression of the target gene with a guide RNA consisting of crRNA and tracrRNA complex. C. The VPR CRISPRa system with a single guide RNA (sgRNA).
In the VPR activation system, dCas9 is fused at the C-terminal end to three transcriptional activators (VP64, p65 and Rta)6. Guide RNAs target upstream of the transcriptional start site (TSS) of a desired gene, bind the dCas9-VPR, guide the complex to the DNA target site, then the transcriptional activators are proximal to the TSS for up-regulation of the target gene.
CRISPRa guide RNA can be either synthetic CRISPR RNA (crRNA) complexed with a trans-activating CRISPR RNA (tracrRNA) (Figure 1B) or a single guide RNA (sgRNA) where the crRNA has been fused to the tracrRNA creating a chimeric structure (Figure 1C).
CRISPRa requires guide RNA designs in proximity to the gene’s promoter region or the transcriptional start site (TSS) to result in activation.3,5 Designing functional gRNAs can be challenging, as TSSs are not always well annotated or may be inaccessible due to presence of other protein factors, and cryptic or alternative promoters may be utilized. Edit-R CRISPRa guide RNA designs come from a published CRISPRa v2 algorithm based on machine learning8. The algorithm used FANTOM and Ensembl databases to more accurately predict the TSSs and incorporates chromatin, position, and sequence features to accurately predict highly effective guide RNAs. Some genes (6.8%) were identified as having alternative transcriptional start sites. For a small number of genes (0.1%) more than two TSS were identified. The publication lists 10 gRNA designs per TSS, ranked in order of predicted functionality.
Similar to studies published using expressed sgRNAs2,9, we routinely see either increased gene activation when using a pool of crRNA (Figure 2A) or gene activation that is equivalent to the most functional individual crRNA (Figure 2B). Additionally, we examined the level of overlap between the published algorithm designed guide RNAs and found that for majority of genes (> 70%) there is either no overlap or only minimal overlap of the guide RNA target sites. For these genes, we typically see an increase of the level of activation when pooling the gRNAs. Only for a small subset of genes (about 12%) there is an overlap of all 4 designs at the TSS, for which we find that the pool typically works as well as the most effective gRNA. Overall, pooling is a good strategy if maximal gene activation from a single guide RNA reagent 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.
Figure 2. 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 (25 nM) targeting EGFR (A) or POU5F1 (B). The pre-designed crRNAs were used either individually or pooled (to a total concentration of 25 nM). 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 ∆∆Cq method using GAPDH as the housekeeping gene and normalized to a non-targeting control.
There are many methods to confirm gene activation including RT-qPCR, western blots or immunofluorescence analysis. Typically, the fastest and easiest method to measure the relative change in target gene expression levels between samples treated with a non-targeting control and CRISPRa guide RNAs is RT-qPCR. RT-qPCR analysis can be completed with either the SYBR green method or probe-based gene expression assays. One thing to note when performing RT-qPCR for gene activation is the gene expression level may be undetectable before CRISPRa. In this case, when using the ∆∆Cq method of analysis, an arbitrary value representing the detection limit of the qPCR instrument is used as a place holder for "non-detectable" as a non-zero value is necessary to perform the calculation. In most cases this value will be between 35 and 40 depending on the number of programmed cycles and the instrument Cq determination method. It is recommended to add additional cycles (up to 45 total) to standard qPCR cycling conditions.
Figure 3. A. Human iPSC (Thermo Scientific Cat #A18945) were nucleofected with dCas9-VPR and sgRNA expression vectors according to manufactures protocols using 96-well shuttle solution P3. B. U2OS cells stably expressing integrated dCas9-VPR were plated at 10,000 cells/well and transfected with synthetic crRNA:tracrRNA positive control #1 targeting POU5F1 (25 µM) using DharmaFECT 4 Transfection Reagent (0.15 µL/ well in a 96 well plate). For both experiments (A. and B.) cells were harvested 72 hours post-transfection 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.
An important consideration when designing CRISPRa experiments is that the level of gene activation correlates with the basal target expression level of each respective cell type. For genes that have low basal expression, higher activation (100-10,000-fold) is observed whereas for genes that are moderate to high expressed have lower fold activation (generally less than 100-fold). Before setting out to perform CRISPRa it is worthwhile to determine the basal expression level for your cell line of interest to set expectations on the activation you can achieve. For example, the gene POU5F1 encodes for a transcription factor (OCT4) that is known to be highly expressed in pluripotent cells like iPSCs but is typically low expressed when cells are differentiated. POU5F1 is one of the genes that we recommend as a positive control, but this would not be a good choice when working with stem cells. Here we show relative gene activation of POU5F1 in iPSCs (Figure 3A) and a U2OS-dCas9-VPR integrated cell population (a bone osteosarcoma cell line) (Figure 3B). As anticipated, we see low transcriptional activation of POU5F1 in the iPSCs (~1.2 fold) but high transcriptional activation in the bone osteosarcoma cells (~600 fold).
There are many options and considerations for CRISPRa experimental conditions. Generally, the most robust gene activation is observed when working with a stable population of dCas9-VPR cells. For extended timepoint assays (more than 96 hours) lentiviral sgRNA is recommended. For short-term assays (less than 96 hours) synthetic crRNA:tracrRNA or plasmid sgRNA give equivalent gene activation. See Table 1 for more detailed component source options, delivery conditions, and recommendations.
The Edit-R predesigned CRISPRa guide RNAs represent the top four guide RNA designs from the Horlbeck et. al. v2 algorithm, and, when applicable, the top four guide RNAs for an alternative TSS. These are labeled as P1 and P2, respectively. If the Edit-R CRISPRa reagents for your gene have both P1 and P2 guide RNAs, it might be beneficial to test both for your experiment. If the CRISPRa guide RNAs for your gene do not have a P1 or P2 designation, then only a single start site is defined for that gene. For the small number of genes with more than two TSS identified, we only offer P1 and P2 as catalog items but can manufacture the guide RNAs as a custom request based on designs from the published algorithm8.
The Edit-R dCas9-VPR reagent is available as a purified plasmid or lentiviral particles in an expression vector with your choice of promoter.
To order CRISPRa predesigned S. pyogenes synthetic crRNAs or lentiviral sgRNAs for human or mouse protein coding genes you can start with the gene search on the Dharmacon website. After you select your gene and the crRNA/sgRNA product type, simply add to your cart. You will also find Edit-R guide RNAs for cleavage and knockout in the list of products, so be sure to select the items that are labeled “CRISPRa!”
There are multiple Edit-R CRISPRa guide RNA formats to choose from:
Offering coverage of the human and mouse genomes, these synthetic RNAs are modified for nuclease resistance and available as pooled or individual crRNA reagents.
Lentiviral particles or glycerol stocks of vector-based single guide RNA are predesigned for genome-wide coverage of human and mouse genes
Lentiviral particles or purified plasmid that express nuclease-deactivated Cas9 fused to transcriptional activators. When complexed with a guide RNA will trigger an endogenous gene’s expression.
CRISPR-Cas9 for gene overexpression and down-regulation
Synthetic & lentiviral reagents for endogenous gene overexpression
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