CRISPR-Cas9 systems are best known for gene editing applications, but non-cutting CRISPR versions now modulate gene expression (CRISPR modulation or CRISPRmod).
When deactivated Cas9 is fused to transcriptional activators and directed by the guide RNA in a sequence-specific manner to promoter regions, it can lead to target gene activation (CRISPRa). Multiple CRISPRa systems exist and are successful components of gain-of-function studies in a pooled screening format. However, many biologically relevant phenotypes cannot be quickly sorted, making arrayed screens a better approach.
Synthetic guide RNAs have distinct advantages for gene-function analysis
Horizon scientists developed synthetic guide RNA, chemically modified with two 2'-O-methyl nucleotides and phosphorothioate backbone linkages (2xMS) at the 5’ end of the crRNA and 3’ end of the tracrRNA. These guides work well with second-generation CRISPRa systems: the VPR system that uses a canonical tracrRNA or SAM system that utilizes tracrRNA containing MS2 aptamer sequences. These synthetic guide RNAs show similar or better activation levels compared to control expression vectors. Multiple synthetic gRNAs targeting a gene’s promoter region act in concert with dCas9 activators to enhance target gene activation. Furthermore, synthetic guide RNAs are well-suited for multiplexing. Multiple genes can be activated in the same cells, making these reagents an excellent choice for pathway analysis or cellular network discovery.
Synthetic guide RNA results in robust transcriptional activation with the VPR and SAM CRISPRa systems
(Figures are adapted from Horizon's publication "CRISPR-mediated transcriptional activation with synthetic guide RNA." in The Journal of Biotechnology.) The protein component of the dCas9-VPR CRISPRa system consists of a catalytically inactivated Cas9 (dCas9) fused with transcriptional activators, VP64, p65 and Rta (VPR). A canonical guide RNA, shown as crRNA:tracrRNA, binds genomic DNA upstream of the protospacer adjacent motif (PAM) to bring activators upstream of the transcriptional start site (TSS), initiating gene activation. MS modifications are indicated with asterisks. The synergistic activation mediator (SAM) consists of three components: (1) a catalytically inactivated Cas9 (dCas9) fused with transcriptional activator, VP64, (2) an MS2-p65-HSF1 fusion of transcriptional activators, and (3) a guide RNA containing an MS2 aptamer on stem-loop 2 of the tracrRNA. The guide RNA with MS2 aptamer, shown as crRNA:SAM tracrRNA, binds the dCas9-VP64 and the aptamer binds the MS2-p65-HSF1 construct; this complex containing the combination of these activators binds genomic DNA upstream of the proto-spacer-adjacent motif (PAM) in the transcriptional start site (TSS) region to activate the target gene. U2OS-dCas9-SAM (gray bars) and U2OS-dCas9-VPR (black bars) cells were transfected with pooled, synthetic crRNA:SAM tracrRNA and crRNA:tracrRNA, respectively. Transcriptional fold activation was measured 72 hours post-transfection using RT-qPCR and normalized to NTC.
Delivery of dCas9-VPR mRNA and synthetic guide RNAs for transient gene activation
dCas9-VPR mRNA enables temporary delivery of the activator with the synthetic guide RNAs where viral transduction is not feasible. This method can further be coupled with the selection of the cells containing the dCas9-VPR mRNA, either through a puromycin selection marker or EGFP reporter to enrich for activation.
Synthetic guide RNA can be used with dCas9-VPR mRNA for gene activation. Workflow for CRISPRa using dCas9-VPR mRNA with synthetic guide RNAs. U2OS cells were co-transfected with dCas9-VPR-EGFP mRNA and crRNA:tracrRNA targeting IL1R2. Cells were sorted 24 hours post-transfection into the following categories: Negative, Dim, or Top 10%. Transcriptional activation was measured using RT-qPCR. U2OS cells were co-transfected with dCas9-VPR-puromycin mRNA and crRNA:tracrRNA targeting IL-1R2, TFAP2C and TTN. 24 hours post-transfection, cells were selected with puromycin for 24 hours, and transcriptional activation was measured.
Proof-of principle arrayed CRISPRa screen
Synthetic guide RNA can be placed in multi-well plates with uniform cell delivery either by lipid transfection or electroporation. In dCas9-VPR integrated cells, synthetic RNA guide can cause significant CRISPR gene activation as early as 24 hours after delivery, peaking at 48 to 72 hours and persisting even at five days. This time frame of two-five days, without the need to split cells, is relevant for many standard cell-based assays and allows for automation and application in arrayed cell screening, such as in the case of conventional siRNA screens.
Our scientists have performed proof-of-principle arrayed screens using CRISPRa crRNA libraries to upregulate the expression of 153 cytokine receptors and identified positive and negative regulators of IL-6 cytokine secretion.
CRISPRa arrayed screen with synthetic crRNA identified IL-6 regulatorsU2OS-dCas9-VPR cells were transfected with the CRISPRa library.153 cytokine receptor targets in an arrayed fashion, each consisting of a pool of four predesigned synthetic crRNAs targeting each gene. Cell supernatant was harvested 72 hours post-transfection, and IL-6 was measured by ELISA assay. Fold change, normalized to NTC, of secreted IL-6 for the cytokine receptor library screen is presented from high to low and colored based on the p-value (positive regulators as red and negative regulators as blue).
The use of synthetic guide RNA allows for CRISPRa to be used in high throughput, arrayed screening applications where more complex phenotypic readouts can be used to complement viability and drug resistance assays typically used in a pooled screening format.
Written by Dr. Žaklina Strezoska
Žaklina Strezoska is the Director of Biology R&D at Horizon Discovery in Lafayette, Colorado. She leads a team of experienced molecular and cellular biologists and oversees projects focused on innovation and new product development for gene modulation and genome engineering. She received her Ph.D degree in Molecular Genetics at the University of Illinois at Chicago and completed her postdoctoral research at Brown University.
Read the full publication in The Journal of Biotechnology
CRISPR-mediated transcriptional activation with synthetic guide RNA. Strezoska, Ž., et al. J. Biotechnology. 319: 25-35, 2020. doi: 10.1016/j.jbiotec.2020.05.005
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