DyadGuide dual-sgRNA; advancing multiplex CRISPR versatility and performance

CRISPR is a widely used, programmable genome-editing system in which a single guide RNA (sgRNA) directs Cas9 nuclease activity to a complementary DNA sequence, enabling targeted cleavage and introducing repair-driven modifications (e.g., indels, knock-ins, or transcriptional modulation using dCas-effector variants).

Multiple delivery modalities exist for CRISPR components. Cas9/dCas9-effectors can be delivered as a protein (Cas9 only), mRNA, or via lentiviral vectors for stable genomic integration. Likewise, sgRNAs can be introduced through lentiviral expression systems or as synthetic RNAs. Synthetic sgRNAs offer advantages for multiplexed applications, as they can be readily pooled. However, this approach is transient and therefore not ideal for sustained gene perturbation, and delivery to difficult-to-transfect cell models can be challenging. In contrast, expression-based systems rely on transcription of individual sgRNA with an RNA polymerase III (Pol III) promoter, most commonly the U6 promoter, allowing for long-term and stable expression of the sgRNA.

More recently, expression systems containing consecutive Pol III promoters, each driving an individual sgRNA, have been developed to express two or more sgRNAs. However, these approaches focus exclusively on sgRNA delivery and require Cas9 to be introduced separately. In addition, we and others have observed that sgRNAs in these systems are not always expressed or functional at equivalent levels, resulting in inconsistent gene modulation or editing outcomes.

To address these limitations, the Dharmacon™ Reagents team has developed a novel expression system, incorporating an engineered bidirectional promoter that enables balanced expression of two sgRNAs. This design supports consistent and uniform gene modulation or editing, regardless of strand each sgRNA is expressed from. Furthermore, the compact architecture of this system allows it to be packaged together with Cas9 or dCas9 effectors into a single All-in-one vector eliminating the need for separate Cas9 delivery or expression.

Robust gene modulation and editing with the DyadGuide™ dual-sgRNA platform

The DyadGuide dual-sgRNA platform employs an engineered bidirectional U6 promoter to drive efficient and uniform expression of two sgRNAs (Figure 1A). This design enables more consistent gene modulation or editing compared with conventional sequential Pol III promoter expression strategies.

When we compared gene repression using several sequential Pol III promoter strategies against the DyadGuide bidirectional promoter in a CRISPRi All-in-one system, sequential promoter designs produced inconsistent and less potent repression. In contrast, the DyadGuide bidirectional promoter delivered robust knock-down that was independent of sgRNA orientation (Figure 1B). Importantly, this improved performance is not limited to CRISPR modulation with CRISPRi or CRISPRa (not shown). We observed the same advantage in CRISPR knock-out (CRISPRko) experiments: sequential Pol III promoter systems again showed variable efficiency, whereas DyadGuide maintained consistent and efficacious gene disruption across sgRNA orientations. Together, these results demonstrate that the DyadGuide dual-sgRNA system delivers consistently superior performance across CRISPR applications, enabling reliable and position-independent gene modulation.

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Figure 1. The DyadGuide dual-sgRNA expression platform outperforms consecutive Pol III promoters.
A. The DyadGuide dual-sgRNA platform utilizes a bidirectional U6 promoter to drive efficient and uniform expression of both sgRNAs, overcoming limitations seen when sgRNAs are expressed by multiple sequential promoters. B. U2OS (10,000 cells/well), were plated in 96-well plates, allowed to adhere for 24 hours, and transduced with CRISPRmod CRISPRi All-in-one Lentiviral DyadGuide™ dual-sgRNA particles containing sgRNAs targeting both PPIB and SEL1L, All-in-one vectors containing sequentialU6 promoters (combinations of Human U6 (hU6) and mouse U6 (mU6) or hU6 and bovine U6 (bU6)) driving expression of sgRNAs targeting both PPIB and SEL1L, or CRISPRi All-in-one lentiviral particles containing sgRNAs targeting SEL1L at MOIs of 0.3. CRISPRmod CRISPRi All-in-one Lentiviral DyadGuide dual-sgRNA particles containing two non-target control (NTC) sgRNAs were used as a control. 24 hours post-transduction, cells were selected with 2.5 µg/µL puromycin for 5 days. 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 ACTB as the housekeeping gene and normalized to an NTC. Robust target gene repression of both targets was observed with the CRISPRmod CRISPRi All-in-one Lentiviral DyadGuide dual-sgRNA system, comparable to levels achieved by the single sgRNA CRISPRmod CRISPRi All-in-one lentiviral system. However, in cells transduced with sequential U6 promoter lentiviral particles, lower knock-down efficiencies for the sgRNA driven by the second U6 promoter were observed, particularly noticeable when the sgRNA targeted PPIB.

Synergistically enhancement of gene modulation

As shown above, the DyadGuide dual-sgRNA system enables researchers to simultaneously target two different genes for more complex combinatorial studies, but also enables the targeting of a single gene with two distinct guide RNAs to enhance gene modulation. Previous work demonstrated that gene activation with CRISPRa can be amplified using multiple sgRNAs directed at the same locus.

To assess this, we compared targeting the POU5F1 transcription start site with an individual sgRNA delivered with our CRISPRa All-in-one system or with two sgRNAs using DyadGuide dual-sgRNA. While each individual sgRNA targeting POU5F1 increased expression of POU5F1 mRNA (Figure 2A) and its encoded protein OCT4 (Figure 2B), the DyadGuide CRISPRa dual-sgRNA approach further enhanced POU5F1 expression and resulted in higher OCT4 protein levels in U2OS cells.

These results demonstrate that the DyadGuide dual-sgRNA platform can synergistically enhance gene activation and protein expression, highlighting its effectiveness for both single-gene and multi-gene targeting.

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Figure 2. The DyadGuide dual-sgRNA expression platform to express two sgRNAs targeting the same gene can synergistically enhance gene activation.
U2OS (10,000 cells/well) cells were plated in 96-well plates, allowed to adhere for >4 hours, and transduced with CRISPRmod CRISPRa All-in-one Lentiviral DyadGuide™ dual-sgRNA particles containing two sgRNAs targeting POU5F1 or CRISPRmod CRISPRa All-in-one lentiviral particles containing a single sgRNA targeting POU5F1 at MOIs of 0.3. CRISPRmod CRISPRa All-in-one Lentiviral DyadGuide dual-sgRNA particles containing two non-target control (NTC) sgRNAs were used as a control. 24 hours post-transduction, cells were selected with 2.5 µg/µL puromycin for 5 days. A. Total RNA was isolated at Day 8 and relative gene expression was measured using RT-qPCR. The relative expression of each gene was calculated with the ∆∆Cq method using ACTB as the housekeeping gene and normalized to an NTC. Robust, synergistic POU5F1 gene activation was observed with the CRISPRmod CRISPRa All-in-one Lentiviral DyadGuide dual-sgRNA system, greatly exceeding the levels achieved by the single sgRNA CRISPRmod CRISPRa All-in-one lentiviral system. B. Replicate wells were fixed in 4% PFA and stained with OCT4 (encoded by POU5F1) antibody (STEMCELL) and counterstained with Hoechst. Similar to the mRNA levels, OCT4 protein levels were much stronger in wells transduced with the CRISPRmod CRISPRa All-in-one Lentiviral DyadGuide dual-sgRNA system driving two sgRNAs against POU5F1 than in the wells transduced with the single sgRNA CRISPRmod CRISPRa All-in-one lentiviral system.

Complex pathway analysis with the Strict-R inducible Cas9 Lentiviral system

Advanced pathway analysis often necessitates simultaneous knock out of two genes within the same cellular context. However, dual target knock-out may produce strong phenotypes that lead to growth disadvantages that result in difficulty propagating the cell line after transduction and antibiotic selection. Thus, some studies may benefit from combining the Lentiviral DyadGuide dual-sgRNA system, which enables efficient multiplex gene targeting, with the tightly regulated Strict-R™ inducible CRISPR system, which provides precise temporal control for complex functional studies.

To demonstrate this capability, we generated a Strict-R inducible Cas9 Hs936.T stable cell line and then introduced the DyadGuide system with sgRNAs targeting both MAP2K1 and MAP2K2, alongside corresponding individual sgRNA controls (Figure 3A). The sgRNAs in this experiment were designed using the Edit-R™ design algorithm, which has shown to produce robust protein knock-out with an individual sgRNA against a gene of interest.

Upon induction of Cas9 expression with doxycycline and Shield1, single sgRNAs targeting either MAP2K1 or MAP2K2 led to clear disruption of the respective protein, with no detectable effect in uninduced cells, confirming tight regulation of the Strict-R inducible system (Figure 3B). Importantly, cells expressing the DyadGuide dual-sgRNA construct showed simultaneous knock-out of both MEK1 and MEK2 proteins following Cas9 induction (Figure 3B). These results highlight the utility of the DyadGuide dual-sgRNA platform for combinatorial gene knock-out studies and its compatibility with Strict-R Inducible CRISPR platforms for controlled, time-resolved pathway analysis.

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Figure 3. Edit-R Lentiviral DyadGuide dual-sgRNA platform achieves inducible protein knock-out of two targets when combined with Strict-R Inducible Cas9 Lentiviral system.
A. Hs936.T (50,000 cells/well) cells were plated in 24-well plates, allowed to adhere for >4 hours, and transduced with Strict-R inducible Cas9 Lentiviral particles at an MOI of 0.3. After 24 hours, media was aspirated and replenished with fresh medium containing 200 µg/mL hygromycin B. Cells were selected for 14 days in hygromycin-containing medium to generate a cell line with stable integration. Strict-R inducible Cas9 Hs936.T cells (50,000 cells/well) were then plated in 24-well plates, allowed to adhere for >4 hours, and transduced with Edit-R Lentiviral DyadGuide dual-sgRNA particles containing sgRNAs targeting both MAP2K1 and MAP2K2 at MOIs or Edit-R lentiviral sgRNA particles targeting either MAP2K1 or MAP2K1. Edit-R Lentiviral DyadGuide dual-sgRNA particlescontaining two non-target control (NTC) sgRNAs were used as a control. 24 hours post-transduction, cells were selected with 2 µg/µL puromycin for 5 days. Once a stable cell line expressing both the Strict-R inducible Cas9 and the desired sgRNAs were obtained, cells were plated in 6 well plates at a density of 100,000 cells per well and either maintained in standard cell growth medium or cell growth medium supplemented with 500 ng/mL doxycycline (dox) and 500 nM Shield1 to induce Cas9 expression. Cells were induced for 96 hours, replenishing medium with or without dox and Shield1 after 48 hours. B. Cells were then scraped in PBS and lysed in RIPA buffer for Western blot analysis. 10 µg total protein was loaded per lane, and proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Nitrocellulose membranes were incubated with MEK1 (Thermo 13-3500, 1:500), MEK2 (Cell Signaling #9147, 1:500), and GAPDH (Thermo AM4300, 1:5,000) primary antibodies for 2 hours at room temperature, washed in TBS-T, and incubated with goat-anti-mouse (Thermo 31431, 1:10,000) or goat anti-rabbit (Thermo A16096, 1:10,000) HRP-conjugated secondary antibodies for 1 hour at room temperature, developed in ECL solution, and imaged on a Thermo iBright FL1500.

Unlock more powerful and complex CRISPR experiments with the DyadGuide dual-sgRNA platform.

By combining balanced, bidirectional sgRNA expression in a vector that is compatible with a CRISPR All-in-one design, the DyadGuide dual-sgRNA platform enables consistent gene modulation, robust knock-out, and seamless multi-targeting of one or two genes. Whether enhancing gene activation, achieving reliable dual knock-outs, or performing time-controlled studies with the Strict-R inducible systems, DyadGuide delivers the efficiency, flexibility, and reproducibility researchers need to accelerate discovery and confidently dissect complex biological pathways.

You can order the DyadGuide dual-sgRNA system here. For additional questions, guidance, or support, please contact our technical support team at technical.horizon@revvity.com.