Strict-R temporal control of CRISPR systems

Enhanced dual-level control of inducible CRISPR systems for regulating functional gene expression

CRISPR knockout using Cas9 is a powerful approach for disrupting gene function through targeted DNA double-strand breaks. Furthermore, CRISPR-Cas9 has been widely adapted for use in genetic engineering and transcriptional modulation using either wild-type Cas9 or deactivated Cas9 (dCas9) fusion systems to enable CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) applications. However, few of these systems provide researchers with the ability to control the timing of knockout or CRISPR-mediated transcriptional modulation. Strict-R is a new small molecule-inducible system for potent and stringently regulated CRISPR activation, CRISPR interference and CRISPR knockout.

Development of the Strict-R inducible CRISPR systems

Dual-layered control for precise and leak-free gene regulation

CRISPR knockout (CRISPRko) enables precise, permanent disruption of gene function through targeted DNA cleavage, while CRISPR modulation uses a catalytically inactive dCas9 to regulate gene expression without altering the DNA sequence. By using dCas9 fused to transcriptional activators or repressors, researchers can selectively up- or down-regulate target genes for applications in functional genomics, pathway mapping, and therapeutic research.

While tetracycline (Tet)-regulated systems provide inducible expression, they often suffer from leakiness in the OFF state, complicating interpretation of results. To overcome this limitation, we engineered a dual-layer regulatory mechanism that combines Tet-controlled transcription with post-translational regulation via an FKBP12-derived destabilizing domain (DD).

In the Strict-R inducible CRISPR systems, transcription of the transgene is repressed and the transgene protein is unstable in the absence of doxycycline and the Shield1 ligand. Only when both small molecules are present robust, immediate, and temporal/inducible expression of Cas9 or dCas9 fusion proteins is induced. This dual control dramatically reduces background activity while maintaining full activity, ensuring stringent control of ON and OFF state across:

• Strict-R Inducible CRISPRa Lentiviral System: Inducible CRISPR activation using dCas9-VPR
• Strict-R Inducible CRISPRi Lentiviral System: Inducible CRISPR interference using dCas9-SALL1-SDS3
• Strict-R Inducible Cas9 Lentiviral System: Inducible CRISPR knockout using Cas9 nuclease

Across multiple cell lines and targets, the Strict-R platform demonstrates potent, tunable gene modulation with minimal basal leakiness, delivering precise temporal control for advanced functional genomics studies.

Transcriptional and post-translational control of Cas9 effectors with the Dharmacon™ Strict-R™ Inducible CRISPR lentiviral system

Diagram of the Strict-R™ Inducible CRISPR lentiviral systems regulating expression utilizing both transcriptional and post-translational control. In the absence of doxycycline and Shield1, the system is “OFF”. Leaky bursts of transcription from the TRE3G promoter result in the translation of Cas9 or dCas9 effector fused to a FKBP12-derived destabilizing domain (degron) that tags the protein for rapid proteasomal degradation. The addition of doxycycline induces potent transcription from the TRE3G promoter and the addition of Shield1 stabilizes the Cas or dCas effector leading to gene activation/repression or knockout depending on the Strict-R technology employed.

Dharmacon™ Strict-R™ Inducible CRISPR Lentiviral System Workflow

Schematic overview of the Strict-R inducible CRISPR system workflow. Cells are first transduced with the Strict-R CRISPR lentiviral construct, followed by transduction of the guide RNA. Upon induction with doxycycline and Shield1, the system enables targeted gene activation or repression or knockout, allowing for subsequent phenotypic analysis. Table 1 summarizes the available selection markers corresponding to each variant of the Strict-R CRISPR system.