Stricter Precision: Enhanced Dual-Level Control of Inducible Cas9 Systems for Regulating Functional Gene Expression

CRISPR-Cas9 has been widely adapted for use in transcriptional modulation and epigenetic engineering with deactivated Cas9 (dCas9) 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 CRISPR-mediated transcriptional modulation. Here we describe a new small molecule-inducible system for potent, stringently regulated CRISPR activation, CRISPR interference and CRISPR knockout. First, we developed the Dharmacon™ Strict-R™ CRISPRa dCas9-VPR lentiviral system. Here, the dCas9-VPR CRISPRa effector is fused to an FKBP12-derived destabilizing domain and expressed from a Tet-inducible promoter to prevent transcription and stable translation of dCas9-VPR in the absence of the doxycycline and Shield1 ligand.  We show that in combination, the Tet-On system and the FKBP12-derived destabilizing domain minimize basal target gene activation (leakiness) while maintaining robust induction across gene targets and cell types. The inclusion of the destabilizing domain enables a degree of temporal control that is not possible with the Tet-On inducible system alone. We then demonstrate how this dual regulated dCas9-VPR effector can be applied in human induced pluripotent stem cells (hiPSCs) to overexpress key proneural factors. Next, we applied similar strategies to engineer the Strict-R inducible CRISPR interference system for precise and tunable gene repression, and the Strict-R inducible CRISPR knockout system for temporally controlled gene knockout. These Revvity technologies provide researchers with validated reagents for tightly controlled expression of various CRISPR systems, enabling precise investigation of time-dependent gene pathways that will accelerate discoveries in developmental biology, improve disease models, and contribute to therapeutic applications.  

Learning Objectives:

1. Inclusion of the FKBP12 destabilizing domain provides exquisite temporal control over CRISPR systems.

2. Rapid induction with highly cell-permeable small molecules across a wide range of cell models.

3. Dual transcriptional and post-translational control greatly reduces leakiness of the Tet-inducible system without compromising potency of CRISPR perturbation.