The study presents chemically controlled and CRISPRi circuits designed to reduce leakiness while preserving strong induced activity in cultured cells.
Evidence
This platform experiment tested Tet-inducible, Branaplam-regulated, and dual-control CRISPR modules across multiple cell lines and targets.
Caveat
The evidence is from engineered cell-system performance, not organism-level disease modeling or clinical use.
Simplified
technologies provide unique capabilities for modeling disease and understanding gene-to-phenotype connections. In cultured cells, chemical-mediated control of Cas9 activity can limit off-target effects and enable mechanistic study of essential genes. However, widely-used Tet-On systems often show leaky Cas9 expression, leading to unintended edits, as well as weak activity upon induction. Leakiness can be problematic in the context of Cas9 nuclease activity, which may result in cumulative DNA damage and degradation of the target cell genome over time. To overcome these deficiencies, we have established transgenic platforms that minimize Cas9 functionality in the OFF-state along with maximized and uncompromised ON-state gene editing efficiency. By combining conditional destabilization and inhibition of Cas9, we have developed an all-in-one (one or multiple guide RNAs and Cas9) ultra-tight, Tet-inducible system with exceptional dynamic range (ON vs. OFF-state) across various cell lines and targets. As an alternative to Tet-mediated induction, we have created a Branaplam-regulated splice switch module for low-baseline and robust Cas9 activity control. Lastly, for circumstances where DNA damage needs to be avoided, we have constructed a dual-control, Tet-inducible CRISPRi module for tight and potent transcriptional silencing. This upgraded suite of inducible CRISPR systems has broad applications for numerous cell types and experimental conditions.
Key numbers
100×
Dynamic Range of Editing
Comparison of ON vs. OFF state activity for CD81 depletion.
~78%
Knockout Efficiency in KOLF2.1J Cells
Measured after treatment with Dox and Shield1.
Full Text
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CRISPR Gene Editing