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Updated: Jul 24, 2025

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines
Published on: June 2, 2018
Taming CRISPRi: Dynamic range tuning through guide RNA diversion
Bob Van Hove1, Lien De Wannemaeker2, Isolde Missiaen2
1Centre for Synthetic Biology, Ghent University, Coupure links 653, 9000 Ghent, Belgium; Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
Abstract:
CRISPRi is a powerful technique to repress gene expression in a targeted and highly efficient manner. However, this potency is a double-edged sword in inducible systems, as even leaky expression of guide RNA results in a repression phenotype, complicating applications such as dynamic metabolic engineering. We evaluated three methods to enhance the controllability of CRISPRi by modulating the level of free and DNA-bound guide RNA complexes. Overall repression can be attenuated through rationally designed mismatches in the reversibility determining region of the guide RNA sequence; decoy target sites can selectively modulate repression at low levels of induction; and the implementation of feedback control not only enhances the linearity of induction, but broadens the dynamic range of the output as well. Furthermore, feedback control significantly enhances the recovery rate after induction is removed. Used in combination, these techniques enable the fine-tuning of CRISPRi to meet restrictions imposed by the target and match the input signal required for induction.
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