Taming CRISPRi: Dynamic range tuning through guide RNA diversion

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.

Automated summary

CRISPRi is a powerful technique for gene repression, but it can cause unintended effects in inducible systems. This study evaluated three methods to enhance the control of CRISPRi. Mismatches in the guide RNA sequence, decoy target sites, and feedback control were found to improve the controllability, linearity, and dynamic range of CRISPRi. These techniques enable fine-tuning of CRISPRi for specific applications.