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Tunable, self-contained gene dosage control via proteolytic cleavage of CRISPR-Cas systems

Katz, N.; An, C.; Lee, Y.-J.; Tycko, J.; Zhang, M.; Kang, J.; Bintu, L.; Bassik, M. C.; Huang, W.-H.; Gao, X. J.

2024-10-09 synthetic biology
10.1101/2024.10.09.617463 bioRxiv
Show abstract

The ability to modulate gene expression through modular and universal genetic tools like CRISPR-Cas has greatly advanced gene therapy for therapeutics and basic science. Yet, the inherent stochasticity of delivery methods cause variation in target gene expression at the single-cell level, limiting their applicability in systems that require more precise expression. Thus, we implement a modular incoherent feedforward loop based on proteolytic cleavage of Cas to reduce gene expression variability against the variability of vector delivery. We target a genome-integrated marker and demonstrate dosage control of gene activation and repression, post-delivery tuning, and RNA-based compatibility of the system. To illustrate therapeutic relevance, we target the gene RAI1, the haploinsufficiency and triplosensitivity of which cause two autism-related syndromes. We demonstrate dosage-controlled gene activation for both human and mouse Rai1 via viral delivery to patient-derived cell lines and mouse cortical neurons. Overall, we established a robust dosage control circuit for uniform gene expression, beneficial for basic and translational research.

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