Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting

The advent of targeted genomic and transcriptomic editing technologies has shown great promise for cellular engineering. In particular, RNA-guided RNA cutting by Cas13d has emerged as a promising tool for engineering cellular transcriptomes for biotechnological and therapeutic applications. However, off-target cutting by Cas13d (so-called "collateral damage") has limited the use of this system. Moreover, it is unclear how this collateral damage is affected by levels of Cas13d protein and guide RNA. To address this, we developed expression-tuning gene circuits to change these levels in a precise and controlled manner. We found that both specific and nonspecific RNA cutting depended on Cas13d and guide RNA levels, and that nonspecific RNA cutting might contribute to target RNA reduction. To optimize this system, we developed new Multi-Level Optimized Negative-Autoregulated Cas13d and crRNA Hybrid (MONARCH) gene circuits that can achieve a high dynamic range of cutting while minimizing basal on-target RNA reduction and nonspecific RNA cutting. Furthermore, we developed mathematical models to investigate how these advantages arise. Thus, our work allows for a better understanding of how collateral activity relies on Cas13d and crRNA levels, providing practical solutions for fine-tuned RNA downregulation.