Both nonspecific and specific RNA reduction is influenced by Cas13d and guide RNA levels.
Precise expression-tuning gene circuits reveal that RNA cutting by Cas13d is affected by the abundance of both Cas13d and guide RNA.
Nonspecific RNA cutting may contribute to the reduction of on-target RNA.
New gene circuits, termed , achieve a high dynamic range with minimal collateral activity.
MONARCH circuits show low basal on-target RNA reduction in human kidney and green monkey cells.
These findings suggest potential for RNA-guided RNA cutting systems in transcriptome engineering.
Simplified
The invention of RNA-guided DNA cutting systems has revolutionized biotechnology. More recently, RNA-guided RNA cutting by Cas13d entered the scene as a highly promising alternative to RNA interference to engineer cellular transcriptomes for biotechnological and therapeutic purposes. Unfortunately, "collateral damage" by indiscriminate off-target cutting tampered enthusiasm for these systems. Yet, how collateral activity, or even RNA target reduction depends on Cas13d and guide RNA abundance has remained unclear due to the lack of expression-tuning studies to address this question. Here we use precise expression-tuning gene circuits to show that both nonspecific and specific, on-target RNA reduction depend on Cas13d and guide RNA levels, and that nonspecific RNA cutting fromcleavage might contribute to on-target RNA reduction. Using RNA-level control techniques, we develop new() gene circuits that achieve a high dynamic range with low basal on-target RNA reduction while minimizing collateral activity in human kidney cells and green monkey cells most frequently used in human virology. MONARCH should bring RNA-guided RNA cutting systems to the forefront, as easily applicable, programmable tools for transcriptome engineering in biotechnological and medical applications. trans Multi-Level Optimized Negative-Autoregulated Cas13d and crRNA Hybrid
Key numbers
70%
Maximum Target RNA Reduction
Reduction achieved from the basal level in 2.0 cells.
3–5%
Basal Effect Reduction
Basal effect observed in 1.0 and 2.0 systems.
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