Optimizing a CRISPR-Cas13d gene circuit for tunable target RNA downregulation with minimal collateral RNA cutting

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 damage, 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 target RNA reduction depends sensitively on both Cas13d and guide RNA levels, part of which might result from collateral damage of Cas13d complexes activated by target cutting. Using RNA-level control techniques, we develop new Multi-Level Optimized Negative-Autoregulated Cas13d and crRNA Hybrid (MONARCH) gene circuits that achieve a high dynamic range of target reduction while minimizing basal target cutting and collateral damage in human kidney cells and green monkey cells most frequently used in human virology. MONARCH systems should bring RNA-guided RNA cutting systems to the forefront, as easily applicable, programmable tools for transcriptome engineering in biotechnological and medical applications. ### Competing Interest Statement The authors have declared no competing interest.