Programmable CRISPR-Cas Repression, Activation, and Computation with Sequence-Independent Targets and Triggers

The programmability of CRISPR-derived Cas9 as a sequence-specific DNA-targeting protein has made it a powerful tool for genomic manipulation in biological research and translational applications. Cas9 activity can be programmably engineered to respond to nucleic acids, but these efforts have focused primarily on single-input control of Cas9, and until recently, they were limited by sequence dependence between parts of the guide RNA and the sequence to be detected. Here, we not only design and present DNA- and RNA-sensing conditional guide RNA (cgRNA) that have no such sequence constraints, but also demonstrate a complete set of logical computations using these designs on DNA and RNA sequence inputs, including AND, OR, NAND, and NOR. The development of sequence-independent nucleic acid-sensing CRISPR-Cas9 systems with multi-input logic computation capabilities could lead to improved genome engineering and regulation as well as the construction of synthetic circuits with broader functionality.