CrRNA:tracrRNA-based binary logic gate design as a tool for synthetic biology

A logic-gate-based system for modifying gene expression or modifying a genome, the system comprising:

• (A) at least one orthogonal pair of hybridizing synthetic nucleic acid constructs, each of the at least one orthogonal pair comprising:
• (i) a CRISPR nucleic acid comprising a repeat sequence and at least one spacer sequence having substantial complementarity to a target nucleic acid, the CRISPR nucleic acid operably linked to a first promoter;
• and
• (ii) a trans-activating CRISPR (tracr) nucleic acid comprising a sequence that is complementary to the repeat sequence of the CRISPR nucleic acid of the same orthogonal pair of hybridizing synthetic nucleic acid constructs, the tracr nucleic acid operably linked to a second promoter, which when both (i) and (ii) are expressed in a cell, a synthetic CRISPR nucleic acid-tracr nucleic acid (crRNA-tracrRNA) hybrid is formed having a secondary structure comprising a lower stem, and optionally, an upper stem and which hybrid forms a complex with a Type II or Type V CRISPR-Cas effector protein, and
• (B) at least one nucleic acid operably linked to a third promoter and encoding a Type II or Type V CRISPR-Cas effector protein, that forms a complex with at least one synthetic CRISPR nucleic acid-tracr nucleic acid hybrid formed by the at least one orthogonal pair of hybridizing nucleic acid constructs;
• wherein the first promoter, second promoter, and third promoter are different promoters, and
• wherein the CRISPR nucleic acid of each orthogonal pair of hybridizing synthetic nucleic acid constructs and a tracr nucleic acid of any other of the at least one orthogonal pairs of hybridizing synthetic nucleic acid constructs comprise at least one non-natural mismatch between the repeat sequence of the CRISPR nucleic acid of each orthogonal pair of hybridizing synthetic nucleic acid constructs and the sequence that is complementary to the repeat sequence of the CRISPR nucleic acid of a tracr nucleic acid of any other of the at least one orthogonal pair of hybridizing synthetic nucleic acid.