CRISPR NAND Gate: rescuing a cis-repressed gRNA using antisense sRNA

The ability to build multi-input, multi-state devices from the wide range of sensors and actuating genetic circuits that are currently available is, at present, hindered by the lack of available regulatory networks. Here we present an approach for building a NAND gate (a functionally complete gate) through the design of a cis-repressed guide RNA (crgRNA). This is a gRNA which is inactive when expressed alone due to the inclusion of a cis repressing element which complements the “spacer” or DNA binding region of the gRNA. To rescue the crgRNA’s function (repression of a downstream gene through dCas9 binding), an antisense sRNA is expressed which complements the cis-repressing element. This releases the active gRNA to form its native structure and repress expression. Binding of the asRNA to the crgRNA forms a NAND gate as expression is only repressed in the presence of both the crgRNA and the antisense RNA. The library of variants presented include placing the cis-repressing element either at the 5’ or 3’ end of the crgRNA and placing a variable number of bulges in the cis repressing element to destabilise the inactive structure. This has the potential to form complex networks as the antisense crgRNA pairs can be targeted to a range of promoters expressing other antisense RNAs or crgRNAs as well as actuating genes.