A CRISPR/Cas system and method for editing or regulating transcription of a genome of a cell are provided, wherein the system includes a Cas endonuclease fused with one or more degron sequences and at least one activatable cognate single guide RNA harboring an inactivation sequence in a non-essential region of the cognate sgRNA, wherein said inactivation sequence comprises one or more endonuclease recognition sites of, e.g., a ribozyme.

The present disclosure is directed to polycistronic guide RNAs, DNA encoding polycistronic gRNA, multiplex CRISPR vectors, a plurality of component DNA fragments for assembly into a DNA encoding a polycistronic gRNA array, a plurality of primer pairs for making a plurality of component DNA fragments to be assembled into a DNA encoding a polycistronic gRNA, and methods of making multiplex CRISPR vectors. The current disclosure is directed to multiplexed CRISPR technologies that have great potential for pathway engineering and genome editing. In the current disclosure describes efficient assembly of tRNA/Csy4/Ribozyme-based gRNA arrays which can be produced in a quick and effective process.

A method for the production and use of scalable co-transcriptional RNA strand displacement (ctRSD) circuits using RNA toehold exchange gates is described. The ctRSD circuits described address the limitations of existing DNA-based strand displacement circuits by isothermally producing circuit components via transcription.