Header and Body 3


This technology uses catalytically inactive CRISPR-dCas9 that is guided by gene specific sgRNAs to design genetic circuits. The technology includes five significant improvements to RNA-guided circuitry. The first improvement is the generation of point mutants of dCas9 that reduce cellular toxicity, which increases cellular growth and reduces dCas9 silencing. The next improvement includes strategies to introduce non-linearity to signal responses by using “decoy” DNA sites or sRNAs to degrade RNAs. This increases the robustness of the system by reducing noise. The third improvement is a map of nucleotide substitutions that can be made in sgRNAs without reducing function. This diversity of sgRNAs reduces the rate of homologous recombination when many sgRNAs are expressed. The fourth improvement is the use of a heterologous T7 RNA polymerase to drive circuit expression. The use of a heterologous polymerase means that the system can be rapidly used across many different organisms without significant adaptation. Finally, the inventors developed a software that automates the design of RNA-guided genetic circuits. The software uses simple user-defined inputs and outputs a wiring diagram, sgRNA sequences, and predictions of circuit performance.