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Technology

The inventors have developed a novel, non-toxic dCas9 complex that enables construction of complex biological circuits in bacterial cells. To decrease non-specific binding, and thus reduce toxicity, the inventors have engineered a mutant variant of dCas9 that can no longer recognize the PAM sequence, yet still retains DNA binding capabilities. This Cas9 variant is fused to a transcriptional repression factor, Ph1F, using a linker peptide. Thus, when Ph1F-binding DNA sequences are placed in front of cognate promoters, the fused dCas9 complex can effectively and specifically repress the expression of target genes. Approximately 104 copies of fused dCas9 protein can be expressed in a cell without compromising cellular health. Furthermore, fusion of dCas9 to Ph1F improved cooperativity compared to the existing dCas9 system, which is critical for building intricate gene circuits.