CRISPR transcriptional repression devices and layered circuits in mammalian cells

Engineered biological circuits offer insights into underlying biology of living cells and potential solutions to a range of medical and industrial challenges. To date, however, an impediment to engineering larger and more complex circuits in any living organism is the lack of an efficient framework for generating a sufficient number of composable devices. Recently, the Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms. CRISPR system provides new promises for generation of the transcription frameworks for diverse device library as it can be easily programmed by altering only the guide RNA (gRNA) sequence that complexes with Cas9 and targets the complementary DNA sequences. Therefore, we generated CRISPR-based devices in human cells that operate by targeting catalytically inactive Cas9(Ca9sm) and gRNA to a specifically designed hybrid promoter architecture that we call CRISPR responsive promoters (CRP). CRPs contain CRISPR target sites flanking mini-CMV promoter and upstream Gal4VP16 target sites allowing both activation by Gal4VP16 and repression by CRISPR targeting. In the presence of Cas9m and gRNAs complementary to target sites at CRP, the recruitment of the complex to CRP, represses an enhanced yellow fluorescent protein (EYFP) expression by steric blocking of the assembly of initiation complex. Examining such devices in human embryonic kidney (HEK) cells demonstrated up to 100-fold repression of EYFP output. Next, we examined the ability to create layered CRISPR-based circuitry in human cells. For such purpose, we rendered U6 promoters that drive gRNA expression repressible by CRISPR system and call the new promoter (CRU6). We then created a cascaded circuitry by connecting two of such devices. First, a gRNA-b will be expressed from a CRa-U6 promoter. Subsequently, the Cas9m and gRNA-b recruitment to CRP-b promoter represses EYFP expression. In the presence of an upstream device that contains a gRNA-a expressed from U6 promoter, targeting of Cas9m-gRNA-a to CRa-U6 promoter represses gRNAb expression and removes the repression on EYFP expression. By examining such circuit in HEK293 cells, we could achieve a functional cascade and derepression in EYFP output. In summary, we provide the first evidence that Cas9 system can be layered to generate circuitry of interconnected devices in human cells and propose a novel architecture to generate a large library of transcriptional devices for such purposes.