CRISPR-Cas type II-based Synthetic Biology applications in eukaryotic cells

Abstract

The CRISPR-Cas system has rapidly reached a huge popularity as a new, powerful method for precise DNA editing and genome reengineering. In Synthetic Biology, the CRISPR-Cas type II system has inspired the construction of a novel class of RNA-based transcription factors. In their simplest form, they are made of a CRISPR RNA molecule, which targets a promoter sequence, and a deficient Cas9 (i.e. deprived of any nuclease activity) that has been fused to an activation or a repression domain. Up- and downregulation of single genes in mammalian and yeast cells have been achieved with satisfactory results. Moreover, the construction of CRISPR-based transcription factors is much simpler than the assembly of synthetic proteins such as the Transcription Activator-Like effectors. However, the feasibility of complex synthetic networks fully based on the CRISPR-dCas9 technology has still to be proved and new designs, which take into account different CRISPR types, shall be investigated.

Keywords: CRISPR; Cas9; Synthetic Biology; gene circuits; guide RNA.

Figure 1.

CRISPR-Cas9-based DNA degradation pathway. A long pre-crRNA chain, which contains pieces of previously-encountered foreign DNA, is processed into Cas9-crRNA:tracrRNA molecules. They bind and cut foreign DNA upon recognition of the protospacer adjacent motif. Double-strand cleavage triggers the degradation of foreign DNA.**

Figure 2.

Synthetic transcription factors built on the dSpCas9:gRNA system. The fusion of an activation domain such as VP64 (A) or a repression domain like KRAB (B) to dSpCas9 confers to the dSpCas9:gRNA system the ability to regulate transcription initiation from eukaryotic promoters. A line ending with an empty circle is used here (an in the next Figures) to depict the recruitment of RNA polymerase II molecules (RNAP) to the DNA.**

Figure 3.

Examples of gRNA used as a scaffold. (A) An scRNA is built by extending a guide RNA with aptamers that are anchor points for RNA-binding proteins. The latter are fused either to repression or activation domains and regulate transcription initiation. (B) The Casilio system gives its name to the usage of dSpCas9:gRNA in conjunction with Pumilio/FBF proteins that are fused to an activation or a repression domain. Best performance was achieved by extending the gRNA with 5 PBSes, as shown here.**

Figure 4.

An AND gate as a bladder cancer cell classifier. (A) In absence of any of the 2 input signals, both hUPII and hTERT promoter are inactive and the SpCas9:gRNA complex targeting the lacI gene is not synthesized. LacI, constitutively produced by the SV40 promoter, binds the synthetic CMV-lacOp promoter and turns off the expression of red fluorescence proteins (RFP), the circuit output. Hence, if the classifier is inserted into non-bladder cells or healthy bladder cells, no fluorescence can be detected. (B) When both input signals are present, the SpCas9:gRNA system is assembled. Upon lacI gene cleavage by SpCas9, RFP is expressed in considerable quantity by the CMV-lacOp promoter. Therefore, bladder cancer cells induce the production of a high red fluorescence signal from this circuit. Green arrows indicate promoter activation, red lines ending with a bar represent repression of transcription, dashed lines ending with a circle stand for gene expression.**

Figure 5.

CRISPR-dCas9-based inducible systems. (A) In presence of GA, GAI dimerizes with GID1 and the VPR activation domain can recruit RNA polymerase II to the promoter targeted by dCas9:gRNA. In logic terms, this is a buffer gate. (B) Only under simultaneous induction with GA and ABA, both GAI-GID1 and ABI-PYL1 dimerize. This double heterodimerization bridges dCas9:gRNA to the VPR and results in promoter activation. In this configuration, a single chemical is not enough to trigger protein expression. Hence, the whole system behaves as an AND gate.**