Structure

The dCas9 Toggle Switch represents the core of our system and will allow switching between 2 stable states of gene expression. It consists of 2 gene classes which we simply call the ON and OFF genes. Each of these two gene classes comprises a strong constitutive promoter with a RNA polymerase II (RNAPII) binding site and multiple copies of the same guide RNA (gRNA) target site downstream next to it. All genes in a class are controlled by the same regulatory region so that their gene products are expressed simultaneously. At least one gene in each class code for a RNA construct consisting of a gRNA sequence flanked by two autonomously folding catalytic RNA sequences (Ribozymes) which leads to the production of free gRNA when transcribed. Other genes in the ON genes class code for proteins which should be only expressed in the ON state of the system. Vice versa OFF genes encode OFF state proteins. In theory these could be any number and combination of genes needed to define one of the states. However, we will only use one additional ON gene and no additional OFF gene. This ON gene contains a GFP coding sequence and will be therefore used as a reporter for the functionality of our system.

Function

The system is controlled from outside of the cell via 2 synthetic pathways leading to the production of either an ON or an OFF free gRNA. If in case of a ON signal the ON free gRNA is produced it will bind to dCas9 (or derivative) and direct it to its target sites in the regulatory region of our OFF gene, thereby blocking gene expression. However the not blocked ON genes will still be able to be expressed in high amounts and will, besides GFP, produce ON free gRNA on their own. This constant stream of new ON free gRNA will deny the expression of OFF free gRNA even after the original ON signal decays and will therefore hold the system in the ON state. The system will only switch into the OFF state if an external OFF signal produces an OFF free gRNA which binds to the target sites in the ON gene promoter region stopping the constant flow of ON free gRNA and replacing it by its own gene product: OFF free gRNA respectively. .

Experimental Parameters

• We will try expressing GFP in the same ORF with the ON RNA construct, but because folding of the ribozymes could lead to a mRNA not capable of translation this might not work.
• In order to balance OFF gene and ON gene products we will also test the effects of expressing two copies of the OFF RNA construct in our OFF gene.
• We will test whether targeting the dCas9 to the coding strand or to the template strand plays a role in our system.
• We will find out if a minimal dCas9 (a dCas9 which has its originally catalytic DNA breaking domain removed or altered in such a way it can not by back mutation become functional again) can achieve also a acceptable level of gene expression for our system to function properly.
• We will use constitutive promoters with different strengths in our ON and OFF genes to find out which of them works best in our system.
• We will use different numbers of copies of gRNA target sites with spacers between them in the promoter regions of our ON and OFF genes to see if that correlates with the strength of repression.
• We will try fusing a repressor (e. g. a chromatin remodeler recruiter) to the dCas9 to further reduce promoter leakage.

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