Description summary:

CRISPR-Cas9 has already revolutionized synthetic biology. To build upon this
development we aim to implement digital-like circuits in yeast using a CRISPR-associated RNA scaffold system (Zalatan et al, 2015). Recently, a study published the use of modular CELLO software automating the design of DNA circuits using transcription factors in E. coli. As a proof of concept we will modify CELLO to use our dCas9 transistors in yeast for a so-called half-adder system, using AND and XOR gates, that we can then experimentally assess. With this approach we hope to pave the way for even more complex biological circuits in yeasts.

What have we done?

We started brainstorming in December and quickly decided to work on the creation of a biological circuit.
We were inspired by the EPFL’s 2015 iGEM team, who worked on bioLOGIC. This system uses a catalytically dead version of Cas9 fused an RNA Polymerase recruiting element (VP64). Depending on the identity of the promoter that dCas9-VP64 binds a promoter, it will either be repressed or activated.

We created brainstorming groups to find applications of the project. The idea of creating a half-adder stood out from the rest. We found out about a program called Cello that automates the design of DNA based logic circuits.

At this point, we split into two groups. The first group worked on the design of the system, the second on the understanding of Cello’s software in order to implement it with our system.

The project was defined as to create simple gates using biological parts. We want to use d-Cas9 to target specific sequences and be able to activate or repress the expression of a targeted gene. We want to implement our system in yeast as they are well representation of mammalian cells and easy to handle. With this system we were to create an half-adder with correspond to and XOR and AND gate. In another part, we want to implement our system to Cello software to help us and others to create simple d-Cas9 based genetic gates.

We stumbled upon a paper concerning a more intuitive way to activate and inhibit genes with dCas9 we thought about improving our project using its results.

This paper describes synthetic dcas9-based transcriptional programs in yeast. Instead of having the dcas9 unit fused to an activator or repressor protein, the guided RNA is extended to include an effector protein recruitment site, that way scaffold RNAs that encode both target locus and regulatory action.

This system offers some advantages compared to previous ones. First of all, we will not have toxicity problems with dCas9 and, in the same time, it offers more complexity of circuits by being rather modular, we need only to change the guide RNA to have a new transistor.