Description: 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 the modular software CELLO which automates 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.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 with an RNA Polymerase recruiting element (VP64) to create transistors, and depending on the identity of the promoter that dCas9-VP64 binds, it will either be repressed or activated.At first, we created brainstorming groups to find applications of the project. The idea of creating a half-adder stood out from the rest for its possible applications as well as its suitability as a proof of concept. Later, we discovered 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 wanted to use d-Cas9 to target specific sequences of promoters and therefore be able to activate or repress the expression of the genes controlled by them. In order to build biosensors, we imagined a system that allows our gates to respond differently to various environments, such as presence of galactose. We also want to implement our system in yeast as they are well representation of mammalian cells and easy to handle. With this system we aim to create an half-adder which correspond to a XOR and an AND gate linked together.As mentioned before, we also plan to modify CELLO to be able to design genetic circuits in yeast using it. Fortunately, CELLO has a modular nature, allowing us to do this easily. CELLO has a User Constraint File that enables users to pass the program information about this system it is designing the circuit for. This file includes information pertaining to the species, the reactivity of gates to inputs, and the plasmids used. In order to obtain this new information, we plan on characterizing our system and gates using photometric experiments.During the process of designing our system, we stumbled upon a paper outlining a more intuitive way to activate and inhibit genes with dCas9, and we decided to improve 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 guide RNA is extended to include an effector protein recruitment site, so that scaffold RNAs that encode both target locus and regulatory action.Using a dCas9 based system with scaffold guide RNAs offers numerous advantages with regards to previous biological circuit designing systems. Firstly, using gRNAs as parts of gates, instead of transcription factors reduces toxicity related to transcription factor density in the nucleus. In addition, our system can be even more complex than systems based on transcription factors since the amount of connections between gates are not limited by the amount of transcription factors available. Finally, the use of scaffolding RNAs simplifies design, since we can have just one dCas9, and it will also hopefully lead to more predictable repression and activation in the system.
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Updated at: 8/29/16