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EPF-Lausanne Bioworks
An orthogonal complex system in a single cell
Project Description
Logic gates are the main components of every modern digital circuit. Digital circuits work with binary values that are either 0 or 1 and we can think of this values as representing a false or true state respectively; physically 0 and 1 (false or true), are represented by 0V or 5V. Logic gates are ‘’black boxes’’ that can take many binary inputs and gives generally one binary output. For example the NOT gate takes one binary input and invert it: if the input is 0 the output will be 1, while if the input is 1 the output will be 0. The NOT gate is one of the simplest logic gates we can imagine but many other gates with different functionalities exists. For example the AND gate takes two inputs and gives an output: the output is true (i.e. the output value is 1) if both the inputs are true, otherwise the output is false (i.e. the output value is 0). Another common example is the OR gate, which takes two inputs and gives an output: the output is false if both inputs are false, otherwise the output is true. By combining different logic gates in sequence and in parallel (each gate having its own characteristic truth table) it is possible to create digital circuits with complex behaviours.
The aim of our project, is to create a general framework allowing simple design of digital circuits inside living cells, using dCas9 proteins with specific gRNAs as activators or inhibitors of gene transcription.
Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease enzyme which can target nearly any DNA sequence complementary to its guide RNA (gRNA). The original function of the CRISPR-Cas9 system in bacteria is to cleave foreign DNA after positive match. However, a dead version of Cas9 (dCas9) unable to cut the DNA can be used as a repressor (by preventing the binding of the RNA polymerase to promoter sequences) or as an activator (our dCas9 is fused with a a polymerase recruiting element).
Our project relies on a three plasmids system. The first one will produce dCas9, another will gather gates acting like a gate array and the last one will act as a linker for the gates and can represent the programm.
This modified Cas9 will form a complex with a chosen produced gRNA (guideRNA) that will allow the complex to complementary bind to the plasmids. That way dCas9 will induce the production of other gRNAs or inhibit them. The output of a gate would then be the production or not of gRNA that will go back to the linker that will induce also a gRNA to continue the program etc…
