The Quest for New Receptors
In many fields of science and technology it is often necessary to analyze the composition of a solution to screen for specific compounds. While to us humans this poses a very difficult challenge, in vivo molecular recognition is a task carried out constantly by all living cells. Membrane-bound receptors recognize specific molecules by binding them via a lock-and-key mechanism. When the right molecule interacts with the binding site of the receptor, a cellular reaction is stimulated. These complicated genetic systems evolved over billions of years to recognize molecules that were relevant for survival. Therefore, the range of molecules that natural receptors can recognize is very limited.
What Team MSP is trying to achieve with its iGEM 2019 Project is to broaden the spectrum of surface receptors available to the scientific community by engineering a synthetic genetic system in Saccharomyces cerevisiae which can automatically evolve new surface receptors capable of recognizing specific, user-defined, molecular targets and provide their quantification in real time.
Our long term goal is to build a standardized tool that researchers can use to generate new surface receptors which are best suited to their investigative needs.
Our Plan to Build the Next Generation of Surface Receptors
The goal is ambitious, but worry not: We have a plan!
In order to generate a new surface receptor for a given target molecule, a few genetic components will be necessary.
As a starting point, an initial receptor which later will be evolved towards the proper recognition of such molecule is needed. Therefore, the first component will be the DNA sequence of a surface receptor with a non-functional binding site. Initially, this receptor will not be able to recognize any molecule and therefore activate any downstream signaling pathway. The goal of our system is to randomly mutate the DNA sequence corresponding to the target site of the selected receptor until a sequence is obtained which leads to a structural change in the binding site so that the target molecule, which will be present at high concentrations in the growth medium, will successfully bind to it. Binding will also produce a visual signal for proper identification of colonies with successfully mutated receptors.
The random mutations will be induced by action of a CRISPR-based dCas9-Fok1 fusion endonuclease. This protein will be instructed by an engineered gRNA to recognize specific sequences found in synthetic introns which will be introduced at multiple sites in the sequence of the receptor’s binding site. This will allow dCas9-Fok1 to generate double-strand breaks (DBS) in the active site without risk of modifying its own recognition sites on the DNA. DSBs will be repaired by the cell’s DNA repair mechanisms, which is known to frequently introduce random mutations in the repaired sequence.
Once the optimal sequence for the receptor will be obtained, the CRISPR system will need to stop inducing mutations to the active site. Therefore, the receptor is engineered in a way that, when activated by the target molecule, it releases a so-called anti-CRISPR protein (LmAcrIIA2). When free, LmAcrIIA2 binds dCas9-FokI on the DNA recognition domain, preventing its binding to the receptor’s DNA and the induction of mutations.
The anti-CRISPR protein will also target a second CRISPR-based dCas9 fusion protein, which was employed as an expression inhibitor for a color producing protein. This way, when the receptor is activated, a color will be produced corresponding to colonies expressing the desired receptor. What the release of LmAcrIIA2 will also cause is the exposure of a protein affinity tag, previously hidden by the anti-CRISPR itself, which will be used to purify the final receptor once obtained: the receptors bearing the wrong sequence will not be activated, will not have the tag exposed and therefore will be discarded by the affinity purification.