As Christopher Voigt explains it, his lab at the Massachusetts Institute of Technology has been “working on new experimental and theoretical methods to push the scale of genetic engineering, with the ultimate objective of genome design.” It’s genetic engineering on a genomic scale, with the expectation for major advances in agriculture, materials, chemicals, and medicine.
As they’ve gone along, Voigt’s group has also been assembling the toolbox needed for anyone to begin considering genetic engineering projects in a very big way. In one of his latest papers, published in Molecular Systems Biology in November, Voigt and Alex Nielsen describe what’s possible when multi-input CRISPR/Cas genetic circuits are linked to the regulatory networks within E. coli host cells.
We talked with Voigt about this collision that’s taking place between CRISPR technology and synthetic biology, the tools he’s making available through Addgene, and where all of it is likely to lead us in the future.
Building Circuits from CRISPRs
Addgene: What can CRISPR do for synthetic biology?
Voigt: The big promise is orthogonality. You can create an almost limitless number of DNA binding proteins with Cas9 guided by different promoters. The challenge in building genetic circuits is getting enough regulatory proteins that don’t interfere with each other. CRISPR is so orthogonal and programmable that you can make a very large number of regulators. That basically means larger genetic circuits that could conceivably be as large as natural regulatory networks.
Addgene: What kinds of things could we do as genetic circuits become larger and more complex?
Voigt: If you look at all the products of biology - even a piece of wood - there’s a lot of control spatially as to what’s happening at different points within the wood. There are different types of cellulose fibers, pattern, shape. All of that is controlled by regulatory networks in the plant. If we want to access that type of material - or do what biology can do - it’s good to be able to build regulation thats as sophisticated as a natural network. We could build a material as sophisticated as wood. To build at that scale, we also have to think through programming cells.
In the context of therapeutics, if you want to program a cell to identify and target a malignant cell, then you’d need the ability to turn on genes at different times. It requires more complex synthetic networks.
Addgene: What hurdles did you have to overcome and what hurdles remain?
Voigt: There are a couple of hurdles we haven’t yet overcome. For this particular paper, there actually were not many hurdles. It was pretty straightforward surprisingly. So I think a nice thing about CRISPR is how much easier it is to work with than regulatory proteins. There are still things we haven’t solved that stop it from being used. One thing is that Cas9 is toxic. When cells carry CRISPR circuitry, it can slow their growth. Something has to be done about that.
The second problem is fundamental. We built a NOR gate because they are used to create any program you can imagine. The Apollo 11 mission circuitry that took a spaceship to the moon was built on NOR gates. It’s a fundamental unit. We did demonstrate that you can use the CRISPR chemistry for a NOR gate. But there are a couple of problems. You couldn’t do it without repeating the same guide RNA twice. The second problem was the shape of the response function or how the output changes as a function of input. Usually that function is in the shape of an S; there is a narrow threshold like a switch that turns on or off. CRISPR shows a more continuous response - not switch-like at all.
It’s also difficult to build larger circuits with many guide RNAs connected because the signal degrades at each layer. If we could fix these things, then I think there is no limitation regarding the size of the networks you could build.
Addgene: Is this what you are working on now?
Voigt: We are working on this now, we have ideas, but so far nothing that has worked. I don’t have the solution. Our paper shows we can easily make orthogonal variation. The first one worked in each case. Sometimes in CRISPR people talk about off-target interactions. That’s relevant if you want to make sure you don’t target anywhere else, but for synthetic circuits, they are extremely orthogonal. It’s a different perspective and you can layer them for more complicated functions. Those things are easy with CRISPR.
Addgene: You’ve made many deposits to Addgene over the years. Can you describe the toolbox you are putting together?
Voigt: We’ve been trying to get everything to the scale of building synthetic genomes or genome-scale construction projects. Not from the ground up, but so that genetic designs of that scale could be created. Someone could take on an engineering project including hundreds of genes and regulation and resource control - everything that’s necessary on that scale.
If you look at what we’ve already put in, the fundamentals are there. There haven’t been enough genetic parts for complexity. We have characterized almost 600 terminators. That gives us enough hardware to control hundreds of genes. We’ve created enough regulatory elements, CRISPRs, repressors, activators. We’ve been putting together very large toolboxes and making them available.
We’ll also be submitting parts from another paper for resource allocation. Those will allow you to set the amount of transcriptional and translational activity a cell has and control how much it taxes the resources of a cell. The objective is to give people the complete toolbox for taking on very large genetic engineering projects.
Addgene: How easy is it to do this now?
Voigt: One of the things we’ve been doing is developing software to allow people to design systems of this scale. That will be coming out over the next year. Then there will both be a combination of parts in Addgene along with the software to tell you which parts are required to get the function that you want. The last few years, we’ve really been making tons of parts and characterizing the software to help you put it together. The software will be available on GitHub, an open source code repository.
Addgene: Over the longer term, where is this all heading?
Voigt: The top products out of biotechnology today are things like butanol - very simple molecules - a few carbons and oxygens. Nothing too much more sophisticated. There are also proteins, like antibodies. But we are really just using what biology has given us. Wood is a complicated material and we can access it, but we are stuck with what biology has given us. Over the next 100 years, we are really going to be able to design new materials and chemicals as sophisticated as those that living cells and animals make. That will be an outcome of genetic engineering.
Thank you to Chris Voigt for speaking with us!
Find the Plasmids:
Nielsen AA, Voigt CA. Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Mol Syst Biol. 2014 Nov 24;10:763. doi: 10.15252/msb.20145735. PubMed.