Luis Ruben Soenksen

Luis Soanksen: All right. Thank you so much. My name is Luis Soanksen. I graduated here, PhD mechanical engineering, but I also have been working at the Wyss Institute in collaboration with Jim Collins on a variety of topics, including another presentation you will see later on in wearable synthetic biology. But this is one that we wanted to present to you because we believe it’s actually pretty cool. It’s this concept of CRISPR Responsive Smart Materials. Luis Soanksen: As Rachel already described, for the last maybe 50 years or so, people have really eagerly experimenting with biomaterials. Reality is that we have had a ton of advancement, from composites, metals, polymers, synthetic and functionalized materials, that at the end of the day, accomplish a certain goal.

00:56 - Reality is that still the idea of bringing new biocompatible materials capable of responding to specific biological triggers is still something that is a priority. Luis Soanksen: Our very, I guess, peculiar way of addressing this, and we don’t want to claim this really that we want it to solve all the materials problem, was can we create programmable, nucleic acid programmable, materials that somehow can respond to us and to the environment to do interesting things? The way we thought it was fun to accomplish this is through this idea of embedding programmable DNA nucleus’s, which is this CRISPR enzymes, into materials with interesting chemistries to accomplish such a thing. If you guys… If anyone here is interested on these results, everything that I’m going to present here, it has already been published in these publication Science that we recently got out. Luis Soanksen: The basic principle is, CRISPR enzymes are highly programmable nucleus’s. So they have a guide that you can program to really detect nucleic acids in a very specific way.

02:10 - Then if you embed those into materials, specifically polymers, that have either tethered or that are already tethered or cross-linked with DNA, then suddenly you can activate those materials and change their properties. Luis Soanksen: Specifically, the things that I’m going to show today is couple of demonstrations that we did across different polymer chemistries, specifically*** PEG hydrogels, polyacrylamide DNA gels and carbon black DNA gels. So just for you to sort of… Just for you to kind of understand a little bit better this diagram in the first block, that says PEG DNA gels for example, there you can have a matrix that is made of primarily PEG, but you can have these single stranded DNA that has cargoes tethering to them. Those, as you will see later, it can be anything from enzymes to fluoro** force to small molecules that then we can cleave in order to make materials that release things into the environment. Luis Soanksen: But in other chemistry such as the middle one that says polyacrylamide DNA gels, we can actually have DNA as the fundamental crosslinking unit so that you can actually trap things inside the material or have a very densely cross-link material.

03:25 - But the moment that you cleave those through the activity of CRISPR enzymes, which again are programmable, then you can subtly actuate those materials to swell more or to be more permeable, basically affecting their mechanical properties, at least in the sense of permeability. And the third one I will discuss today is this idea of other more interesting, potentially conductive materials. So these carbon black DNA gels. Carbon black is a material that is highly… Can be made to be highly conductive. And if you, because of functional groups that it presents, it actually has the capacity to crystalling tightly with single stranded DNA. Luis Soanksen: So just by combining carbon black with DNA, you can actually make these polymers, complex polymers, that are conductive, but yet they are mechanically attached together by these DNA strands, hopefully to create potentially electronics that can be modulated by the activity of CRISPR.

04:22 - Luis Soanksen: All right, so schematically this is what we accomplish and I’ll show you data in a moment. We were able to demonstrate that for these materials, specifically these polymeric hydrogels, we are able to, on demand, release our cargoes, which include enzymes, small molecules, but also cells. We’re able to create conductive materials that are up on sensing of nucleic acids in an environment can detach from electrodes, basically short-circuiting or in this case opens your quitting electronic circuits to be measured. But also as medium to change permeability as we discussed previously. In order to do other interesting things like short-circuiting, RFID antennas and other things that are kind of more in the realm of devices to make it more visible of how you could use this for diagnostics and other purposes.

05:19 - Luis Soanksen: So I know that this is kind of a data dense slide but just bear with me in a minute. So here we have this PEG matrix and again we have tethered… We were able to tether molecules here in panel B for example, you see a fluorophore that is on demand release from these hydrogel up on the addition of a single stranded DNA. That is our target. So in this case, a gel has this molecule, and he has embedded cast off a enzyme, which is a type of CRISPR enzyme and it has been programmed to detect the specific target, DNA target. And as you can see, depending on the presence of a specific target as compared to a scramble DNA in the environment, you can have very different release profile. We did the same for enzymes.

06:09 - So you can not only imagine to release a small molecules into the environment, but also enzymes into the surrounding medium. Luis Soanksen: And that, as you know, other [person tissues 00:00:06:20] have alluded to could have very interesting implications because you can release peroxidases or other things into the surrounding buffer to have more interactions and do more catalytic activity, for example. The interesting part about CRISPR is that it’s highly… It’s not only that it’s highly programmable, but it’s also very orthogonal. So here for example, we created these hydrogel in panel D to detect four different types of, actually five different genes in that are involved in the resistance of Methicillin-resistant Staphylococcus aureus, which is the superbug that you find in hospitals.

07:03 - And you can see that depending on the different target that you expose this to, you have a different amount of activity, which is really nice. In terms of the electronic circuits, what we did is we created these carbon black DNA hydrogels that we basically just deposited into interdigitated electrodes. As you can see here, those are just, they look like little dots, dark dots in these electrodes. Luis Soanksen: What is interesting is that when you embed these electrodes into an environment that has, for example, the target that you want to detect these dots just like dissolve or detach from the electrodes much more frequently when you have an environment that has that target. So here, for example, you can envision a circuit that is just…

07:51 - Stands there, but the moment you interact with it and or the environment interacts with that electrode and that environment has the nucleic acid target that you’re interested on detecting then itself, the circuit itself, the conductive pathways on those circuits will change and that’s what we have demonstrated here. Obviously this is not perfect technology. For example, many of these interactions happen at the interface between the interdigitated electrode and the bolt material. Really destroying a [beed of 00:08:22] of hydrogel on itself it can be a lengthy process for enzymes, but in this case we’re able to achieve these results in a couple of hours, which is interesting. Luis Soanksen: Now, a big application that we thought was interesting was for, not only for drug delivery, but also for release of viable cells that for example, can be immune cells. In these experiments, for example, we created these hydrogels that were able to encapsulate viable mononuclear STEM cells on them. And so those…

08:56 - In our publication we’ll have other sort of gels as you can see, but these look very well in the projector. We have these let’s say hydrogels, those could be potentially implanted into an animal, into a human, and those will actually hold STEM cells on them that may be programmed or not to do whatever. But at the end of the day, the idea is that you don’t want those cells to be necessarily circulating all the time or at any point, but rather only when certain cues are sort of presented to them. Luis Soanksen: And in this case, nucleic acids, were the things that we wanted to detect. So if this is immune cells, you can imagine that you can have, if you have a nucleic acid that is relevant in the detection of certain virus or certain bacteria that you want those immune cells to be released on demand.

09:43 - Also for cancer therapeutics this seemed to be interesting. And so what we’ve demonstrated here is that if we just present these gels with the scrambled DNA, don’t really dissolve that much in, for example, in this first hour, which is this first section in the left. But as you are presenting those with more and more of these specific DNA trigger, then those gels just dissolve out and those release those cells. And the last section of that panel just shows that kind of like a viability test on the cells just to ensure that the actual process of releasing them is not killing those cells, which is still screen does. So you see there. Luis Soanksen: So I know that this is all like different layers of complexity, but something that we thought was interesting is since we have the capacity to create and to sort of solve dissolve shells to can we use these shells in there to do more interesting stuff in terms of flow.

10:46 - And so there are some people in the last 20 years that have created this stop flow essays were changing… You have a paper, a lateral flow immunoassay for example, you have a paper strip where you have flow but you can stop that flow by making something cross link along the way during that flow. And that’s exactly what we did here with this, in literature they are called origami micro paths, but basically they are just layers of paper that had been printed with wax to have almost like channels into them defined. Luis Soanksen: And we can put in different layers of paper and all these, these things are paper. You can put different substrates for the crosslinking of these hydrogels.

11:31 - So you can have the precursors, the polymers, you can have dyes, you can have buffer, everything that you need but what we did here is create this assembly that has all the precursors and everything to create those gels as flow is happening. But we’re in the presence of a target the CAS enzymes will be activated basically cleaving everything that makes possible for these systems to cross link basically allowing for flow to happen if there is a presence of a target and flow to be stopped in the absence of a target. As you can see in the panel on the right, for example, in the presence of the enzyme you can see in the bottom that buffer flows and you can see in these lateral flow section in the bottom that there is discolored dietaries flowing through and you can see the matrix there of the cells. Those just clean in a way. Luis Soanksen: Whereas in the upper section when there is no target and therefore the CAS is not activated, then you’ll get hydrogel forming, stopping the flow. And so as you can see, you can have just a very easy colorimetric signal out of these same sort of materials being broken down or not. But you can also imagine… Sorry.

12:49 - Measuring conductivity out of this because as a buffer is flowing, and these buffers are usually PBS or just they have salts on them, you can actually think about measuring the conductivity of these micro paths as a measurement of distance of how much buffer flows. And that’s exactly what we did in order to just almost solidify this concept that we could use these very intercommunicated systems, the chemical, the antemetic aspect of CRISPR, the genetics out of it, but also sort of the permeability aspect of our materials and then potential electronic uses. Luis Soanksen: We did a couple of demonstrations where we actually just in colorimetric experiments, which is this panel A, we’re able to show that we were able to create a very cheap diagnostic for Ebola that was able to detect 11 attomolar… Up to 11 a attomolar concentrations of Ebola trigger. And that’s pretty incredible because that’s pretty much ball parking day in where PCR is and this is just in a piece of paper, right? And just in smarter materials.

13:57 - So it’s interesting application of this pathway that you’re pursuing. But sometimes people want to have these integrated into electronics to do more automated reporting. Luis Soanksen: And so I need to stop now but the idea here is that we integrated this piece of paper also with RFID tags to short circuit a small RFID tag and that suddenly that’s what hit us. Because when you are able to modulate materials in this way that are potentially conductive or not, then you can really have these new interfaces between biology and electronics in a way that we didn’t expect too. So again, if anyone is interested about this, everything is published already in Science and we’re about to release these nature protocols so that anyone can come to replicate these types of results.

14:49 - Luis Soanksen: I want to thank everyone that was involved in this and thank you so much. .