RNA Control and Regulation

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A Conversation with David Bartel

INTERVIEWER:RICHARD SEVER

Assistant Director, Cold Spring Harbor Laboratory Press

David Bartel is a Professor of Biology, a Member of the Whitehead Institute, and an Investigator at the Howard Hughes Medical Institute at the Massachusetts Institute of Technology.

Richard Sever: You spoke yesterday about microRNAs, given gene, but just because you’re hitting a whole bunch the small RNAs that regulate the activity of genes post- of them? transcriptionally. You made the point that the effects of Dr. Bartel: I think that’s the easiest way to think about it. knocking down these microRNAs could be really debili- In some cases, people have found single individual targets tating. That was kind of a shock to me because people are where that 50% down-regulation has a dramatic effect. But always saying that it’s a fine-tuning of gene expression. even there, they have hundreds of other targets that are Can you say a little bit more about that? conserved more than they would expect by chance. Cer- Dr. Bartel: I like the term “tuning” rather than “fine-tun- tainly, over the course of evolution we know that biology ing” because it actually is more of a tuning. It turns out that cares about many more than single targets, and, again, these microRNAs do have very striking phenotypes when each of them is regulated by a relatively small amount of you knock them out, but even before we knew the knock- tuning. So, that’s our current view of what’s occurring. out phenotypes, we knew that they recognized many tar- ’ gets. We know that there are 90 families of microRNAs Richard Sever: For those of us stuck in the 90s, how highly conserved from humans to fish. Each of those 90 does that compare with the way we think of transcription ’ have on average about 400 preferentially conserved tar- factors? Do you think that we shouldn t be singling these gets. That adds up to more than half of the human genes out as different in the way they work? that are conserved targets of microRNAs. On average, Dr. Bartel: Depending upon the transcription factor, you ’ they re targeted by four or five different microRNAs. might also see these minor effects on many targets; it may They [the microRNAs] have these very widespread not be that much different. There are some transcription effects. factors that will have a 40- or 50-fold effect on the tran- – It is true that for each microRNA target interaction, that scription of a certain mRNA, but I think, more generally, — — [regulation] can be a rather subtle maybe 20% down- transcription factors might also have widespread effects— regulation, sometimes 30%, sometimes more. But because on the level of microRNAs—and may not be a lot different they have so many targets, when you knock out the micro- for a lot of those interactions. RNA in mice—which is what many labs have been doing; they’ve knocked out one or several members of the same Richard Sever: The way the microRNAs act is by exert- family of microRNAs—and when they look at the pheno- ing their effects on messenger RNA [mRNA]. There’s two types, for these 90 conserved families nearly every one of possibilities there: stopping its translation, and degrading them where knockouts have been reported on, they cer- it. You’ve made the point that actually it’s much more of tainly do see a phenotype. They’ve looked at over half of one than the other. them and seen phenotypes. We already know that 15 of Dr. Bartel: What we see is that—with one exception— these microRNA families, when you knock them out, you every place where we’ve looked, over two-thirds of the have embryonic lethality or perinatal lethality, so that’s repression could be explained by the microRNA recruiting pretty severe. And there are others that also have [other] factors that deadenylate the RNA and then cause the RNA very severe phenotypes like blindness, deafness, infertili- to get destabilized. Often, over 90% can be explained by ty, seizures, epilepsy, cancer, etc. There’s a huge range of this degradation mechanism, leaving somewhere between phenotypes—and many of them very severe—from the 10% (sometimes less) up to a third with this additional microRNAs. So, we know that they’re playing very im- mode of repression that’s through translational repression portant roles. —inhibiting, presumably, translation initiation. So, in Richard Sever: Are you saying that the severity of the general, the majority of the effects that we see are through phenotype is not because of the quantitative effect on any this mRNA destabilization.

© 2019 Bartel. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted reuse and redistribution provided that the original author and source are credited.

Published by Cold Spring Harbor Laboratory Press; doi:10.1101/sqb.2019.84.039313 Cold Spring Harbor Symposia on Quantitative Biology, Volume LXXXIV 259

The exception, which is really interesting, is that in early transcription, and so the way they do it is by changing the zebrafish [embryos], you completely miss what the micro- tail length. They have this phenomenon called cytoplas- RNAs are doing by looking at the changes in mRNA mic polyadenylation, which will extend the length of the levels. Whereas everywhere else—it’s kind of nice—you tail, and that will cause a massive increase in translation. can perturb the microRNA and be able to know what the They also have other ways of shortening the tail, so they targets and the effects of the microRNAs [are] just by can adjust the amount of protein output [in either looking at changes in mRNA, which is much easier to direction]. do than looking at changes in protein or ribosome-protect- Richard Sever: Do you get an oscillation there? ed fragments. But, in the zebrafish [embryos], that would not work at all. That was shown by Antonio Giraldez’s lab Dr. Bartel: Yes, [but more generally,] depending on —that in the early zebrafish embryo, the effects of the the gene, you can just get different output at different microRNAs could only be seen when you look at the points in early development through this polyadenylation changes in ribosome-protected fragments. mechanism. So, then the question is, “Why does it [this coupling Richard Sever: That’s this early phase of development in between tail length and translation efficiency] go away?” zebrafish. Is it early phases of development in other or- And the reason we think it goes away is that later in de- ganisms or is it something unique to a particular lineage? velopment, transcription is already started up, and that’sa Dr. Bartel: It’s a good question. I think that the same will great way to regulate genes. So, you don’t need this tail- probably also hold in frogs and in flies, but I’m not sure length control to regulate translational output; instead, that people have done those experiments. We do under- what the cells do use is transcription, and they also use stand what’s going on in zebrafish, and we think—based mRNA stability. That’s what the microRNAs are doing; on what we see in these other organisms—that it’ll also they’re changing the stability of the mRNAs—and that’s hold there. what we see in all these postembryonic contexts. The effects of the microRNAs is that they recruit a When the cell is using mRNA stability to regulate protein called TNRC6 [trinucleotide-repeat–containing genes, then it’s not such a great strategy to only translate 6] that in turn recruits the deadenylation complexes, the the mRNAs with long tails, because those are the mRNAs Pan2–Pan3 or Ccr4–Not complexes, and that causes the that just came into the cytoplasm, whereas the RNAs that poly(A) tail to get shorter. Once the tail gets to be a certain had been there a long time have shorter tails, and the cell length, you get decapping and decay of the mRNA. would not want to discriminate against them—it wouldn’t What we see in early zebrafish is that the microRNAs want to do this “age discrimination” against the older cause the tail to get shorter, and in that developmental mRNAs. If it’s had those RNAs stable, it wants to use context—in that regulatory regime—what we find is that them; it wants to use the RNAs with short tails just as mRNAs with short tails are translated much less efficient- much as it wants to use those with long tails—if it’s using ly than mRNAs with long tails. There’s this very strong mRNA stability as a mechanism for gene regulation. So, coupling in the early zebrafish, before gastrulation, where the idea is that when the cell switches over to this mech- short-tailed mRNAs are translated much less efficiently anism of using mRNA stability to regulate genes—which than long-tailed [mRNAs]. What’s interesting is that that they use a lot; you have these massive differences in goes away at 6 h postfertilization, which is the [time of] mRNA stability—then the cells switch away from control- gastrulation of the fish. At that point, the microRNA also ling translation based on the length of the tail. causes the tail to get shorter, but in that context, a short tail Richard Sever: When you were looking at the length of leads to [mRNA] degradation. the poly(A) tails, you were discriminating between steady Richard Sever: Sticking with the pregastrulation scenar- state levels and a better experimental approach to make ios, is there an a priori reason for that? Going back to this sure you know what the length is and when. Can you tell kind of tunability, if you’re stopping a translation rather us a little bit about that? than degrading the RNA, does that give you more ability Dr. Bartel: Sure. What I described so far was in the early to tune? Is it reversible? zebrafish embryo, and there it’s not really a steady state Dr. Bartel: I think you’re onto it there. I’ll just add that we situation. You have development happening, and you can see this same sort of transition in translational control in see these tail-length differences very readily for [the tar- frogs and in flies, and this coupling between the length of gets of] the microRNAs. But interestingly, if you just look the tail and the efficiency of translation—which is very in mouse 3T3 cells—the fibroblast cells of mouse—and strong in the early embryo of frogs, flies, fish—is also very you’re just growing cells at steady state, and you have a strong in oocytes, and then goes away around gastrulation. microRNA that you’ve induced that is there at very high The reason that we think that there is that coupling be- levels, and then you just look at the poly(A)-tail lengths of tween the length of the tail and the efficiency of translation the mRNA targets of that microRNA, surprisingly, at is that this is a time in development—at least up until about steady state, you see no difference. The distribution of 3 h postfertilization—in which there is no transcription. tail lengths is the same whether or not the mRNA is a The mRNA is all maternally inherited, and yet the embryo target of the microRNA or not. That caused us to realize cells still need a way to regulate genes. They can’tdoitby that we shouldn’t really be looking at steady state mea-

A Conversation with Adrian Krainer

INTERVIEWER:ANKE SPARMANN

Senior Editor, Nature Structural & Molecular Biology

Adrian Krainer is the St. Giles Foundation Professor at Cold Spring Harbor Laboratory.

Anke Sparmann: You were awarded the 2019 Break- “flavors,” if you will. They’re synthetic short nucleic ac- through Prize in Life Sciences together with Dr. Frank ids, single-stranded. They have chemical modifications, Bennett of Ionis Pharmaceuticals for the development of and they can be designed to destroy the target RNA. antisense oligonucleotide drugs to target RNA splicing They will home in on an RNA through base-pairing inter- and the incredible success story of SPINRAZA, the first actions, so they can be very specific. If the chemistry is drug approved for spinal muscular atrophy. Can you start designed in such a way that the duplex is recognized by by telling us about this devastating disease and the mo- endogenous RNase H enzymes, then they destroy the lecular mechanism underlying it that you discovered? RNA target. In our case, we use a different type of oligonucleotide Dr. Krainer: SMA, or spinal muscular atrophy, is a motor design that binds to the RNA target by the same sort of neuron disease. It’s very severe, and it mainly affects in- physical chemical interactions, but it doesn’t lead to its fants and young children. There are milder forms of the destruction. Instead, it blocks the binding of RNA-binding disease, with delayed onset, which affect older patients, proteins. If you place an oligonucleotide in the correct including adults. Depending on the type of SMA, it leads place, then you can block the binding of a protein that to progressive muscle weakness and paralysis, and it can be affects splicing in some way. In our case, we were looking lethal. It’s inherited as an autosomal recessive, Mendelian to block the binding of a splicing repressor, so that the kind of disorder. The disease was well-characterized, and exon that’s nearby can now be recognized more efficiently the responsible gene was identified in 1995. Sometime by the splicing machinery. Now splicing looks more like it later—4 years or so—it became clear that a defect in splic- does in the SMN1 gene, even though we’re targeting the ing is related to the disease. We began to work on that SMN2 gene that is still present in patients. If you deliver because our interests in my lab have always been on the drug to the right cell types, the cell now knows how to RNA splicing—both the fundamental science and the re- allow this gene to express higher levels of functional pro- lationship to disease. tein. That’s the molecular mechanism of action of the There are two genes that are closely related. One is miss- oligonucleotide. ing or defective in patients; the other gene functions as a kind of backup. It can express the correct protein, but in Anke Sparmann: You were involved from the start fig- fairly low amounts due to the type of splicing that the uring out how this works, but then also all the way through transcript undergoes. So, we began to characterize that to actually drug development. What were the major chal- ’ process. Weweren t the ones who described this difference lenges throughout that whole process? in splicing, but we were interested in that general problem. Between the two genes, there are very few nucleotide dif- Dr. Krainer: There were many. This started as a very ferences, but one in particular had been pointed out in the basic science kind of effort, and we made mechanistic exon that is inefficiently spliced. So, we studied that prob- observations that inspired a way to try to correct the defect, lem: What is it about that nucleotide? What is normally and we went through successive modalities for doing being recognized in the transcript by various factors? We that. We learned things along the way. The ultimately worked on that for a couple of years, and then we began to successful approach was a bit simpler than the way we think about how to correct the splicing of the SMN2 [Sur- had started. Importantly, we began to collaborate with vival of Motor Neurons 2] RNA in order to allow the gene Frank Bennett at Ionis Pharmaceuticals in 2004. We had to produce higher levels of functional protein. a lot of discussions and decided to use a particular kind of chemistry and to go with the approach that I just de- Anke Sparmann: How does this drug that was eventu- scribed, which is to find oligonucleotides that will block ally developed actually work? How does it correct the the binding of a repressor. splicing? We did that very systematically. There was a postdoc Dr. Krainer: It’s a kind of drug called an antisense who joined the lab at that time, Yimin Hua, who did pretty oligonucleotide. Those come in different modalities or much all the early work, the key preclinical experiments.

© 2019 Krainer. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial License, which permits reuse and redistribution, except for commercial purposes, provided that the original author and source are credited.

Published by Cold Spring Harbor Laboratory Press; doi:10.1101/sqb.2019.84.039461 276 Cold Spring Harbor Symposia on Quantitative Biology, Volume LXXXIV

We did have a lot of advice from Ionis people who were regulation, because the way we approach the problem is doing real pharmacology, but initially we were doing bio- all based on insights about the mechanism. In this case, we chemistry, then cell-based experiments. Later, we set up were targeting a splicing repressor binding site. A few mouse models. This took quite a number of years. years earlier, we didn’t even know these molecules exist- I don’t know how to define when exactly we started ed, so one first had to discover that and understand some- working on this. We can put the start date when I, and thing about how they work. We continue the basics, but my trainees, began to work on splicing, which is much we’re also pursuing projects in which we try to apply earlier. On SMA specifically, we began around 2000 or similar or related approaches—blocking splicing com- 2001, with the work of a postdoctoral fellow, Luca Car- ponents or RNA-binding proteins—in order to change tegni. The antisense screen, as it ultimately was carried splicing, and also other RNA processing, such as non- out, began in 2004. The SPINRAZA molecule had other sense-mediated mRNA decay. We’re exploring several names earlier, but we published it in 2008. As things were potential targets that could lead to therapeutics for various progressing quite well, particularly when we began to do diseases. mouse-model experiments and seeing pretty dramatic re- Anke Sparmann: In this basic kind of research, what is sults in terms of splicing and protein and phenotype, Ionis the next thing that’s going to happen in splicing? got quite serious about undertaking the clinical development and picking among the many different oligonucleo- Dr. Krainer: That’s moving along on many different tides that were effective, to look for the one that would be fronts. When I started in this field, we were just doing most specific and had minimum toxicity at high doses, etc. cell-free splicing. It was all biochemistry. I started work- That’s a separate effort in pharmacology, for which they ing on the development of systems for that as a graduate had a lot of experience. student. There was a lot to do, a lot of biochemistry to The next step was clinical trials. Those were initially identify components. Of course, other labs were using sponsored by Ionis. About a year later, Biogen teamed up genetic approaches and model organisms. Nowadays, with them. The clinical trials were done in a variety of there are many more disciplines that are contributing to clinical centers and hospitals in several different countries. understanding the whole process: quantitative approaches, The key trials—Phases I through III—took about 5 years, bioinformatics, genomics, transcriptomics. A lot of tech- which I think all went pretty smoothly. It ended up taking a niques have been invented since I started in this field, so year less than had been planned, because the results along there are always new ways to revisit an approach. There are the way were very encouraging. It was possible to com- many powerful cell biology approaches, as well. plete the trials so that the patients still remained as part of One sees steady progress on many fronts. Every once in an open phase extension study, beyond the original clini- a while, there are breakthroughs, so things advance more cal trial. There are still ongoing clinical trials, but the ones rapidly. The structural biology approach has had a tremen- that were key for obtaining the approval of the drug took dous impact in recent years with cryo-EM [electron mi- about 5 years. croscopy]. Seeing snapshots of spliceosomes in action felt like the field suddenly moved forward 10 or 20 years. One Anke Sparmann: Not that long ago, thinking of RNA as could appreciate details, some of which were already a drug molecule was not really out there. What were the known, but now you could really see it in real time. It changes that made this possible? was no longer an indirect demonstration or hypothesis. Dr. Krainer: It’s all gradual. Like every new modality, There’s a lot of work that needs to be done using newer there’s a concept, and then there’s the problems and reduc- approaches like that to get new insights. ing it to practice. There are always stumbling blocks. Typ- I think there will be a lot of surprises, and all these ically, delivery of a new type of drug is something that things inform how you might do therapeutics develop- requires a lot of effort. We were lucky that by the time we ment where splicing underlies the overall approach. There started working on this there were already several years of are efforts to develop small molecules. It’s not our work, experience with antisense oligonucleotide pharmacology. but developments in the field to also modulate splicing, They had gone through many chemistries. There was clin- and so we need to understand better how these molecules ical experience, not so much with splice modulation, but are actually doing that. What’s the mechanism of action? nevertheless with the related chemistries. A lot of that How specific are they, and how applicable is that approach knowledge—maybe more than 20 years of accumulated to other targets, other splicing events? Structural insights knowledge—is what makes these types of things possible. from the spliceosome can inform those efforts and vice Monoclonal antibodies went through something similar. versa. There was a description and one could see right away the Anke Sparmann: Are there any problems with off-target potential, but to turn those into a drug took many years. effects of these kinds of drugs? Now, it’s much more routine. Dr. Krainer: Any drug obviously has that. With antisense Anke Sparmann: You’re looking at other diseases to oligos, because they’re based on base pairing, you target. What are you moving onto? obviously have to pick sequences that are not repetitive. Dr. Krainer: Part of the lab still continues to study the SPINRAZA, in particular, binds to a sequence that’s basic fundamental aspects of splicing mechanisms and unique: It’s only present in intron 7 of the SMN genes

A Conversation with Feng Zhang

INTERVIEWER:STEVE MAO

Senior Editor, Science

Feng Zhang is the James and Patricia Poitras Professor of Neuroscience at the McGovern Institute for Brain Research, Associate Professor in the Departments of Brain & Cognitive Sciences and Biological Engineering at the Massachusetts Institute of Technology, a Core Member at the Broad Institute of Harvard and MIT, and a Howard Hughes Medical Institute Investigator.

Steve Mao: Would you mind telling us about the new take advantage of endogenous promoters to drive tissue- CRISPR [clustered regularly interspaced short palindro- specific expression. Before working on gene editing, I mic repeats] system that has a great potential to be repur- worked on a system called optogenetics. Optogenetics posed as a new tool for genome editing? allows us to stimulate brain cells using light, but the bot- tleneck of optogenetics is there are so many different types Dr. Zhang: Our work looks at the diversity of CRISPR of brain cells. How do we specifically control one type of systems. CRISPR is not a single system; there are many cell and not other brain cells? One way to do that is if we different types. This new system is something where a are able to introduce these light-sensitive channelrhodop- transposable element called Tn7 has, over the course of sin protein genes into specific promoter regions so that evolution, co-opted a CRISPR so that it can use the RNA they’re only expressed in the cell type of interest. That targeting mechanism of CRISPR to spread itself to viruses has been a major challenge. Neurons are postmitotic, so or plasmids. By studying the molecular mechanism of the traditional way of incorporating DNA through homol- ’ this, we realized that it s a potentially programmable ogous recombination is very inefficient. We needed a new way to be able to introduce DNA into the genome. One way to do it. of the major hurdles of gene editing is we can cut DNA, One of the potential applications of CRISPR transpo- but introducing DNA into the genome in a precise way has sase is to introduce genes into specific sites. If you wanted been challenging. Using these transposable elements that to control parvalbumin interneurons and not perturb ex- ’ are RNA-guided, there s the potential to develop a new citatory cells at the same time, you can use CAST to genome-editing tool. introduce channelrhodopsin into the parvalbumin promot- Steve Mao: Basically, this is a new CRISPR system that is er region. If you want to visualize a particular type of cell not functioning as an adaptive immune system, but instead in the intestine, you can use CAST to introduce GFP it’s co-opted by T7-like transposons. It’s RNA-guided, [green fluorescent protein] into the promoter region as a ’ and it can insert a large fragment of DNA into a precise unique marker for that cell type. That s one way to use it. location. You’ve shown that it can be reconstituted in vitro, From a therapeutic perspective, there are also exciting meaning you only need minimum host factors. You also applications. Many genetic diseases are caused by single- showed that it can be repurposed in an E[scherichia] coli nucleotide polymorphisms [SNPs]. What that means is system. Do you think that it will work in mammalian that within some exon of an important gene, there is a system? small mutation. People are working on ways to use gene editing to correct these mutations, but the way that the Dr. Zhang: That’s a good question. We’re now exploring existing gene-editing systems work is that you have to many of these different CRISPR-associated transposase introduce specific guide RNAs tailored for individual mu- systems, or what we call CASTs. We are very hopeful tations. Even though conceptually it’s all doable, from a that we’ll have something that can work efficiently in practical standpoint in terms of developing drugs, you mammalian cells so that we can use it for a broad range have to have many different compositions to target the of applications. same disease. CAST can provide an alternative approach to treat disease because if there are multiple mutations that Steve Mao: What specific application can this system be affect the same exon, rather than fixing an individual mu- used for that current systems like Cas9 or Cas12 cannot? tation we can use CAST to incorporate an intact exon and Dr. Zhang: I wanted to have a way to be able to introduce that can address any mutations affecting the same exon. genes into specific positions in the genome so that we can You end up with one composition that can treat a number

© 2019 Zhang. This article is distributed under the terms of the Creative Commons Attribution-NonCommercial License, which permits reuse and redistribution, except for commercial purposes, provided that the original author and source are credited.

Published by Cold Spring Harbor Laboratory Press; doi:10.1101/sqb.2019.84.039560 302 Cold Spring Harbor Symposia on Quantitative Biology, Volume LXXXIV

Published by Cold Spring Harbor Laboratory Press. This is a free sample of content from Cold Spring Harbor Symposium on Quantitative Biology. Volume LXXXIV: RNA Control and Regulation. Click here for more information on how to buy the book. A CONVERSATION WITH FENG ZHANG 303 of different mutations in the same disease group. These are We decided not to use naturally existing cytosine deami- some of the exciting applications. nases because most of the known cytosine deaminases In agriculture, it’s also very exciting: the ability to be work on single-stranded substrates. RNA is naturally sin- able to introduce genes into the same region so that when gle-stranded. If you have a cytosine deaminase, it will be you are breeding these crops, the genes don’t get segre- hard to achieve specificity on a targeted RNA. gated. We can significantly increase the pace at which we Instead, we took a directed evolution approach. We hy- can develop new crops. These are just some of the pothesized that maybe you can turn ADAR [adenosine applications. deaminase, RNA-specific], which normally deaminates adenosine, into a cytosine deaminase. ADAR works on Steve Mao: You mentioned that a lot of these diseases are double-stranded RNA and only deaminates adenosine caused by those SNPs. You are one of the developers of that’s mismatched in a bubble, mispaired with a cytosine. the DNA and RNA base editor systems. Can you tell us That’s how you get single-base specificity. We found out something about the Cas13 RNA base editor? What’s the that you can actually do that with ADAR. You can turn it current progress in this field and what’s the future of this into a cytosine deaminase by just having a mispair with a field? cytosine. You get a C–C bubble. After 16 cycles of direct- Dr. Zhang: Another thing that we have been working on ed evolution, we were able to get a CDAR [cytosine deam- in the lab is to develop new ways to edit RNA. There are a inase, RNA-specific] that has a similar level of activity as couple of advantages to editing RNA. First of all, there are a natural ADAR. We’re pretty excited about that. diseases that are caused by single genetic mutations. For Steve Mao: A lot of your systems have very cool names, those, the treatment strategy would be to convert that dis- like REPAIR. Do you have a cool name for the C-to-U ease-causing variant back to what is found in the majority system? of healthy people. If we’re able to do it precisely and efficiently at a DNA level, then correcting DNA makes Dr. Zhang: Yeah. We’re calling it RESCUE: RNA editing a lot of sense. for specific C-to-U exchange. But then there are other diseases where you may want to Steve Mao: There’s another acronym from your lab. It’sa introduce a risk-modifying allele. For those, the thinking system called SHERLOCK [specific high-sensitivity en- is a lot more complicated. Even though we know that it zymatic reporter unlocking]. That’s actually a slightly dif- confers either increased or reduced risk for some aspect of ferent system. It’s not trying to repair, manipulate, or edit a disease, those variations can often have other more com- the DNA or RNA. Instead, you’re trying to detect the plicated interactions that we don’t know about. For those, nucleic acids. Can you tell us something about that, espe- putting in a DNA change is probably less ideal, because cially its potential for diagnosis? what if it causes a catastrophe and you need to reverse it? RNA editing provides that possibility. You can reverse the Dr. Zhang: SHERLOCK is a diagnostic system that we change. developed by taking advantage of a property of Cas13. Another really exciting way to use RNA editing is Cas13 is an RNA-guided RNA nuclease, but unlike Cas9, changing proteins transiently so that we can modulate it doesn’t cleave just the target nucleic acid. Once it rec- cellular signaling. A number of studies have shown that ognizes the target RNA, it can then also go and cleave if you can modulate the Wnt pathway or the Hippo path- many other RNAs. What that means is that there is ampli- way, you can drive regeneration in liver to get hepatocytes fication in the nuclease activity of this enzyme. We to grow, or we can regenerate photoreceptor cells. When thought maybe you can use this as a way to develop an modulating these proteins, you don’t want the modulation amplifying diagnostic. to be permanent because you will probably end up with a One of the latest iterations of the technology we devel- tumor. You actually want it to be just for a short enough oped is a paper strip test. You can use urine, saliva, or period of time so that you get enough regeneration, but no blood, and then you just put in Cas13 protein and the more than that. These are the reasons that we’re develop- RNA guide that you designed to recognize a Zika virus ing RNA-based systems. or Ebola or influenza or a bacterial pathogen. Within the So far we have developed this one system that we call same reaction, there’s a shorter reporter RNA that has a REPAIR [RNA editing for precise A-to-I replacement], biotin and another molecule called FAM attached to the which allows us to convert adenosines into inosines. Ino- two ends. If Cas13 found the virus or the pathogenic sine is an RNA base that basically functions in the same sequence, it will cleave the pathogenic sequence but it way as a guanosine in splicing and also translation. That will also activate this collateral activity to cleave these means if we can reverse specific adenosines into a guano- reporter RNAs. Then you have biotin and the FAM sepa- sine-like behavior, then we can correct the protein product rated from that RNA linker. or the splicing result that comes from a specific variant. Then you flow this reaction on a paper strip. It ’s not too We’re continuing to work on other types of editors. One different from a pregnancy test strip. It’s got two lines. The of the things that we have been putting the most effort into first line has streptavidin on it, so you’ll bind to biotin. recently is making a C-to-U editor. C-to-U editors allow us Then the other line has an antibody that binds to FAM. to address a different set of changes, but also are very When you flow the reaction, if the pathogenic sequence applicable for modulating protein phosphorylation states. you’re trying to find is not there, then the reporter is going

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