Blog / Making the Cut

Why preprints in biology?

Jacob Corn

I’m going to take a step away from CRISPR for a moment and instead discuss preprints in biology. Physicists, mathematicians, and astronomers have been...

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I’m going to take a step away from CRISPR for a moment and instead discuss preprints in biology. Physicists, mathematicians, and astronomers have been posting manuscripts online before peer-reviewed publication for quite a while on arxiv.org. Biologists have recently gotten in on the act with CSHL’s biorxiv.org, but there are others such as PeerJ. At first the main posters were computational biologists, but a recent check shows manuscripts in evo-devo, gene editing, and stem cell biology. The preprint crowd has been quite active lately, with a meeting at HHMI and a l33t-speak hashtag #pr33ps on twitter.

I recently experimented with preprints by posting two of my lab’s papers on biorxiv: non-homologous oligos subvert DNA repair to increase knockout events in challenging contexts, and using the Cas9 RNP for cheap and rapid sequence replacement in human hematopoietic stem cells. Why did I do this, and how did it go?

There’s been some divisive opinions around whether or not preprints are a good thing. Do they establish fair precedence for a piece of work and get valuable information into the community faster than slow-as-molasses peer review? Or do they confuse the literature and encourage speed over solid science?

In thinking about this, I’ve tried to divorce the issue of preprints from that of for-profit scientific publication. I found that doing so clarified the issue a lot in my mind.

Why try posting a preprint? Because it represents the way I want science to look. While a group leader in industry, I was comfortable with relative secrecy. We published a lot, but there were also things that my group did not immediately share because our focus was on making therapies for patients. But in academia, sharing and advancing human knowledge are fundamental to the whole endeavor. Secrecy, precedence, and so on are just career-oriented externalities bolted on basic science. I posted to biorxiv because I hoped that lots of people would read the work, comment on it, and we could have an interesting discussion. In some ways, I was hoping that the experience would mirror what I enjoy most about scientific meetings – presenting unpublished data and then having long, stimulating conversations about it. Perhaps that’s a good analogy – preprints could democratize unpublished data sharing at meetings, so that everyone in the world gets to participate and not just a few people in-the-know.

How well did it go? As of today the PDF of one paper has been downloaded about 230 times (I’m not counting abstract views), while the other was downloaded about 630 times. That’s nice – hundreds of people read the manuscripts before they were even published! But only one preprint has garnered a comment, and that one was not particularly useful: “A++++, would read again.” Even the twitter postings about each article were mostly ‘bots or colleagues just pointing to the preprint. I appreciate the kind words and attention, but where is the stimulating discussion? I’ve presented the same unpublished work at several meetings, and each time it led to some great questions, after-talk conversations, and has sparked a few nice collaborations. All of this discussion at meetings has led to additional experiments that strengthened the work and improved the versions we submitted to journals. But so far biorxiv seems to mostly be a platform for consumption rather than a place for two-way information flow.

Where does that leave my thoughts on preprints? I still love the idea of preprints as a mechanism for open sharing of unpublished data. But how can we build a community that not only reads preprints but also talks about them? Will I post more preprints on biorxiv? Maybe I’ll try again, but preprints are still an experiment rather than a resounding success.


PS – Most journals openly state that preprints do not conflict with eventual submission to a journal, but Cell Press has said that they consider preprints on a case-by-case basis. This has led to some avid preprinters declaring war against Cell Press’ “draconian” policies, assuming that the journals are out to kill preprints for profit motives alone. By contrast, I spoke at some length with a senior Cell Press editor about preprints in biology and had an incredibly stimulating phone call – the editor had thought about the issues around preprinting in great depth, probably even more thoroughly than the avid preprinters. I eventually submitted one of the preprinted works to a Cell Press journal without issue. Though I eventually moved the manuscript to another journal, that decision had nothing to do with the work having been preprinted.

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Thinking about threats

Jacob Corn

In early February the Worldwide Threat Assessment listed CRISPR as a dual-use technology, one that could be used for either good or bad (think nuclear power)....

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In early February the Worldwide Threat Assessment listed CRISPR as a dual-use technology, one that could be used for either good or bad (think nuclear power). At the end of that same month, a delegation from the intelligence community asked to meet with me.

Five years ago, I would never have dreamed of typing that previous sentence. Now it’s a day in the life.

I flew back from a Keystone meeting a day early (this one was my science vacation, just for fun) to meet with the group. I really didn’t know what to expect from the delegation. I had visions of people dressed in severe suits who wore dark glasses indoors and could read my email at the press of a button. Instead, I was treated to a lively, well-rounded group of scientists, ethicists, and economists. I could have imagined any one of them walking the halls of UC Berkeley as faculty. Though one’s business card was redacted in thick black sharpie (no joke).

We were  joined by Berkeley’s Director of Federal Relations and had an outstanding discussion lasting a few hours. I learned a lot from the group about how government educates itself and came away very impressed with the people the U.S. government charges with looking in to emerging technologies.

But I do have a point to make in bringing up this unusual visit.

As scientists, I think we should work responsibly with our new gene editing capabilities and be honest about the potential dangers. The whole point of next-gen gene editing is that it’s fast, cheap, and easy. I have undergraduates volunteering in my lab who edit their first genes within a month of joining. That’s exciting, but can also be scary. I agree with the threat assessment that CRISPR could be used for bad things, since it’s just a tool. In fact, it might even be possible to accidentally use CRISPR in a bad way. Think AAV editing experiments designed for mice that accidentally also target human sequences.

But like I said, gene editing is just a tool. A hammer can build a house, but it can also hit someone on the head. Likewise, gaining one tool doesn’t make everything easy. Try building a house with only a hammer.

Just because we now have democratized gene editing doesn’t mean that bad things will start popping up left and right. Bacterial engineering has been around for a long time, but it’s still hard to do bad things in that arena. There are many other barriers and bottlenecks in the way, and the same is true for bad guys who might try gene editing.

So what should we do? As gene editors, I think we should closely and enthusiastically engage with appropriate agencies. This includes federal and state bodies, and even local groups like campus EH&S. We should also be instilling a culture of responsibility and safety in the lab, even above and beyond normal safety. It’s one thing for a postdoc to remember their PPE, but it’s another thing to think to ask, “Should I talk to someone before I do this experiment?” Security through obscurity is not the way, but sometimes it really is better to first talk things through in a very wide forum. Remember the outcry about the H5N1 flu papers… 

The idea is not to scare people. The technology isn’t scary, and gene editing really isn’t new. It’s just easier and cheaper now, which changes the equation a bit. We should be open about risks and proactive about managing them, otherwise they’ll be managed for us.


Apologies for the long delay between posts. I was teaching this last semester and also trying to get three papers out the door. 

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WaPo Guest blogging

Jacob Corn

Instead of our regularly scheduled (in reality intermittently posted) blog on this site, I’ll direct you to a guest blog I wrote for the Washington Post...

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Instead of our regularly scheduled (in reality intermittently posted) blog on this site, I’ll direct you to a guest blog I wrote for the Washington Post. The topic is one I’ve touched on before: the biggest impact of Cas9 will not be in the clinic, but in its ability to accelerate fundamental biological discovery across numerous labs. Which is not to say that curing genetic disease is a small thing. It’s a sea-change for our relationship with our own genomes and something I’m personally very passionate about. But I think it’s undeniable that thousands of labs all using democratized gene editing to make new discoveries will snowball in a big way.

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SCR-What?

Jacob Corn

If you’re following the use of gene editing for sequence replacement via aka homology directed repair (HDR), you probably noticed the two papers in ...

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If you’re following the use of gene editing for sequence replacement via aka homology directed repair (HDR), you probably noticed the two papers in Nature Biotech simultaneously reporting the use of a ligase IV inhibitor called SCR7 to improve the process. The idea was to suppress non-homologous end joining (NHEJ), thereby tipping the balance towards HDR. This sounds great in theory and the results looked pretty good in the two papers. 

But now there’s some confounding news out from Tocris Bioscience, first reported by Derek Lowe (to the best of my knowledge). Tocris is pretty careful and did their homework when making their own SCR7 for sale. And it turns out that the published structure of SCR7 is somewhat at odds with the actual compound. SCR7 instead turns into a pyrazine. 

scr7 SCR7
scr7pyrazine pyrazine

We tried SCR7 long ago (actually a while before the NBT papers came out), but never really had any luck with it in a variety of settings. When the papers appeared we did a double-take and re-tried using the exact same sources and similar conditions, again with no luck. It’s true that we typically do HDR using single stranded donors in human cells, which is not exactly the use case described in the NBT papers. But now I’m wondering what really underlies the observed SCR7 activity. Is it NHEJ inhibition? If the compound lot originally identified as a LigIV inhibitor was also actually the pyrazine, then it’s a pretty clear cut case and one can just substitute the altered structure for the original. But if the LigIV inhibitor is not the HDR enhancer, then things quickly get confusing. We’ll certainly go back and try again with the defined pyrazine, but it’s likely that the compound in the first two papers and from the original supplier were just the pyrazine. All of this on the heels of a papers out in NAR and Nature Communications indicating that SCR7 doesn’t do much at all and one should instead use a Rad51 activator called RS-1. The plot thickens.

 

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Cas9 flaps

Jacob Corn

To date, most of my blog posts have talked about big picture things surrounding the business of science. But today it’s time for real data. Out today in Nature...

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To date, most of my blog posts have talked about big picture things surrounding the business of science. But today it’s time for real data. Out today in Nature Biotechnology is our paper on new insights into Cas9 mechanism and their use to enhance precise sequence replacement in human cells.


 

TL;DR Cas9 “flaps” out part of the DNA target and you can take advantage of that to increase the frequency of HDR.


 

We started with a very simple question: what happens between Cas9 cutting and the appearance of genome edits? This could be answered in a variety of interesting ways, including elucidation of DNA repair mechanisms. We chose to go even simpler and figure out how Cas9 and sgRNA interact with and dissociate from a DNA target.

Our first surprise came when we found that catalytically inactive dCas9 takes a very long time to dissociate from DNA. As in, six hours or more! Far longer than one might expect from an enzyme that’s basically a retargetable restriction enzyme. Our second surprise came when we found that active and inactive Cas9 behave identically in this respect – even after Cas9 cuts a DNA target, it stays bound for six hours. This length of time was significant to us, since it made sense of a mystery. When one makes double strand breaks in eukaryotic cells using radiation, they are repaired very rapidly. But when Cas9 makes double strand breaks it instead takes (wait for it) about six hours for them to be repaired.

flappycas9

Using labeled DNA molecules, we found that Cas9 globally holds on to both sides of a cut duplex. That is, neither half duplex floats away. But we next wondered about the more fine-grained nature of the complex after cutting. That’s when we got our third surprise. It turns out that when Cas9 holds onto the four strands of the cleaved duplex (think about that one for a moment), it’s only holding on tight to three of them. The DNA strand opposite the PAM on the non-target strand (the one not bound by the sgRNA) is flapping in the breeze. So much so that in vitro we could anneal a complementary piece of ssDNA. dCas9 does the same thing, but since it doesn’t cut the target DNA there’s now an intact DNA bubble formed opposite the sgRNA. If that description is leaving you cross-eyed, here’s a picture of what I mean (gray is Cas9, blue is the sgRNA, yellow is the PAM, red are the active sites, and white x’s are the place where Cas9 is tightly holding the target).

The ability to anneal complementary single stranded oligonucleotides to a DNA flap put us in mind of recent work from Nancy Maizels’ lab, which described a nick repair structure in which single stranded repair templates annealed to one side of a flap. Turning to human cells, we asked if oligonucleotide donors capable of annealing to the Cas9 flap could be used for sequence replacement. And we were very pleasantly surprised. The Cas9 flap is on the order of 20-30 nucleotides, and indeed we found that editing donors complementary to the flap by about 30 nucleotides consistently worked the best of all donors. Absolute frequencies varied, but at one locus we reproducibly observed up to 60% sequence replacement. Making the complementary region shorter decreased efficiency as one would expect, and making it longer also caused a decrease, we think because it would need to intrude into the intact duplex outside of the flap.

Finally, we decided to try a crazy experiment. We wondered if the bubble formed by catalytically inactive dCas9 could be used to anneal a single stranded donor in cells, and if that cell could be tricked into using it for repair. By targeting three dCas9s to a precisely spaced region matching the length of a single stranded donor and all on the same strand of DNA, we did observe sequence replacement rates of a bit less than 1%. This is by no means a large number, but it was attained without any of the error-prone repair that normally accompanies Cas9 cutting. We still don’t know the mechanism underlying dCas9 editing, but it could be very useful to tackle genetic diseases in which sequence replacement holds some kind of fitness advantage but error-prone repair of the gene would be disastrous (e.g. if breaking the gene is worse than leaving the mutation alone).

I’m very curious about the unexpected flappy mechanism of Cas9 and wondering if it plays some part in Cas9’s role in CRISPR anti-viral biology. I’m also more and more intrigued in the mechanism by which single stranded donors are used by the cell. Eric Hendrickson and Nancy Maizels have some great stories on that front, and my lab has some data that seem to shed some light. On a more practical note, Cas9 is currently very good at breaking genes, but its ability to precisely introduce mutations has lagged behind. I’m optimistic that these simple approaches to designing a sequence replacement experiment we discovered, which don’t rely on chemical manipulation of cells, can be useful in tackling a variety of genetic diseases. More on that soon.

This manuscript was put together by a stellar team: postdoc Chris Richardson, technician Jordan Ray (very soon on his way to grad school), former lab manager Gemma Curie, and postdoc Mark DeWitt. Congrats to them!

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Both fast and safe?

Jacob Corn

By now I’m sure most people who might read this blog have read about the clinical trial tragedy in France, in which several people were seriously injured (one...

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By now I’m sure most people who might read this blog have read about the clinical trial tragedy in France, in which several people were seriously injured (one left brain dead) during a FAAH inhibitor dose escalation study. To the best of my knowledge, this is the worst such event during a clinical trial since the cytokine storms associated with TGN1412. And highly apropos to gene editing, let’s not forget Jesse Gelsinger.

 I bring up this horror because genome editing is still new but racing towards the clinic. Anyone motivated to help patients with new therapies, whether based on genome editing or otherwise, should closely examine clinical failures to learn as many lessons as possible. I’m sure a detailed analysis of the trial will be forthcoming, but what could gene editors learn from an disastrous enzyme inhibitor neuro trial?

Foremost, I think we need to think hard about safety testing for in vivo gene editing. There are currently several unbiased methods to assess off-target editing events, and some of these methods look pretty good as labs pit them against one another. If working in an ex vivo setting where one can sequence modified cells, one could certainly assure oneself that a reagent is clean. And an early safety study could test how edited cells that do get cleared from the body are tolerated.

But what about therapies that would modify a gene in situ? Differential dosing of an editing reagent might affect more or fewer cells. But it could also induce more or fewer off-targets. And unlike a traditional therapeutic, after editing has happened that patient can’t go off treatment. Instead, any negative effects become part of an individual’s genetic makeup and would need to be treated like a brand-new genetic disease.

These kinds of fundamental questions make me somewhat nervous when I hear groups declare that they’ll take Cas9 into the clinic by 2017. Therapeutic discovery takes courage, since there are real people hoping for cures at the end of the road. Those patients should be both the motivator and brakes for new therapies – how can we help them as quickly as possible while simultaneously taking care that we don’t make things worse?

I want to get there as rapidly as anyone, but while the first group to take Cas9 into the clinic will no doubt be famous in the biomedical community, the first seriously negative event (god forbid) would be infamous in a much broader sense. We need to give dots all the i’s and cross all the t’s along the way.


A few hours after I wrote this, I started to wonder if I come across as a Chicken Little on this blog. That’s probably a bit funny to those in my lab, since I’m usually relentlessly optimistic about gene editing and encourage them to be boldly entrepreneurial in their research. Maybe there’s something about the internet that brings out my opposite side.

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Translating the statement on human gene editing

Jacob Corn

Scientists, bioethicists, policy makers, and more met Dec 1-3 at the national academies for the International Summit on Human Gene Editing. The topics were...

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Scientists, bioethicists, policy makers, and more met Dec 1-3 at the national academies for the International Summit on Human Gene Editing. The topics were well chosen, the speakers were eloquent, and the summit ended with a responsible and forward thinking statement about the future of gene editing.

But what does it mean? The summit statement is very clear to a scientist, but in some places is opaque as mud to the lay person. For a topic such like human gene editing, it’s important that everyone know where we are and where we’re going. And so, here’s a translation of the international summit statement on human gene editing. This won’t be quite as simple as Randall Munroe’s Thing Explainer, but I’ll strive for simplicity. The original statement is in blockquote italics, while the translation is in normal text. 


TL;DR Gene editing is going to be great, but we should hold back for now on changing humans in ways that would be passed on to future generations.


 

On Human Gene Editing:
International Summit Statement
Scientific advances in molecular biology over the past 50 years have produced remarkable progress in medicine. Some of these advances have also raised important ethical and societal issues – for example, about the use of recombinant DNA technologies or embryonic stem cells. The scientific community has consistently recognized its responsibility to identify and confront these issues. In these cases, engagement by a range of stakeholders has led to solutions that have made it possible to obtain major benefits for human health while appropriately addressing societal issues.

Fundamental research into the ways by which bacteria defend themselves against viruses has recently led to the development of powerful new techniques that make it possible to perform gene editing – that is, precisely altering genetic sequences – in living cells, including those of humans, at much higher accuracy and efficiency than ever before possible. These techniques are already in broad use in biomedical research. They may also enable wide-ranging clinical applications in medicine. At the same time, the prospect of human genome editing raises many important scientific, ethical, and societal questions.
After three days of thoughtful discussion of these issues, the members of the Organizing Committee for the International Summit on Human Gene Editing have reached the following conclusions:

Biology has done a lot of good for human health through things like new medicines. But science moves fast, and sometimes we learn how to do something before we’ve really had a chance to talk about whether we should do it. Newly developed ways to edit genes fall into that category. Using gene editing, it is now easy to precisely change the DNA of a cell or even an animal. This could include curing genetic disease in humans. Gene editing can be done in specific organs in adults, so that people with a genetic disease are themselves cured in a specific way but don’t pass the cure on to their children. But gene editing could also be done in eggs just after fertilization, which means both an entire baby would be edited and that baby would eventually pass the edits on to their own children. This is a big deal, and we should talk about the ramifications of this ability.

1. Basic and Preclinical Research. Intensive basic and preclinical research is clearly needed and should proceed, subject to appropriate legal and ethical rules and oversight, on (i) technologies for editing genetic sequences in human cells, (ii) the potential benefits and risks of proposed clinical uses, and (iii) understanding the biology of human embryos and germline cells.  If, in the process of research, early human embryos or germline cells undergo gene editing, the modified cells should not be used to establish a pregnancy.

Basic research should not be hindered, but needs to follow the rules. Human gene editing research should go ahead in three areas: i) how to do gene editing in humans to cure disease, ii) where gene editing would be most useful and what might go wrong during editing, iii) how do we humans even have babies in the first place and why do some people have trouble having babies? If scientists use gene editing to ask basic research questions about the biology of eggs, or sperm, those edited eggs or sperm should not be used to make a baby. This means that edits will not be passed on between generations.

2. Clinical Use : Somatic. Many promising and valuable clinical applications of gene editing are directed at altering genetic sequences only in somatic cells – that is, cells whose genomes are not transmitted to the next generation. Examples that have been proposed include editing genes for sickle-cell anemia in blood cells or for improving the ability of immune cells to target cancer. There is a need to understand the risks, such as inaccurate editing, and the potential benefits of each proposed genetic modification.  Because proposed clinical uses are intended to affect only the individual who receives them, they can be appropriately and rigorously evaluated within existing and evolving regulatory frameworks for gene therapy, and regulators can weigh risks and potential benefits in approving clinical trials and therapies.

Gene editing could be used in the treatment of many diseases. This could involve using gene editing as a tool to make a new drug, or actually editing cells in patients who have a genetic disease. In this latter case, gene editing should only be used in cells that are not eggs or sperm. This means that individual patients will receive the benefits of the treatment, but the gene edits will not be passed on to their children. Restricting gene editing to cells other than eggs and sperm is important because we don’t yet understand the long-term risks associated with gene editing. There is a decent medical regulatory framework in place that could be applied to gene editing medicines, and we should build experience in gene edits that are not passed between generations.

3. Clinical Use: Germline. Gene editing might also be used, in principle, to make genetic alterations in gametes or embryos, which will be carried by all of the cells of a resulting child and will be passed on to subsequent generations as part of the human gene pool. Examples that have been proposed range from avoidance of severe inherited diseases to ‘enhancement’ of human capabilities. Such modifications of human genomes might include the introduction of naturally occurring variants or totally novel genetic changes thought to be beneficial. Germline editing poses many important issues, including: (i) the risks of inaccurate editing (such as off-target mutations) and incomplete editing of the cells of early-stage embryos (mosaicism); (ii) the difficulty of predicting harmful effects that genetic changes may have under the wide range of circumstances experienced by the human population, including interactions with other genetic variants and with the environment; (iii) the obligation to consider implications for both the individual and the future generations who will carry the genetic alterations; (iv) the fact that, once introduced into the human population, genetic alterations would be difficult to remove and would not remain within any single community or country; (v) the possibility that permanent genetic ‘enhancements’ to subsets of the population could exacerbate social inequities or be used coercively; and (vi) the moral and ethical considerations in purposefully altering human evolution using this technology.

Gene editing could also be used to change human sperm, eggs, or early embryos. If this were done, the gene edits would be passed down to the edited person’s children, and are thus trans-generational. This could be done to cure disease, but there’s also a possibility of making gene edits in an attempt to improve humans, for example by making an edit associated with lower cholesterol. There are many potential problems to think about, such as: i) what happens if the trans-generational gene editing procedure goes wrong or not all cells get edited? ii) what happens if a trans-generational gene editing procedure goes right, but bad things happen over the long term between generations because we don’t understand human genetics very well? iii) what will future generations think about being the product of gene editing? iv) once we make trans-generational gene edits, it will be very hard to go back because people intermingle so often. v) trans-generational gene editing could be very unfair, and might extend “rich get richer” societal problems into our very genes, and vi) trans-generational edits could change our own evolution more than societal influences, and it’s not clear that we actually want to or should do that.

It would be irresponsible to proceed with any clinical use of germline editing unless and until (i) the relevant safety and efficacy issues have been resolved, based on appropriate understanding and balancing of risks, potential benefits, and alternatives, and (ii) there is broad societal consensus about the appropriateness of the proposed application. Moreover, any clinical use should proceed only under appropriate regulatory oversight. At present, these criteria have not been met for any proposed clinical use: the safety issues have not yet been adequately explored; the cases of most compelling benefit are limited; and many nations have legislative or regulatory bans on germline modification. However, as scientific knowledge advances and societal views evolve, the clinical use of germline editing should be revisited on a regular basis.

Therefore, we should not do trans-generational editing at this time. But we should keep asking ourselves hard questions about trans-generational editing on both scientific and societal levels:  i) how safe is trans-generational editing and do the benefits outweigh the risks? ii) does everyone agree that we should do trans-generational editing? iii) how would we regulate trans-generational editing?

4. Need for an Ongoing Forum. While each nation ultimately has the authority to regulate activities under its jurisdiction, the human genome is shared among all nations. The international community should strive to establish norms concerning acceptable uses of human germline editing and to harmonize regulations, in order to discourage unacceptable activities while advancing human health and welfare.
We therefore call upon the national academies that co-hosted the summit – the U.S. National Academy of Sciences and U.S. National Academy of Medicine; the Royal Society; and the Chinese Academy of Sciences – to take the lead in creating an ongoing international forum to discuss potential clinical uses of gene editing; help inform decisions by national policymakers and others; formulate recommendations and guidelines; and promote coordination among nations.
The forum should be inclusive among nations and engage a wide range of perspectives and expertise – including from biomedical scientists, social scientists, ethicists, health care providers, patients and their families, people with disabilities, policymakers, regulators, research funders, faith leaders, public interest advocates, industry representatives, and members of the general public. 

We need to keep talking about gene editing. And by “we”, that means everyone, even across national boundaries. And everyone in all walks of life need to be involved in the conversation. Gene editing has the potential to have a huge impact on medicine and could finally cure genetic diseases. But we must move carefully and with lots of thought, so don’t rush into things.

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An interesting thought about Precision Medicine

Jacob Corn

I was recently in The Netherlands for a few days, at an event for ERIBA. One of several interesting conversations at dinner merited a speculative blog post....

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I was recently in The Netherlands for a few days, at an event for ERIBA. One of several interesting conversations at dinner merited a speculative blog post.

The topic under discussion was precision medicine (a recurring theme on the blog), and accidental motifs that could develop while trying to ensure that new drugs end up in the hands of those most likely to benefit. As a reminder, the purpose of precision medicine is the development of protocols such as biomarkers that predict drug efficacy, with the goal of matching patients to medicines. The use of Trastuzumab in HER2-positive breast cancer is a classic example.

Now all medicines must go through three phases of clinical trials, culminating in demonstration of efficacy in Phase III. Companies often develop biomarkers for use during clinical trials (these can but do not necessarily take the same form as the final precision approach), in the hopes that enrolling the “right” people will improve chances for a successful trial. It’s not kosher to run a trial and then post-hoc decide that responders and non-responders can be separated to yield statistical significance – it’s something you need to do right up front.

But even before the frequent use of molecular diagnostics, patients in trials tend to be biased in an important way – they are often very sick (especially in oncology). Which brings us to the dinner topic at ERIBA: is disease severity an accidental diagnostic in modern precision medicine? Are the marker levels chosen to accompany targeted medicines restrictive to the point where they unintentionally select only the sickest patients? This might be OK on the level of an individual drug, since it strives to only give drugs (expensive and with potentially nasty side effects) to those most likely to respond. But in aggregate across the industry, does it quietly bias away from moderate disease and towards the worst severity? Are we working our way into a corner that appropriately pairs medicines only at the extreme?


PS – Yes, it’s been a long time since the last post. I’ve been juggling a lot of travel with getting three papers out the door. The rest of the year will probably continue to be light on the blog. But teaching next semester will reduce my travel and give me plenty of opportunities to write blog posts while procrastinating lesson plans. 

 

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Where will be the biggest impact of Cas9?

Jacob Corn

As I’ve written about before, CRISPR/Cas9 is taking off. And that’s been picked up in the popular press by the likes of The Economist, The Wall ...

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As I’ve written about before, CRISPR/Cas9 is taking off. And that’s been picked up in the popular press by the likes of The Economist, The Wall Street Journal, and Wired. Cheap, fast, and easy gene editing is indeed changing the world. But not in the way you might immediately think.

At the IGI, my lab is working on improving gene editing technology, particularly the ability to change existing sequences or insert new ones (so-called HDR as opposed to NHEJ-mediated disruption of sequences), and we’re using that tech to develop reagents that can cure genetic diseases. But we’re also using Cas9 for fundamental biology, to discover new things about the world around us.

And that’s where the big, long-term impact lies. Through translational research, gene editing shows promise to completely change our relationship with genetic disease. Those potential cures are a big deal, and the IGI is committed to helping usher in that future. But researchers everywhere can also now use gene editing tech to ask fundamental questions about how biological systems work. And they can do it in areas that were previously technically difficult or impossible. This turns an exclusive practice, where people with questions had no way to get answers, into an inclusive one, where many questions lead to many answers. I often call this the “democratization” of gene editing.

The very power of democratization lies in the unexpected benefits of basic research. CRISPR/Cas9 gene editing tech itself came from asking basic questions about bacterial immunity, not from searching for the next best thing in gene editing. Some people are excited about Cas9 because it allows them to do their research in model systems much more quickly – flies, worms, mice, and so on. That work is leading to some incredible discoveries, no doubt. But researchers now have the ability to perturb or insert genetic sequence into systems where that was previously difficult or impossible – butterflies, the malaria parasite, goats, plants, and more. That means we’ll learn more about how these systems work, with the possibility of incredible surprises and exponentially expansion of our knowledge of the wealth of biology.

That’s the big long-term benefit of Cas9 genome engineering – the exponential growth of knowledge about the world around us by enabling fundamental research. Why should a non-scientist care about non-model organisms and seemingly quirky discoveries?

Here’s an example happening right next door to me: A colleague at Berkeley is working on a particular type of crustacean that happens to regenerate limbs incredibly well. Lop one off, and it grows right back. Can you imagine the future possibilities if we figured out the fundamental biology behind this incredible regeneration and if it could be translated to humans? But this crustacean previously “had no genetics”, meaning it was impossible to ask which genes give the thing its regenerative ability. Working with the IGI, my colleague has recently gotten Cas9 editing to work in his favorite organism, meaning he can now go figure out what gives it the power to regrow limbs. He can also ask questions about body patterning (how do limbs end up where they are), neurobiology (how does it see things around it), and even behavior (are instincts linked to genes?).

Speaking of behavior, Hopi Hoekstra is asking mind-blowing questions about gene-behavior links in another non-model organism, the Peromyscus mouse. Through incredibly hard work she’s homed in on a genetic region linked to burrowing behaviour, and the last time Hopi visited Berkeley she told me that she’s hoping Cas9 will let her get all the way to the causative gene.

As a technical note, the Cas9 RNP (in vitro transcribed guide RNA + purified Cas9 protein) seems to be the enabling change for non-model systems. No need to figure out promoters, terminators, expression conditions, etc for your favorite organism. Microinjection or electroporation delivers the RNP into eggs/zygotes/embryos/etc, and from there Cas9 just acts like the targeted nuclease that it is, with double strand break repair accomplished by the host cells. My lab has figured out ways to make Cas9 and in vitro transcribe guide RNAs in high throughput – check out our protocols on protocols.io and feel free to get in touch if you have questions. Also be on the lookout for our 2nd annual CRISPR/Cas9 how-to workshop in summer of 2016.

So Cas9 is changing biological research in more ways than you might think. Yes, it’s going to lead to incredible therapies. Yes, it’s going to lead to new insights into the way our own bodies and cells work. But it’s also going to continually surprise us, because now we’ll be doing much more than looking under the lamp post.

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A wish list for science

Jacob Corn

File this one under “crotchety grumblings.” I started this post as a wish list for the future of CRISPR/Cas9, but came to the realization that ...

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File this one under “crotchety grumblings.” I started this post as a wish list for the future of CRISPR/Cas9, but came to the realization that it could apply to just about any sexy field. So instead, this is a wish list for biology in general. These points are related to the dichotomy between the business of science and the pursuit of knowledge. I’m sure some of these points have come up in previous posts, but I want to get this off my chest in one place. I understand that the business of science has very logically led us to a point where the items below are natural and common, but it doesn’t make them right. I’m also sure that most of these have been discussed in one form or another elsewhere, but I’m adding my $0.02. Consider this a reminder for students and postdocs who might be reading the blog.

At heart, all of the below come down to one thing. As scientists, we are trying to discover new things about the world. One assumption to most of our work is that there is an external, objective truth for us to observe. We see that truth through a glass darkly, and hence we attempt to put the external world in a form that can be understood from human context. We can do no else. To purposefully distort that truth does real harm and sets everyone back, but has unfortunately become de rigueur in some contexts.

  1. Rickety Tricks. Doing tech dev or poking in to biology by taking advantage of overly-specific proof of concept just creates frustration and eventually casts a black cloud over whole fields. Science is typically bleeding edge, and it can be very difficult to know the boundaries of an approach while it’s being developed. But that does’t justify purposefully take advantage of “tricks” to look successful while simultaneously knowing that there’s no clear path anywhere other than the one-off proof of concept experiment. This cynical approach might inflate one’s own CV, but generates mistrust for whole fields and poisons the potential for real world application.
  2. Reproducibility. Related to the above, there have been several articles about the reproducibility problem in pre-clinical science. I won’t bother to link to all of them, since by this point the problem is well-recognized and I could only accidentally miss some attributions. The general problem is that perverse incentives for publication mean that many pieces of research are just plain wrong. This isn’t necessarily outright fraud, but it can stem from squinting at the data in just the right way and hoping, because positive results are good but there’s no Journal of Negative Results. I saw much less of this when I was in industry, perhaps because the motivations are much different. If one is just aiming to get a paper out the door, then one might not particularly care if a discovery is Right or Reproducible (though one should!). But in industry, that paper (or internal discovery) is just the start for what might be a very expensive and long-running drug discovery project, and there are many incentives for robustness and reproducibility in that case.
  3. Over Hyping. Be honest those who consume your science (both primary articles and press releases + interviews) and yourself. As we approach an age of open science, it’s becoming easier for non-scientists to access research articles and learn about cutting-edge developments. This is a very good thing, since non-scientists are frequently very motivated, for example by family members suffering from a disease. These people should have access to all the information they might want. But coming from outside the business of science, they might not understand the hype machine. Promising the world based on an incremental advance might increase one’s chances for getting into Nature, but has very real consequences by potentially giving people false hope. I feel very strongly here, since I’ve personally answered emails and telephone calls from people who saw certain overhyped papers and called me to ask about cures for their sick child. It’s heart-breaking. And if compassion for fellow humans doesn’t motivate, consider that the government (including people who set budgets!) have little tolerance for science that promises then doesn’t deliver.

It’s a beautifully sunny day on the weekend, so I’ll stop here before I dip into too much of a funk. But I’ll end by saying that these are things we can fix. There is no Science with a capital S. We are scientists who are building science as we go along! We are a global community – we write our own papers, referee our own papers, write our own blog posts, form our own societies, and organize our own conferences. It’s all up to us.

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Questions and/or comments about Corn Lab and its activities may be addressed to:

JACOB.CORN@BIOL.ETHZ.CH

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