√ Crispr, Human Genetic Modification, A Needed Course Correction

Are designer babies made using CRISPR or other genetic modification technologies closer to reality today? If so, what exactly should we do about it?

Researchers can use CRISPR to genetically modify just about any organism or its cells, but targeting humans is the subject of the most intense discussion including using CRISPR in the human germline for heritable “editing” or genetic modification of humans. This could in theory be done via human embryos or human germ cells with mostly existing technology.

CRISPR studies on healthy human embryos are apparently now being conducted in the UK and Sweden by Kathy Niakan and Frederick Lanner and potentially others (see here and here) strictly for research (not reproduction).

I support the use of CRISPR for early human embryo research, but it needs to be done carefully and with proper bioethical oversight as well as transparency. To my knowledge to date, no team working on CRISPR in health human embryos has been willing to even say what genes they are targeting so that’s a masalah in my view. I would guess at least one group is aiming to knock out key pluripotency genes such as OCT4 and NANOG in otherwise normal human embryos. Will we learn dramatically more about human early embryo development that is distinct from findings in mouse embryos on the same genes? I hope so, but you never know.

We also need to realize that the moment one of these studies on intentional genetic modification of healthy human embryos is published there is a good chance there will be a political firestorm in response from some quarters. This is in part why seemingly pro-heritable human modification arguments popping up need to be responded to and discussed as even the strictly research-focused CRISPR work in healthy human embryos will lead to invocations of germline human modification.

√ Postdoc Job Opening: Cancer Stem Cell Epigenomics Knoepfler Lab

UC Davis School of Medicine

An NIH and foundation-funded postdoctoral fellow position in the Knoepfler Lab at UC Davis School of Medicine is open. The focus of research will be on cancer and stem cell epigenomics. Studies will include functional genomics assays such s ChIP-Seq, chromatin configuration, CRISPR genetics work, and cell biological research.

Qualifications:

Applicants must have a PhD, an MD, or both. Preference will be given to applicants who do not already have postdoctoral experience, but who have a strong track record as graduate students of biological research including publication of their work. Genomics, next generation sequencing, bioinformatics, cancer biology, and/or stem cell experience would be a plus. Excellent written and oral communication skills are required.

To apply: E-mail knoepfler@ucdavis.edu a 1-page overview of research experience and career goals, a CV, and contact information for 3 references.

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√ In First For U.S., Mitalipov Reportedly Crispr’D Human Embryos It Was Great

More CRISPR’d human embryos, but this time in America?

MIT Tech Review is reporting that Oregon scientist Shoukhrat Mitalipov has used CRISPR on human embryos in his lab in the US. Apparently a paper is in the works on this.

While details are sketchy and some specifics remain to be clarified to be sure of what’s the deal here, this Tech Review report appears generally accurate based on what I’ve heard so this appears to be the first reported use of CRISPR on human embryos by an American lab.

Boom, the door is open.

Based on the little we know and rumors out there, it appears likely that Mitalipov’s team, while creating embryos from sperm of men with genetic disease, used CRISPR on otherwise healthy, viable embryos, but that remains to be confirmed.

Tech Review seems to be gushing a bit too much for my taste though on this CRISPR’d human embryos development and making some pretty big assumptions about how it turned out. For instance, this quote sounds like hype to me:

“Now Mitalipov is believed to have broken new ground both in the number of embryos experimented upon and by demonstrating that it is possible to safely and efficiently correct defective genes that cause inherited diseases.”

Safely and efficiently?

Isn’t that “safely” part jumping the gun? You’d have to make a person from the CRISPR’d human embryos to really know if it was safe or not.

Apparently as to the latter claim of “efficiently”, Mitalipov’s team reportedly used CRISPR on “tens” of human embryos and reportedly found better efficiency and lower rates of mosaicism, where down the developmental path days after CRISPR introduction only some cells have edits, while others don’t.

Let’s wait for the data. Also, I’d recommend reading my ABCD plan for handling human genetic modification research, which suggests being transparent about the genes being targeted for one thing.

The US National Academy’s panel on human gene editing outlined many reasons for caution on the use of CRISPR in human embryos but left the door ajar and now Mitalipov seems to have gone through. Since federal funding of embryo modification isn’t clearly allowed in the US, presumably this team used private funding of some kind.

Overall, is this development a good thing?

It’s a mixed bag. I’ll reserve more definitive judgement until the paper is actually published and we can all discuss it. However, it is very important not to hype the use of CRISPR in human embryos as an easy or safe path to preventing genetic disease, and also to point out the existing proven technologies of embryo screening (PGD, PGS) that could be used instead right now to achieve almost all of the same goals.

Meanwhile researchers in China are reportedly doing more CRISPR on human embryos, and researchers in the UK and Sweden have apparently already been CRISPR’ing healthy human embryos based on their respective governmental approvals…strictly for research purposes.

What comes next?

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√ Who Leaked Mitalipov Crispr Human Embryo Paper?

The new CRISPR human embryo paper from Shoukhrat Mitalipov is stirring things up, but then there’s also the murky back story as to how the news of this as yet unpublished paper got leaked in the first place. The actual paper is still not out and from what I understand hasn’t even been officially released by Nature in embargoed form to journalists. So what the heck happened?

Specific details of the Mitalipov paper popped up a couple of days ago on Tech Review and on a UK news outlet (both by the same author, Steve Connor) perhaps as much as a full week before the paper is set to come out. Leaks at the White House and leaks in science?

You can see the “iNews” front page at right. Connor’s piece in Tech Review in my view was a bit too upbeat about the manuscript in terms of “safety” in particular, but I haven’t even seen the manuscript so I can’t be sure and Connor’s scoop on this admittedly was interesting. I’m excited to read the actual CRISPR human embryo paper too and without it much of what is out there remains somewhat unclear.

We all want to know more about the data, but many seem now to be asking the same kind of bigger picture question too, ‘who leaked it?’

In his iNews piece, Connor quotes at least one anonymous source:

“Although Mitalipov and his colleagues are under a strict confidentiality agreement with a leading scientific journal, which has scheduled to publish the work next month [August], we understand from other sources that the study breaks new ground in demonstrating the feasibility of creating genetically modified babies. “I’ve heard Mitalipov has done it. He’s successfully done genetic modification of human embryos. The quality of the work was high,” said one senior scientist who wished to remain anonymous.”

Connors also quoted a Salk Institute scientist, apparently a co-author with Mitalipov, by name about the paper in the Tech Review piece:

“Reached by Skype, Mitalipov declined to comment on the results, which he said are pending publication. But other scientists confirmed the editing of embryos using CRISPR. “So far as I know this will be the first study reported in the U.S.,” says Jun Wu, a collaborator at the Salk Institute, in La Jolla, California, who played a role in the project.”

I asked myself, “How would anyone know to contact this one particular person Jun Wu regarding an unpublished paper not even released by the journal in the first place?”

It’s also notable that Connor’s quote says “scientists” as in plural.

The apparent fact that Nature has not even officially released embargoed copies of the paper to the press and won’t do so until early next week  means that no embargo was broken since no embargo yet exists (weird situation, huh?), but something unusual happened here.

I reached out to Connor, but he was unable to discuss sources regarding this story, which is totally understandable.

Does it really even matter if an important science paper or its key findings are leaked out a few days or a week in advance? If it does matter (and gut feeling is that it does on some levels), what are the risks to science and scientists? Or is it more about the journal itself maintaining control of the timing and the initial media coverage?

What do you think?

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√ Big 5 Questions Anticipating Mitalipov Human Embryo Crispr Paper

Modified open Wikimedia image

The human embryo CRISPR paper with Shoukhrat Mitalipov as senior author is coming soon.

It will reportedly be focused on the use of CRISPR to genetically modify viable human embryos for reversal of a disease-associated mutation. While strangely press already broke early on this paper last week, as much as a week before the paper comes out, and that press suggests very positive data, how clear will that be from the paper itself?

Here are some ideas on the big 5 questions (some include nested questions) likely to come up from this now eagerly-anticipated paper:

• 1. How much residual off-target activity was there and if it was very low, how was that achieved? How low does it have to be where someday this hypothetically could be used for actual reproduction with a strong expectation of safety and efficacy? Was WGS done on many embryos to look comprehensively for unpredicted off-target activity?


• 2. At the sasaran site, were indels ever created instead of the desired precise edits?


• 3. Is the goal of Mitalipov and the larger team on this paper to actually use this approach in the germline for heritable human gene editing within say 5 years? If so, how will it be limited to just that and not other applications such as infertility or trait modification? What kind of ethics review went into this paper and how does it compare to the NAS report on human gene editing?


• 4. Will others use this paper as a foundation to argue for a more permissive policy on human germline edits?


• 5. And/or conversely will there be a political reaction here in the US (and maybe elsewhere) leading to restrictions on human CRISPR research or other areas of innovative research in the US?

Note: this post was written prior to my seeing the human embryo CRISPR paper.

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√ Review Of Mitalipov Paper Crispr’Ing Human Embryos: Transformative Work On The Edge

In the same way perhaps that some excited relatives or parents-to-be both gush and worry about a baby before it is even born, our field has been transfixed for a week by the Mitalipov paper on CRISPR’ing human embryos even though the paper just now came out.

Figure 3a Ma, et al. Nature, 2017

Now that the paper is out, we can take a closer look at this “baby” and for us scientists that involves giving it a critical review. In science, “critical” often means a thorough once over with a somewhat skeptical eye, but not necessarily a negative one. Indeed, my overall take on this paper is positive. It is quite strong technically and has many elements that are innovative even though 3 previous studies have already tried CRISPR gene editing in human embryos of various kinds.

This new paper “Correction of a pathogenic gene mutation in human embryos” is in a different category than the other ones in its approach and implications. It is quite rigorous and contains generally very thorough analyses. There are still some very important open questions and I believe there are some issues with perhaps some small overstatements, but by and large this paper is top-notch.

What are the key take homes from this study? Let’s look at my big five questions and now some attempts at answers.

1. Off-target activity? They didn’t detect any. Overall some statements in the paper are perhaps a bit overexuberant such as statements of “abolished mosaicism” (they actually did find a mosaic embryo). I also do not believe they can be quite so confident about “no off-target activity”, when as best as I can tell they did not look thoroughly in enough embryos and cells and in an unbiased manner at the whole genome to really be sure about this. Still their finding of no detectable off-targets so far is impressive.


2. Indels? The Indels present at the sasaran locus even under ideal circumstances in just under 30% of embryos are a big deal and remain a major problem. See part of Figure 3A above in the experiment where they found the 27% of embryos having Indels.


3. Clinical intent overall and NAS report on human gene editing? These folks make no bones about their hope to one day use this kind of technology for human reproduction with specific clinical goals. While they also included some appropriately cautionary statements about future clinical use, at the same time some language such as envisioned possible “rescue” of embryos was potentially concerning. I am highly skeptical that gene editing in the human germline can make sense as a safe and more effective approach than embryo screening by PGD and PGS.


4. Will this paper embolden others to dive in to this space too? Perhaps it will catalyze more research on CRISPR in human embryos. That could be both good in the sense of learning more, but also risky in terms of not everyone doing such a good job as these authors did in considering ethical implications and even in the technological level they used. Also, where will everyone get eggs and sperm for studies?


5. Will this paper lead to a negative, perhaps political reaction? I do think it is better than 50-50 that there will be some kind of political reaction from conservatives about this development and possibly some kind of proposed restrictions.

And more questions pop up now that I’ve read the paper.

10,000 eggs or embryos? What if to get to a clear answer on whether this technology is safe and effective it takes 1,000 or 10,000 human embryos, and hence eggs? CRISPR’ing human embryos at that scale might be needed to get clearer answers on efficacy and safety. Does the hypothetical potential benefit of pursuing human germline editing justify that? These are not every day run of the mill cells. Procurement and use of human eggs and embryos requires extra consideration.

What about epigenetics? Does CRISPR’ing human embryos lead to epigenetic effects that have biological outcomes, some of which may be negative?

Flying blind OK? Another thing to keep in mind is that if this technology were taken in a reproductive direction, you could not analyze the embryos in depth like they did in this paper. You’d have to largely fly blind. At best you could pluck a few blastomeres off for analysis, but you have to leave most of the embryo behind to actually get a baby.

Overall, this is an impressive paper, but one that also raises the stakes on future CRISPR use in humans.

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√ 4 Key Reasons Mitalipov Paper Doesn’T Herald Safe Crispr Human Genetic Modification

We can be confident that human genetic modification via CRISPR’ing of embryos soon will be safe and effective after that new exciting Mitalipov team paper, right?

Wrong.

The reality is far more complicated and interesting on the tech side.

In a nutshell, I see the paper as a significant scientific, but not necessarily medical advance.

The media coverage overall has painted too rosy and simplistic a picture of the Mitalipov paper. Setting aside tough bioethical and societal issues regarding human genetic modification for the moment in this post, there are 4 major, mostly overlooked technical reasons why the Mitalipov CRISPR human embryo paper is unlikely to herald safe human genetic modification.

1. I dream of Gene-y. The “best” disease-associated gene to sasaran with CRISPR in humans would be one that CRISPR always “hits” perfectly. The Mitalipov team probably gave deep thought to picking the gene they did for CRISPR targeting. It’s a gene going by the name MYBPC3 that is associated with a fatal type of heart defect called familial hypertrophic cardiomyopathy (HCM). That disease association is an important reason for trying CRISPR on the mutation in this gene. However, most likely at least one big technical reason they chose MYBPC3 was because it had so few predicted possible CRISPR off-target sites (meaning other places in the genome found by computer algorithms that CRISPR might stray to and make damage). If I’m right about this, then they were just being smart in that choice and I would have probably done the same thing in their shoes. But the targeting of this hand-picked gene means that the upbeat findings on accuracy with reverting mutant MYBPC3 are probably unlikely to be representative of efforts to “gene edit” disease-causing mutations more generally. By analogy would you like to throw a dart at a dartboard where the bull’s-eye takes up fully two-thirds of the dartboard (MYBPC3?) or where the bull’s-eye is just one thirtieth (some other disease-causing mutations)? For many diseases you may in effect have no choice but to go for the far tougher bull’s-eye because of the nature of the particular gene and its disease-associated mutation.

Isn’t it possible for all major disease-associated gene mutations that CRISPR will work as well (or even better) than MYBPC3? Nope, that’s not the way the real world works unfortunately.

In fact, one of the authors (Jin-Soo Kim of the Institute for Basic Science in Daejeon, South Korea) specifically emphasized to Nature News the low predicted off-target rate of this gene:

“Even so, Kim notes that the CRISPR–Cas9 error rate can vary depending on which DNA sequence is being targeted. The MYBPC3 mutation, in particular, was predicted to produce relatively few opportunities for off-target cutting.”

It is also possible the team picked MYBPC3 because its mutation is very small (only 4 base pairs and in theory easier to repair) or they had the human sperm donor lined up with this mutation.

A combination of factors most likely guided the team in gene choice.

Other gene mutations are going to be far tougher because they will be prone to dramatically more off-targets (see below) and perhaps more Indels (see below). Many mutations are large and complicated as well.

2. Off targets there, but not oft found? There’s also the likely possibility that the team unintentionally missed finding some off sasaran effects of CRISPR that were in their modified embryos, but not found because of how they did the sequencing. The very next quote in that Nature News piece is from Keith Joung on this concern:

“Just because the team did not find off-target changes does not mean that the changes aren’t there, cautions Keith Joung, who studies gene editing at the Massachusetts General Hospital in Boston. “Although this is likely the widest examination of off-target effects in genome-edited human embryos performed to date,” he says, “these investigators would need to do much more work if they wanted to define with certainty whether off-target effects do or do not occur in this context.”

Give the Mitalipov team credit for the screening they did do, which was relatively a lot, but much more is needed to be even close to sure about this, especially if one has clinical hopes as this team does.

3. Indel pain in the neck. The metaphor behind the language and concept of “gene editing”, the preferable phrase to “genetic modification” within the scientific politics of today (admittedly I sometimes use this phrase myself), suggests precision changes as do other metaphors like “genome surgery”. However, even if CRISPR-Cas9 avoids off-targets and sticks to the gene of interest, it can often make these things called “Indels” short for insertions and deletions right in sweet spots in genes. Indels often functionally kill genes entirely rather than precisely changing them. The Mitalipov team found Indels more than 1/4 of the time in embryos even under their most optimized conditions. The rate of Indels needs to be at or very close to zero to begin to have any reasonable chance of clinical safety of using CRISPR in the human germline. Plus, at other mutant genes that may be targeted in human embryos, Indels may be much more commonly created by CRISPR than at MYBPC3. We just don’t know.

4. Mosaic monitor. Mosaicism with CRISPR is where the cells of the embryos after introduction of CRISPR-Cas9 machinery don’t all have the same genome any more. For example, some cells in the same embryo may be normal and some mutant. There’s a genomic gemisch. That’s generally not good for ultimate health so mosaicism would be unsafe for hypothetical clinical applications of CRISPR in humans. One of the most impressive things about the Mitalipov CRISPR embryo paper was that they reportedly got rid of most (just 1 mosaic found) mosaicism in CRISPR’d human embryos. However, this was essentially just a very narrow test case study with one male sperm donor and one or a few women who donated eggs. Thus, the embryos used were very similar. More broadly, there is likely to be substantial variability in propensity to embryo mosaicism in part related to the unknown characteristics of specific gamete donors.

PGD reminder. Beyond the technical challenges, the fact is that almost anything CRISPR could do of medical use heritably in humans is already achievable using embryo screening including by the common, proven method called PGD. Think of it this way by analogy. Let’s say you have 8 books with 4 having errors and 4 not having errors. You have a very reliable way to know which books are which, and you only need 1 correct book. Do you try to correct the 4 errant books knowing that you could easily make more errors yourself in trying to fix the error, or just pick from one of the easily identifiable perfect ones?

Bottom line. For all these reasons, we should all be more cautious in making meaning from this one paper. There’s a long tough road ahead with a marathon of challenges (and the authors rightly acknowledged many of these so kudos to them) if one has clinical aspirations for CRISPR in the human germline.

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