√ Doubts Raised On Key Points Of Nature Paper On Crispr Gene Editing Of Human Embryos

Is it possible that CRISPR gene editing actually didn’t happen in many of the human embryos in that big Nature paper that made such news a couple weeks back?

Some doubts have emerged that call the main conclusions of the paper into question and argue that more definitive studies are needed to be sure.

Egli, et al. preprint Fig 1e-f

An international team of top scientists led by first author Dieter Egli has responded via a preprint on Biorxiv to that Mitalipov team high-profile Nature paper on CRISPR gene editing of human embryos. Egli, et al. raise the possibility that the CRISPR gene editing as reported in the Nature study may actually not have happened, at least not in every case and perhaps not the way the Ma, et al. paper argued it did (via homology directed repair (HDR)-based CRISPR-Cas9 action specifically depending on interaction between normal maternal and mutant paternal chromosomes).

On one level it isn’t so unusual to see a scientific critique of and technical questions raised about a published paper that made splashy news. However, I see this particular case as a striking turn of events because although the new Egli, et al. piece is very collegial and diplomatic, they convincingly lay out a number of rather compelling reasons why the main conclusions of the Ma paper might be incorrect and the reasons why there may not have been CRISPR gene editing in many of the embryos. To be clear, Egli and colleagues don’t seem to be saying the Ma, et al. paper is definitely wrong, but they describe some quite reasonable ways in which the Ma paper could hypothetically have inadvertently reached incorrect central conclusions. To me these possible alternative explanations just simply make a lot of sense and are things that should have been ruled out as alternative explanations.

The preprint author group includes highly respected scientists (Dieter Egli, Michael Zuccaro, Michal Kosicki, George Church, Allan Bradley, and Maria Jasin) and the questions they raise should be taken seriously. On my first reading of their well-written piece, I was already convinced that their concerns may be valid. Early on in the piece they write this:

“Considering the data presented in Ma et al., alternatives to recombination between homologues are possible and would seem more likely, as the cell biology of fertilized eggs would appear to preclude the direct interaction between the maternal and paternal genomes required for inter-homologue HDR. Therefore, clear evidence for a novel linkage of maternal and paternal alleles is an imperative for any embryo that would be considered for future implantation.”

What are the main issues articulated in the Egli paper that raise at least some doubts about the main conclusions of Ma, et al. paper?

First, the Ma paper makes the unusual argument that HDR-driven gene editing occurred after CRISPR-Cas9-induced DNA breaks in the mutant paternal allele essentially exclusively using the normal maternal chromosome as a template within the same 1-cell embryo rather than via an introduced synthetic template. In fact, in some of the Ma paper’s studies no template was included so that CRISPR-Cas9 gene editing had to rely on endogenous DNA in the embryo for HDR. However, Egli, et al. point out that this is exceedingly unlikely because the male and female pronuclei are entirely physically separated in the 1-cell embryo (Figure 1e-f above). How could the maternal and paternal chromosomes have physically come together to mediate this HDR during meiosis? Hypothetically possible? I suppose, but it’s hard to imagine a likely mechanism.

What about HDR later during mitosis? This is theoretically possible as the preprint notes the maternal and paternal genomes come together at that later time: “Merging of maternal and paternal chromosomes does not occur until microtubule action assembles both genomes on a common metaphase plate at the first mitosis…direct interactions between maternal and paternal genomes required for inter-homologue repair do not seemingly occur until embryos enter the 2-cell stage when the two genomes are packaged within the same nucleus.” If gene editing via HDR did occur much later during mitosis (but note that such recombination is thought to occur much less frequently during mitosis than meiosis), the Mitalipov team should have seen dramatically more mosaicism. Importantly, in addition if the gene editing only took place that late, why would they have observed such an apparently large difference in outcomes between MII and zygote injections?

Second, Egli, et al. point out the potential risks of relying as the Ma team did for some assays just on the apparent absence of a detectable mutant allele. Concerningly, there are a number of reasons other than CRISPR gene editing of a mutant allele back to wildtype that could more simply explain why no mutant alleles were detected. The Egli piece argues that one such possibility that wasn’t ruled out is the presence of moderate-to-large deletions resulting from CRISPR activity and eliminating primer binding sites, leading to no amplification of mutant alleles. This may in theory result in only WT alleles being detected leading to the potential incorrect conclusion of gene editing reversion of mutant alleles, when in the fact in that scenario the altered (not repaired) mutant alleles are there but just not detectable.

The preprint mentions unpublished data that such large deletions occur with CRISPR gene targeting in around 20% of gene edited cells. Sequencing to conclusively rule out Indels as a cause of failure to detect mutant alleles in various embryos or cells could be very difficult for a variety of technical reasons, but it could be done. I’m not clear as to whether the extent of genome sequencing in the Ma, et al. paper was enough to be sure.

Third, another alternative possibility discussed is that there sometimes was no contribution of the paternal genome to the zygote and hence no paternal genome present in some later embryos or ES cell derivatives. The Egli, et al. piece describes one possible way that could happen: “Zygotes with a single pronucleus are not uncommon after intracytoplasmic sperm injection, occurring in 10% of fertilization attempts, and are mostly of parthenogenetic origin, containing only the maternal genome.” That makes sense to me as a possible alternative explanation. In addition, it is also possible that “a fraction of embryos derived from successful fertilization with mutant sperm are at more risk of paternal chromosome loss due to the occurrence of the Cas9-induced DSB.” In either case, lack of a paternal genome being present would give the incorrect appearance that mutant paternal alleles had been gene edited back to a WT state. There is some data in the Ma, et al. paper showing paternal contribution to certain embryos/ES cells, but not from enough samples to be sure overall.

I asked Gaetan Burgio, Group Leader in Genetics of Host-pathogens interactions and Genome editing, and Head of the Transgenesis Facility at The Australian National University, for his reaction to the Egli, et al. preprint:

“This preprint manuscript raises serious concerns about Ma et al. paper published in Nature over the finding that the deleterious mutation was corrected by “self repair” from the non-disease copy of the genome. They also argue that many underlying and important changes (large deletions) were undetected after editing in these human embryos.”

While the potential remarkable claim of inter-homologous repair in the Ma, et al. paper could still turn out to be correct, my sense is that it seems more likely that some of the time other events occurred such as those possibilities outlined in the Egli, et al. piece. It concludes:

“In summary, the conclusion of gene correction in human embryos requires further investigation, including direct verification. Efficient inter-homologue recombination in embryos in which the maternal and paternal genomes are undergoing distinct biological programs and in distinct nuclei would be a stunning biological finding.”

It will be important to see how the Mitalipov team responds (e.g. with additional data?) to the Egli, et al. piece. Perhaps there are good reasons why they think they are still correct.

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√ Mitalipov Briefly Responds To Egli Preprint, Stands By Human Embryo Crispr Pub Findings

Shoukhrat Mitalipov points to an image of a CRISPR edited human embryo inside an incubator at the Center for Embryonic Cell and Gene Therapy in Portland, Ore.
Rob Stein/NPR.org

There has been a wave of intense discussions both in the public domain such as on Twitter and behind the scenes over the new Egli, et al. preprint that challenges the main conclusions of the Ma, et al. Nature paper from Shoukhrat Mitalipov’s lab.

Ma, et al. reported CRISPR gene editing of human embryos, arguing for a mechanism of HDR-based gene editing relying on interaction of the maternal and paternal genomes in the early embryos. Egli, et al. presented several alternative explanations — mostly involving what would be artifactual outcomes — for why the Mitalipov team saw what they did in the human embryos.

I’m not an embryologist or recombination guru myself, but I’ve been brushing up on them this week, and still don’t quite get how the HDR between the genomes could have happened. But there is a lot of uncertainty about this situation. For all we know, Mitalipov’s team could generally be right, but we all just need more information to understand why as well as what potentially unusual biology and genetics explains it.

Below is a statement from Mitalipov in response to this situation, where he stands by their main conclusions:

“The study co-authors and OHSU welcome scientific discussion and inquiries of our peer-reviewed study published on Aug. 2 in the journal Nature. Our research uncovered a novel mechanism of DNA repair in human embryos, as well as a method to eliminate mosaicism.

“We recognize that these results must be confirmed by additional studies, and that independent verification of important new findings is a cornerstone of science. We encourage other scientists to reproduce our findings by conducting their own experiments on human embryos and publishing their results.

“We stand by our study’s key finding that human embryos are capable of effectively repairing disease-causing mutations by using a normal copy of the gene from a second parent as a template. We based our finding and conclusions on careful experimental design involving hundreds of human embryos.

“The critique leveled by Egli, et al, offers no new results but instead relies on alternative explanations of our results based on pure speculation. We will respond to their critiques point by point in the form of a formal peer-reviewed response in a matter of weeks.”

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√ Mulling Over That Mitalipov Human Embryo Crispr Pub, Still Many Questions

Modified open Wikimedia image to symbolize an 8-cell CRISPR’d human embryo

What really happened at the DNA level in the experiments in that high-profile CRISPR of human embryos paper from a team led by Shoukhrat Mitalipov at OHSU?

Is the team right that they successfully conducted CRISPR of human embryos to correct a mutant gene, as they reported in their Ma, et al. Nature paper?

Or is the Egli, et al. preprint that came out later in response to the Ma paper more likely to be correct in their implied argument that something else very probably happened instead?

We as a field don’t know yet for sure what molecular events occurred in those embryos, but I believe at this point that the odds are very much that Egli, et al. are right. Hopefully more data will make things clear, but we’ll see. In the meantime, it is useful to think conceptually about what was claimed and place those claims in the context of decades of published biology. That’s what Dieter Egli and colleagues did. More such constructive dialogue and collegial brainstorming about the Ma paper is useful. This kind of process is what science is all about, both pre- and post-publication. If relatively more of it happens prior to publication such as during review, of course, that can save a lot of potential headaches.

The most extraordinary (and fascinating if true) claim made in the Ma, et al. paper was that their use of CRISPR-Cas9 triggered what we might call inter-homolog repair-mediated gene editing and that mechanism was pretty much the only way that CRISPR did its thing in the embryos. What does that mean in English? The idea is that the CRISPR gene correction in the one-cell human embryos was only mediated using the normal maternal chromosome as a template to fix the male chromosome that contained a mutation. If this happened, it is a unique and important finding. However, in their preprint Egli, et al. argued that this proposed phenomenon is very unlikely or maybe even impossible because of the particular spatial nature of chromosome dynamics in the early mammalian embryo.

In that chromosome dance that occurs in the early one-cell embryo, the male and female chromosomes by all accounts remain too far apart to partner up with each other to in turn lead to inter-homologue DNA repair. By analogy imagine two possible dance partners far far away from each other not being able to come together from opposite sides of a ballroom to do a slow dance and engage in conversation. You can’t slow dance with someone 100 yards away, right? Things are going to be even more difficult for inter-homolog repair to occur when the maternal and paternal chromosomes become separated in their own individual pronuclei so in our dance analogy, the two prospective partners would be both far apart and literally in their own bubbles of a sort. It is thought that only much later during metaphase of the first mitosis do the distinct genomes physically interact and if CRISPR acted only at that point or later there could be problems for chromosome segregation and there would almost certainly be more mosaicism.

Mitalipov said a week ago that he still thinks his team is right in an official OHSU statement, but it’s not clear how the CRISPR-Cas9-mediated gene repair could have occurred based on current understanding in the fields of embryology, chromosome dynamics, and DNA repair. So maybe the fields are wrong? That’s not impossible, but the simpler explanation is that the gene editing didn’t happen like they thought.

A new piece from journalist Meghana Keshavan at STAT a few days ago includes fresh quotes from Mitalipov on his team’s paper and the controversy. As quoted in this piece, the first thing that struck me as surprising was that Mitalipov seems very low-key about the whole thing:

“So, I guess this is a pretty startling discovery,” Mitalipov said.

“I actually never knew this would be such a big deal,” Mitalipov said.

“This study has to be tested by time — to learn whether this is a real mechanism or not,” Mitalipov said.

That last quote also suggests much more uncertainty than in the official statement via OHSU. If the central proposed mechanism reported in the Ma paper turns out not to be real, what happens then?

Mitalipov is also quoted there in STAT as trying to explain why others haven’t seen this phenomenon before. Why, for example, in the probably hundreds of thousands of mutant mice made, bred, and studied by researchers over the decades around the world, has no one seen in mice what the Ma paper claims happened in humans? From STAT:

“Mitalipov also has a theory as to why this phenomenon hasn’t yet been seen in other experiments: Most embryonic work is still done in genetically engineered mice. To make good mutant mice, scientists generally sasaran both alleles in a given chromosome — the paternal allele and the maternal allele.

Since both alleles are damaged, there’s no chance for a healthy version to take over and repair (or replace) the broken one. And that, said Mitalipov, may be why no one has previously observed the phenomenon he saw in his lab.

“When you hit both alleles, you can’t see how they repair each other,” Mitalipov said. “So that’s why they never would see it.”

Actually, we generally start with heterozygous (het) ES cells and then het mice. Some researchers more recently are injecting CRISPR-Cas9 into mouse embryos too for gene targeting that could lead to both alleles being mutated, but that’s much less common overall than making and breeding het mice. In other words, we don’t just instantly have all homozygous mutant mice as implied in the above quote.

In addition, the breedings we do should provide plenty of opportunity for maternal correction of mutant paternal alleles, if such a thing was possible. I’ve made quite a few knockout mice and never seen any evidence of spontaneous mutant allele correction. Het x het crosses are the most common we do especially with embryonic lethal mutations, and there would be plenty of opportunity there (and also in Het x WT crosses) for maternal correction of mutant paternal alleles in early embryos (or vice versa). In addition, I’ve never seen anyone else report this kind of spontaneous gene correction via the other corresponding normal chromosome in the germline. Perhaps in his quote Mitalipov was thinking specifically of the kind of mutant mice created using CRISPR in one-cell embryos, which again could sometimes yield two mutant alleles?

Overall, if the early inter-homolog repair between maternal and paternal genomes is a real phenomenon as proposed by Ma, et al., seemingly at least some mouse geneticists would have seen it before and reported it as a novel event, unless it only happens in human embryos and/or only in the context of CRISPR-Cas9. That latter notion of potential CRISPR-Cas9-specificity could in theory be why we haven’t readily seen it in mice, again if it is a real phenomenon. Maybe in the mutant mice to get a spontaneous correction of the mutant gene in question via the corresponding WT allele in embryos, an extra step of a spontaneous DNA double-strand break (DSB) specifically in the mutant allele would be needed to catalyze the process? In the CRISPR context that the Mitalipov team used, Cas9 makes such a DSB at a pre-existing mutant allele. However, in mutant mice in the absence of a nuclease, after the initial mutant allele production phase and during subsequent breedings the mutant allele would not typically then go on to have a new DSB associated with it except in very rare cases, so the embryo may fail to view the gene as damaged and thus not invoke inter-homologue repair? Maybe?

Or maybe in mice, inter-homolog repair does sometimes rarely happen, but we don’t become aware of it because perhaps we just think our mutation didn’t get stably made (or “go germline” as we say), when in fact it was present but then got changed back to WT by inter-homolog repair in one-cell embryos when we bred our initial chimeric mice?

From a bigger picture perspective, inter-homologue repair that evolved specifically for fixing spontaneously damaged alleles in early embryos would risk impairing genetic diversity and leading to loss of heterozygosity. Also, wouldn’t scientists have seen evidence of this in human genetic studies? It should have made for some very unexpected pedigree charts.

Yes, I’m speculating throughout this post and there are a lot of maybes here in trying to model what might have happened in the Ma paper versus what is known from past cell and developmental biology as well as genetics studies. A simpler possibility again is that the Ma paper is just plain wrong, but we don’t know that yet.

Mitalipov has suggested that other labs try to replicate his team’s experiments with human embryos rather than just speculate on what may or may not have happened, but that’s not likely to be easily done. Who else has access to scores or hundreds of human oocytes and/or embryos, and both institutional approval and relevant state and federal laws that allow it? Also, recall that in the U.S. no federal funding can be used for such research and in many other countries there are major constraints or prohibitions on human embryo research. All of this greatly limits possible replication efforts by independent groups.

Unfortunately, the muddy waters related to the claims of the Ma paper aren’t likely to get conclusively resolved one way or another any time soon. A big first step will be to see if the Ma paper team can rebut the concerns of the Egli preprint with new data of their own.

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√ Why We Shouldn’T View The Human Embryo As A Gizmo Even In The Crispr Era

My first job in science was as a lab technician at UCSD School of Medicine and a big part of that job was growing cells called HUVECs or human umbilical vein endothelial cells. We isolated and grew the HUVECs from umbilical cords that we retrieved from the maternity ward of the UCSD hospital, which first entailed getting the placenta and attached umbilical cord as our starting material.

Some people viewed this material as “gross”, but as a newbie scientist I was kind of in awe of the whole process and it made me think about human development starting with human embryos and leading up to birth of babies in a new way. Those HUVECs were integral to proper human development and it was cool even if challenging to grow them.

Now about 25 years later, after working with dozens of human cell types, I have been thinking more about human embryos the last few years, especially as relates to CRISPR, even though I don’t do research on them in my lab.

How should we view a human embryo, both overall societally and within a scientific context?

Is a human embryo just the same as any other research material?

If a lab is working with a 100-cell human embryo is there some kind of equivalency between that and say the dish next to it in that same lab with a cluster of 100 human stem cells? What about 100 human skin cells grown in a lab?

Is a human cell just a human cell?

I don’t think so on either account. However, at times it seems like some research on human embryos (and commentary on it by scientists, philosophers, or others voicing their opinions) reflects a certain nonchalance about it or a sense that no special care or thought was or should be given to this kind of work. I call this a “gizmo” mentality about human embryos. That these are just some “thing” that can be fiddled with and you can always get another one. I believe that kind of mindset should be avoided.

For context, I’m not a particularly religious person so that is not driving my viewpoint here. Also, I personally do not believe that human embryos are people and I’m not a fan of the personhood movement. At the other extreme, human embryos also are not just a collection of random cells either. They aren’t a gizmo.

In my view, research on human embryos, such as introduction of CRISPR into them for gene editing or some other kind of larger scale genetic modification like a “knockout”, requires extra consideration. There should be some advance contemplation about it and ideally consultation with a bioethicist. These extra steps and planning via researchers asking themselves questions and having discussions with others should start early and before you begin the actual embryo research. Better to ask questions early than too late.

For example, where will the human embryos come from and where did the gametes come from to make them?

Were gamete donors or embryo donors properly consented?

Are the embryos “leftovers” from IVF or are they being made purposefully just for research? The latter requires more consideration.

Why are you doing the human embryo research and is there a rock-solid justification for this work? Could you not answer the same research question using mouse embryos for example? You couldn’t address the same research challenge with just stem cells in a dish in the lab?

These aren’t easy questions, but they are important.

Since human gametes and embryos made from them require human donors (and there are risks to donors) and have the potential if implanted to make a human being, you should be extra careful in experimental design, biostats, etc. as well. Also, you need to think in advance about what you will do with the experimental embryos once the project is done if some are to be frozen.

This is all a lot more complicated than just experimenting on every day human cells in a dish, but the extra steps are needed because again human embryos are not your average group of 100 cells. Going the extra mile to do human embryo research properly lowers risks to others and to the research field.

Further, consider how your work might be portrayed in the media, especially if you have some future clinical intent with your human embryo project in years to come such as trying to prevent a genetic disease. Being proactive and thoughtful can help you avoid the risk of being at the center of some controversy. If you have some long-range clinical intent for your work, you should also be very cautious in publicly discussing that and always discuss existing alternatives like PGD for prevention of genetic diseases in future offspring.

It’s encouraging to see that some labs publishing high-profile human embryo papers recently have apparently given it quite a bit of thought and there has been advance institutional review, but some other reports out there and/or statements in the media from those doing such research or individuals commenting on it are concerning. There are clearly some important gaps that need to be addressed in how this work is approached and discussed. Some gizmo mentality can creep in at times in discussing actual human embryo research.

We should also be conscious that as human embryo work such as that using CRISPR becomes more commonplace, as it seems to be trending lately, that doesn’t change the core questions and bioethical concerns. Just because someone for example has CRISPR’d 500 human embryos doesn’t mean those embryos should be viewed as commonplace research materials. Extraordinary mindfulness is always going to be needed for this kind of research even if it becomes more commonly conducted.

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