Now, here is a paper that is causing a stir. Published in Nature Methods, researchers from Stanford, the University of Iowa, and Colombia are reporting that CRISPR treatment in mice introduces far more mutations than thought. The news sent the stocks of companies like Editas and CRISPR Therapeutics dropping yesterday, and has led to consternation around the biomedical research field.
First, what this paper reports: after screening four small guide RNAs (sgRNAs), they picked the one with the most activity to go into mice. The two treated mice (F03 and F05) were whole-genome-sequenced at an average coverage of 50x (controls at 30x), and the two sets were compared for insertions/deletions (indels) and single-nucleotide variations (SNVs). And here’s what showed up:
All pipelines showed that F03 harbored 164 indels and 1,736 SNVs (63 and 885 of these, respectively, associated with known genes). F05 harbored 128 indels and 1,696 SNVs (51 and 865 of these, respectively, associated with known genes). . .The same 117 indels and 1,397 SNVs were detected in both of the CRISPR-treated mice, which indicated nonrandom targeting. . .The mutation rate detected in CRISPR- treated mice was substantially higher than that generated by spontaneous germline mutations (3 to 4 indels and 90 to 100 SNVs, de novo, per generation).
Oh dear. If that holds up, that’s clearly going to be a major difficulty in bringing CRISPR-based therapeutics forward, at least with the current state of the art. Just as worrisome, if not more, is that fact that software predictions of the fifty most likely off-target sites of action did not match any of the variants that were actually seen. So it would appear that we have no idea of what’s going on here. As the paper says, with great restraint, “The unpredictable generation of these variants is of concern.”
Now we get to the arguing, though, because the big question is whether these results are correct. They do not match up well with what’s already in the literature on the subject, that’s for sure. On Twitter, Nicolas Bray brought up one of the concerns. His question is a simple one, but it needs to be answered: the two treated mice were siblings, while the control was (apparently) more distantly related. How many of these variations, then, can be ascribed to what the mice started out with? The control animal was from the same inbred strain, but still – the number of variations seen in this paper is way off what others have reported. Sam Sternberg noted that the paper (and its supplementary information) is not very clear about the relationship between the mice, and also suspects that many of these mutations are from the founders and not the CRISPR treatment per se. Meanwhile, Gaetan Burgio pointed out that the experimental details say that “Briefly, an sgRNA- expressing plasmid had been coinjected, into FVB/NJ zygotes, with the single-stranded oligodeoxynucleotide (ssODN) donor template and Cas9 protein to generate mosaic F0 founders“. Plasmids themselves, he notes, are known to cause mutations, since they have much longer half-lives than RNA or protein, and he says his own lab’s work with direct injection of sgRNA and Cas9 protein showed far fewer mutations, in keeping with the rest of the literature. Another potential problem has been brought up by many observers: this study has an n of 2, with one control animal. That’s a pretty thin platform on which to build the “CRISPR Is Doomed!” monument.
So there are a number of reasons to wonder if these results are real. If they are, other questions arise about the newer Cas9 variant enzymes and various sgRNA designs, but I’m not even going to think about those, to be honest, until this result has been replicated and given a thorough going-over. The implications are too big to start running around in circles just yet. There will be time for that, if needed. . .
Update, in the interests of full disclosure: after publishing this post and looking at the moves in the stock prices of both EDIT and CRSP yesterday and today, I found my own doubts convincing enough to have bought some of both as a short-term trade. I have no idea what their long-term prospects are, but their sudden drops due to this news may not be justified. On the rare occasions that I’ve taken a position in any sort of individual biopharma stock, I’ve noted it on the blog, and will continue to do so. Now you all can watch me lose money in real time. . .