Clearing CRISPR of off-target suspicion

mathcrisprFaster, better, cheaper is a motto adopted by Nasa that perfectly fits CRISPR as well. The most popular technique for genetic modification, in fact, has the reputation of being quick, affordable and precise. This deserved good name was unexpectedly tarnished by a study questioning the technology precision, published in the June issue of Nature Methods. However, reports about CRISPR’s demise have been greatly exaggerated, to paraphrase Mark Twain. Just over a month later, three analyses challenging the controversial study are already available in the pre-publication archive bioRxiv, and Nature Methods has alerted its readers about the criticisms received by publishing an editorial note which could turn into a retraction.

CRISPR is described as revolutionary because, among other things, may find many applications in the medical field by helping to correct genetic illnesses (some twenty clinical trials are already ongoing or about to start). If it actually caused hundreds of unexpected off-target mutations, as Stephen Tsang and colleagues argued in their Nature Methods paper, it would mean that thousands of researchers understand pretty nothing about the functioning of the technique they are using. The therapeutic potential of the approach would plummet, and the path from the lab bench to the patients’ bed would be hindered. But if Nature Methods is wrong and the reported mutations are not CRISPR’s fault, then much ado about nothing: CRISPR remains the best technique for genome editing. The second scenario is much more plausible according to comments released by several specialists of the field, analyses prepared by two biotech companies (Editas and Intellia), and now also the paper posted online on July 5 by researchers from Harvard and MIT. Luca Pinello, who leads a computational laboratory at Massachusetts General Hospital-Harvard Medical School, re-analyzed the original results partnering with the labs of Martin Aryee and Keith Joung. “Our appraisal is that the mice used in the experiment were not similar enough for the purpose of the analysis (particularly the control mouse) and that the mutations reported do not seem to relate to CRISPR activity but to pre-existing differences”, he told us via email. So there is a straightforward explanation for the alleged anomalies, which would clear the technique of any suspicion: it is extremely likely that many sequence variations found in the two CRISPR-treated mice were inherited from a common ancestor and were already shared in common before treatment. The control mouse lacks them not because it was not subject to CRISPR editing, but because it is less closely related genetically to the first two animals than they are to each other. In short, the experiment was not well designed to answer the question: how many off-target mutations are caused by CRISPR? In recent years, several studies have given reassuring answers to the same query. Furthermore, according to Pinello and colleagues’ calculations, the probability that the technique would cause the same unexpected mutations being exactly the same length in two mice is less than one in a trillion. The message sent is loud and clear: the authors of the Nature Methods study should “restate (or at least temper) the title and conclusions of their original paper or provide properly controlled experiments that can adequately support their claims. Not doing so does a disservice to the field”.

 

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