Tuesday, June 9, 2015

@LluisMontoliu guest post! about low off-target #CRISPR rates in embryos.

Lluis Montoliu is very well known to the transgenic mouse community and and expert on all things related to mouse genetic engineering.  Therefore I was very happy when he sent a message to the ISTT mailing list describing the recent in-depth confirmation that yes, CRISPR can have extremely low off-target cleavage rates in mouse zygotes, as alluded to in one of my previous posts (and probably true for human embryos too despite a recent report).   

He has kindly agreed to let me re-post his message on this blog.  Thanks Lluis!  You can also follow @LluisMontoliu on Twitter, and check out his own CRISPR information web site and also his lab's web page.  

Subject: [ISTT_list] Off-target mutations are rare in CRISPR-Cas9-edited animals

Dear colleagues,

Anyone who has already carefully analyzed mice edited by CRISPR-Cas9 will have confirmed the almost absence of off-target mutations, in contrast to what was initially predicted and announced. Off-target mutations appear to be very rare in genome-edited animals, if present at all. We and other have usually taken a shortcut and have opted to analyze a limited number of off-target sites in our genome-edited mice, selecting a few off-target sites (those with higher score, higher probability to be modified) and cloned and sequenced these DNA pieces from all founder animals generated, just to find that none of them appear to be modified.

Now, Bill Skarnes and collaborators (Sanger Inst., Hinxton, UK) have done the proper experiment, the experiment we and other would have liked to do, namely: whole deep genome sequencing on CRISPR-Cas9-edited mice. And they found the same result. Even if you don't select for sites and you review the entire genome there appear to be no off-target sites that are modified by the CRISPR-Cas9 reagents.
Off-target mutations are rare in Cas9-modified mice Vivek Iyer, Bin Shen, Wensheng Zhang, Alex Hodgkins, Thomas Keane, Xingxu Huang & William C Skarnes Nature Methods 12, 479 (2015) doi:10.1038/nmeth.3408 http://www.nature.com/nmeth/journal/v12/n6/full/nmeth.3408.htmlhttp://www.ncbi.nlm.nih.gov/pubmed/26020497

Hence, these amazing tools are far more precise and accurate than initially considered, particularly when these are injected as RNA (orprotein) into zygotes (into fertilized oocytes). Of course, this does not mean that you should not aim to obtain and analyze at least two independent mutant/edited animals to confirm the robustness of the associated phenotype, as you would be doing with any other genome alteration you would be producing.  And, bear in mind, the whole picture might be different in cells, particularly if they are transfected with DNA plasmids transcribing Cas9 constantly and in high amounts,  and hence providing lots of opportunities (and time) for this endonuclease to cut elsewhere, other than the expected targeted sequence. In contrast to what happens in zygotes, where a limited amount of Cas9 RNA (or protein) is used, does the job and vanishes away.

Further enjoy your genome-edited animals!

Dr. Lluis Montoliu
Investigador Cientifico - Research Scientist CSIC Centro Nacional de Biotecnologia (CNB-CSIC) Campus de Cantoblanco C/ Darwin, 3
28049 Madrid (Spain)


  1. How do you square the incredibly low/nonexistent OT mutation rates seen in this Skarnes paper with the very high OT rates seen in the Liang paper using tripronuclear human embryos? Increased gestation time? Different sgRNAs with different OT rates? These explanations seem off to me... I'd love your thoughts. Thanks!

  2. Please read my April 29 blog post about the Liang paper which directly addresses this issue. I am fairly certain the OT rates in the human embryos were actually much lower than they claimed. http://mortlockcrispr.blogspot.com/2015/04/the-reported-off-target-effects-in.html