Measuring off target mutations during CRISPR gene editing

Ygggdrasil

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CRISPR gene editing is a newly developed tool that allows researchers to easily make changes to an organism's DNA. There is much interest in using this technology in clinical applications, but there have been major concerns surrounding the safety of the technology, including whether the gene editing approach leads to unintended, off-target mutations elsewhere in the genome. Today, two independent teams of researchers developed methods to measure the rate of off target mutations due to CRISPR gene editing and published their results in the journal Science. One team studied the question in mice and another team studied the question in rice, and the two studies came to largely the same conclusions.

The study in mice took embryos that had undergone a single cell division, at which point the embryo consists of only two cells. In once cell, they injected the reagents for CRISPR gene editing along with a marker that would color the cells red, while they left the other cell untreated. After the embryo had developed for a few days, they could then take the embryo, disassociate the cells, sort the red (edited) cells from the uncolored (non-edited) cells, then perform DNA sequencing to detect mutations in each population of cells.

They tested various different CRISPR variants, including the classic CRISPR method (which cuts the DNA to introduce errors during repair of the DNA) or newly developed base editors (which directly change DNA base pairs without cutting the DNA). They found that the classic CRISPR method and one type of base editor that can change A-T base pairs to C-G base pairs essentially introduced no detectable off target mutations (the number of new mutations found in the edited cells was indistinguishable from the number of new mutations found in the unedited cells). However, they detected a much higher off target mutation rate for another type of base editor (which changes C-G base pairs to A-T base pairs). The study in rice (which only compared the two types of base editors) found similar results.

The studies indicate that, when correctly deployed, some CRISPR methods can be used for gene editing with minimal risk of off target mutations, and indicated how improvements can be made to some newer base editing technologies (as the C-G base editor was only invented a few years ago, it is likely that improved versions can be developed).

Zuo et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science. Published online 28 Feb 2019. http://science.sciencemag.org/content/early/2019/02/27/science.aav9973

Jin et al. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science. Published online 28 Feb 2019. http://science.sciencemag.org/content/early/2019/02/27/science.aaw7166

Popular press coverage: https://www.wired.com/story/precise-gene-editing-is-trickier-than-expected-but-fixes-are-in-sight/
 
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Where did the large number of off-target mutations reported from classic CRISPR before came from then?
 

Ygggdrasil

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Where did the large number of off-target mutations reported from classic CRISPR before came from then?
In 2017, Nature Methods published a peer-reviewed paper (Schaefer et al.) in which the authors reported that CRISPR–Cas9 caused unexpected off-target changes in mice. Since its publication, we have been contacted by many scientists challenging this work. Five of these critiques are now published, as peer-reviewed Correspondences, in this issue.

As the authors of these critiques point out, and as confirmed by four independent referees who evaluated both the critiques and the original authors' response to them, the study by Schaefer et al. lacked key controls so that it is not possible to ascribe the observed genomic variants, with reasonable confidence, to CRISPR. The paper has now been retracted to maintain the accuracy of the published record.
https://www.nature.com/articles/nmeth.4664

In particular, the retracted paper examined only two gene edited animals, and as a reference, sequenced the DNA of one animal of the same strain. The choice of reference is problematic because there is extensive variation in the DNA even between individuals of the same inbred mouse strain, and it is likely that many of the mutations that the authors of the 2017 study detected were due to this intrinsic variation.

In contrast, the new method uses cells from the same embryo as the reference, which eliminates issues with inter-individual variation. Mutations can also arise spontaneously throughout the course of embryonic development, but the authors of the new study performed careful controls to measure the rate of spontaneous mutation so that they could accurately measure the excess mutations due to gene editing.
 
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