Thursday, June 08, 2017

CRISPR Mediated Off Target Mutations in Mice

In a very recent paper in Nature Methods , researchers used CRISPR technology to repair a gene mutation related to blindness in mice. But what they found was a large number of off target mutations compared to what is typically expected.

An article in The Conversation discusses some of the possible explanations for these findings. Some critics have suggested that the large number of off target mutations could be related to the specific methods used to control the activity of the Cas9 enzyme, which would impact the number of cuts/edits made in the host DNA that occur.

Others have pointed out that there are various flavors of CRISPR, and even temperature can impact enzyme activity and off target impacts, as well as better and worse methods of detection of off target mutations.    

Xiang et al. (2017). Temperature effect on CRISPR-Cas9 mediated genome editing. J. Genetics & Genomics. (Apr 20) 44(4):199-205.

High-fidelity CRISPR–Cas9 nucleases with no detectable genome-wide off-target effects. Nature 529, 490–495 (28 January 2016)

Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases Genome Res. 2014. 24: 132-141

When it comes to food crop applications, critics of CRISPR technology, as well as older recombinant DNA technologies have been largely concerned with genetic disruptions. These criticisms imply that genetic disruptions indicate increased risk to consumers. I think a very relevant question in this regard (give or take the Nature Methods paper) is related to the comparative differences in genetic disruptions between CRISPR mediated genetic improvements vs traditional plant breeding methods including mutation breeding (chemical and radiological mutagenesis used in conventional and organic foods).

Given that previous risk management/regulatory reviews and agencies have found little evidence to restrict or highly regulate traditional and mutagenic crop improvement methods, if genetic disruptions for CRISPR mediated crop improvements are comparable the argument for increased scrutiny of CRISPR based crops is weakened. Previous research indicates that genetic disruptions for traditional plant breeding methods are actually greater than what we observe in recombinant DNA methods.

Batista R, Saibo N, Lourenço T, Oliveira MM. Microarray analyses reveal that
plant mutagenesis may induce more transcriptomic changes than transgene
insertion. Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3640-5. doi:
10.1073/pnas.0707881105. PubMed PMID: 18303117; PubMed Central PMCID: PMC2265136

Baudo MM, Lyons R, Powers S, Pastori GM, Edwards KJ, Holdsworth MJ, Shewry PR. (2006). Transgenesis has less impact on the transcriptome of wheat grain than conventional breeding. Plant Biotechnol J. 2006 Jul;4(4):369-80

 To reiterate two important questions in relation to the Nature Methods paper as it may apply to food seem to be:

1) are the drastically higher than expected off target mutations based on sound methods/application of CRISPR

2) What is the weight of evidence comparing genetic disruptions in CRISPR vs conventional crop improvement methods.

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