standard Short-Half Life Cas9 Improves Mutagenesis

Reducing the half-life of Cas9 has some positive and unexpected impacts on the  efficiency of mutatgenesis, according Tu et al (2017) in a paper they just published in Scientific Reports.

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Macaca fascicularis. Image credit. André Ueberbach

Tu et al were primarily interested in applying Cas9 mutagenesis to non-human primates and from their perspective mosaicism following mutagenesis was a liability.  By mosaicism the authors were referring to embryos having different mutations in different types of cells in the same organism.

While this may not be as much of a concern among insect biologist what Tu et al . found was somewhat unexpected and consequently may be of interests to insect biologists.

Ubiquitin-proteasome pathway

Ubiquitin-proteosome pathway. Image Credit

Tu et al were motivated to find a way to reduce mosaicism in non-human primate embryos and created a short half life Cas9 protein by adding a ubiquitin-proteasome degradation signal to the N-terminus of Cas9.

This modification reduced the half life of Cas9 but did not affect the ability of the protein to cleave DNA.

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Primate early embryogenesis.

Compared to wild-type Cas9, ubiquitin-Cas9 (Ubi-Cas9) has a significantly reduced half life.  At 24hrs post transfection of HEK 293 cells only about half of the Ubi-Cas9 protein was detectable relative to time-0, while 80-80% of the wild type protein was still present at this time.

Tu et al injected 1-cell stage embryos with either wt- or Ubi-Cas9 mRNA or protein, allowed the embryos to develop to the 4-cell stage then isolated and genotyped each cell individually for the presence of mutations.  What they observed was about the same frequency of embryos that had at least 1 mutated cell at the 4-cell stage – about 75%.  However, the number of embryos that had all four cells mutated was significantly higher when Ubi-Cas9 mRNA or protein was used.  In the context of mosaicism, Ubi-Cas9 resulting in fewer mosaic embryos.

It seems that one could also interpret their findings as resulting from increased activity of Ubi-Cas9, resulting in mutagenesis occurring before the first cell division more frequently than wt-Cas9.

Of course it is not known if short half-life Cas9 will have the same effect in other organisms but testing in insects will be important.

Zhuchi Tu, Weili Yang, Sen Yan, An Yin, Jinquan Gao, Xudong Liu, Yinghui Zheng, Jiezhao Zheng, Zhujun Li, Su Yang, Shihua Li, Xiangyu Guo & Xiao-Jiang Li, (2017) Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryo. Scientific Reports 7, Article number: 42081 (2017) doi:10.1038/srep42081

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