HomeInVivo Biosystems BlogCRISPRHey People ….its SSSH! time – Getting Easier Integration via Transgenesis Innovation

Hey People ….its SSSH! time – Getting Easier Integration via Transgenesis Innovation

Contrary to what the librarian says to you when you are part of loud conversation, SSSH! time here is referring to the Self-Selecting Safe Harbor (SSSH) tool invented by Zach Stevenson and crew in the Patrick Phillips Lab at the University of Oregon.

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There is a back story here. We are proud of our people at InVivo Biosystems (IVB).  Some, like me, have been hanging around with IVB for quite a long time. Others, like Zach, come and go, but still leave their mark.

Zachary joined us before Knudra Transgencis merged with NemaMetrix, which today is known as InVivo Biosystems.  He was fairly new to genome engineering, but Zach was a quick study.  He became a master of CRISPR-based transgenesis which he leveraged in his next career move – helping him get into graduate school at the University of Oregon.

Zach and the team at Knudra had tasked themselves with the aim of finding better tools for detecting genome integration. We needed efficient systems that helped identify only the animals that have experienced genomic integration. Better yet, the tool would be most effective if only the desired genome integrated strains could survive after exposure to a toxic compound. During Zach’s time at Kundra, the idea floated around a bit, but it never got the legs of experiment implementation to demonstrate its feasibility.

Once in graduate school, Zach teamed up with Megan J. Moerdyk-Schauwecker and Brennen Jamison in the Phillips Lab to get the real world evidence that demonstrated the idea could work.  Their team chose the hygR gene, to determine if a split-hygR gene could be harvested as tool to identify integration in a safe harbor locus (Stevenson et al. G3. 2020)

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The principle is simple – chop the hygR gene into two parts.  Put the long part into the genome of C. elegans and put the other part in your transgene plasmid. Zach did this at the MosSCI ttTi5605 safe harbor locus. This transgenic target strain contains most of the hygR gene but is missing a critical segment needed for creation of a functional hygromycin B phosphotransferase.

Next, their transgene of interest was made in a plasmid that also contains the missing hygR part. The trick now is to have the same sgRNA site in the plasmid and in the edited safe harbor site. The interaction of the plasmid and the genome when injected with CRISPR reagents renders a region of overlap of about 700 bp on each end of the insertion cargo that allows homology repair to do its magic.

When designed right, you only need one sgRNA to initiate the DNA cuts that trigger efficient homologous recombination repair.  This technique works great in C. elegans transgenesis. Add hygromycin B to the growth plates and only the genomic-integrated animals can survive.  Whether it can work in embryo injections with other organisms remains to determined.

At InVivo Biosystems, we are building on this to use our fast and easy CRISPR-sdm technique to place a small the small split-hygR fragment at any locus of the genome. This will allow us to drive large constructs 5 to 10 KB (and perhaps even 20-100 KB) into any native locus.

Bottom line: Getting some SSSH! time with this split-hygR technique can calm the frustration of the aggravated C. elegans researcher.

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