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What is the new Split hygromycin method? How does it differ from MosSCI? How can we use it to help you?


In 2020 a new method of inserting transgenes into the genome of C. elegans was introduced to the field, and was quickly adopted by InVivo Biosystems. In this article we will discuss the new Split hygromycin method, how it differs from the traditional MosSCI method, and what benefits this new method has both for us in the InVivo Biosystems lab, and for our customers.

The first strategy for introducing DNA into C. elegans was developed in 1991 and consisted of injecting the DNA into the gonad of a hermaphrodite which is eventually incorporated into the nucleus; seventeen years later the MosSCI (Mos1-mediated Single Copy Insertion) method was developed, enabling the insertion of a single copy transgene into a defined location in the C. elegans’ genome (Mello et al., 1991; Frøkjaer-Jensen, 2008). Nearly a year ago Stevenson et al. (2020) presented a new, CRISPR based integration strategy, known as the Split hygromycin method, which has some notable advantages to the previously established approaches.

Traditional Method: Mos1-mediated Single Copy Insertion (MosSCI)

This method works by creating a double-strand break at defined sites (known as a MOS1 site) in the C. elegans genome, and exploiting the cell’s natural repair mechanism – as, when in the presence of an exogenous DNA template, the cell will incorporate this transgenic cargo into the genome. 

Consequently, unlike the original method for incorporating DNA into the C. elegans genome, the MosSCI method enables researchers to make insertions at a known locus. The MosSCI method also allows for the insertion of a broad range of custom cargo, and the inclusion of a selection marker (such as unc-119 rescue) along with your cargo.


New method: Split hygromycin

This method works by utilizing custom CRISPR/Cas9-engineered synthetic split landing pads (SLPs) which include a 3′ hygromycin coding sequence and transcription terminator to create hygromycin resistance, and an sgRNA guide which enables Cas9 to cut the chromosome, creating a double-strand break and triggering homologous recombination repair. During the repair process the transgenic cargo is incorporated into the native locus, and the hygromycin gene is restored. 

Since the selection marker is split and only functions upon integration, researchers are able to avoid selecting heritable extrachromosomal arrays.

Cas9 Insertion

Figure One. Diagrams of how MosSCI method and Split Hygromycin method compare. 

The MosSCI and Split hygromycin methods both have the benefits of: 

  • Transgene insertions that are stable
  • Transgenes are able to be expressed at levels close to endogenous gene expression
  • Transgenes are expressed in the germline.

Advantages of Split hygromycin method

Size of selection marker. ~500 bp for split hygromycin method compared to 2kb for MosSCI (providing ~1.5 kb more space for a desired sequence to work with than MosSCI). The split hygromycin method varies so vastly from the MosSCI because only part of the selection marker needs to be included in the donor homology plasmid. This method utilizes hygromycin resistance instead of unc-119 rescue, and thus is cleaner for neuronally expressed constructs since unc-119 is a neuronally expressed gene. If the target construct is also neuronally expressed, it’s beneficial to use a non-neuronally expressed selection marker, like hygromycin resistance, so that the selection marker phenotype doesn’t interfere with the target construct phenotype. 

Speed. This method has the potential to be faster (~ 3-4 days) than traditional mossci projects because there is no need to heat shock the progeny of the injected worms in order to remove extrachromosomal array animals from the population. This enables the technician to proceed straight to genotyping from selection of hygromycin surviving animals. 

Eliminates the possibility of extrachromosomal array animals in the candidate population. Including the whole array marker in the donor homology template enables animals with the extrachromosomal array to be included in the candidate population. This is the case with the MosSCI method. The reliance on integration for the expression of the selection marker allows for only animals with proper integration to be included in the candidate population. This saves time and resources during the screening process by eliminating the potential for screening and genotyping undesired extrachromosomal array animals. 



The new Split hygromycin method is an exciting advancement that addresses some of the most aggravating aspects of traditional C. elegans transgenics (time-line, limited-sized constructs, potential for extrachromosomal array animals). However, MosSCI remains a good approach for inserting transgenes, and we at InVivo Biosystems will continue to do both depending on the project. 

If you’re interested in learning more about InVivo Biosystems C. elegans transgenic services reach out to one of our experts.


  1. Frøkjaer-Jensen, C., Davis, M. W., Hopkins, C. E., Newman, B. J., Thummel, J. M., Olesen, S. P., Grunnet, M., & Jorgensen, E. M. (2008). Single-copy insertion of transgenes in Caenorhabditis elegans. Nature genetics, 40(11), 1375-1383. https://doi.org/10.1038/ng.248
  2. Mello, C. C., Kramer, J. M., Stinchcomb, D., & Ambros, V. (1991). Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences. The EMBO journal, 10(12), 3959-3970.
  3. Stevenson, Zachary C., Moerdyk-Schauwecker, Megan J., Jamison, Brennen and Phillips, Patrick C. (2020). Rapid Self-Selecting and Clone-Free Integration of Transgenes into Engineered CRISPR Safe Harbor Locations in Caenorhabditis elegans G3: GENES, GENOMES, GENETICS vol. 10 no. 10 3775-3782; https://doi.org/10.1534/g3.120.401400

About The Author

Lauren Resch

About The Author

Alexandra Narin

Alexandra is the Marketing Content Manager and Grant Writer for InVivo Biosystems. She graduated from the University of St Andrews in 2020 where she earned a Joint MA Honours Degree in English & Psychology/Neuroscience with BPS [British Psychology Society] Accreditation. She has worked as a research assistant, examining the LEC's (lateral entorhinal cortex) involvement in spatial memory and integrating long term multimodal item-context associations, and completed her dissertation on how the number and kinds of sensory cues affect memory persistence across timescales. Her hobbies include running, boxing, and reading.

About The Author

Marilee Hoyle

Marilee is a Lab Technician 1 on the Molecular biology team at InVivo Biosystems. She graduated with a Bachelor of Science in the Environmental sciences with an emphasis in Marine conservation at Oregon State University. While at OSU she researched the freshwater prokaryotic and eukaryotic diversity of the Oregon Willamette River Basin. When not at work she likes to spend time with her cats, playing with makeup/fashion, hiking, or tide pooling on the Oregon coast.

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Anna Malinkevich, MS

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