Team:Thrace/Contribution

In order to contribute to iGEM’s Registry of Biological Parts, we decided to enrich a part’s entry through bibliographical research. The parts that we chose to engage with are orthologs of the Cas12a enzyme and specifically FnCas12a (BBa_K2644101), AsCas12a (BBa_K2965021) and LbCas12a (BBa_K2927005). Our project incorporates the Cas12a enzyme in its design, as it utilizes the enzyme to detect the biomarkers and produce a visible result. We hope that our contribution will help future teams to select the ortholog of Cas12a that best suits their project.

Effect of ions on Cas12a’s collateral cleavage activity.

Cas12as’ cleavage activity is highly dependent on the presence or absence of specific ions. Not all orthologs of Cas12a react in the same way to the presence of these ions. In fact, there are some major differences between them.

1. AsCas12a

   • AsCas12a’s activity is modified in the presence of certain ions. In particular, AsCas12a is able to degrade DNA substrate in the presence of Mg2+ and/orMn2+. Mg2+ is required for a crRNA free cleavage activity of DNA substrate. Furthermore, in the presence of Mg2+ buffer, AsCas12a displays a preferred endonuclease activity for ssDNA rather than dsDNA.
   • For circular ssDNA, AsCas12a enzyme has the same activity for the same period of time, no matter which kind of metal ion (Mg2+ or Mn2+) is used. Another ion that promotes the cleavage of ssDNA from AsCas12a is Co2+.

2. LbCas12a

   • LbCas12a’s collateral cleavage activity can be altered in the presence of particular ions. Specifically, Mn2+ ions can activate the crRNA-independent Dnase activity of the enzyme. Mn2+ is not able to trigger LbCas12a-mediated dsDNA cleavage activity. None of the divalent metal ions is able to trigger LbCas12a-mediated dsDNase activity

3. AsCas12a

   • LFnCas12a is a Cas12a ortholog. It’s activity is modified in the presence of ions. Specifically, Mn2+ ions are able to trigger FnCas12a-mediated dsDNA cleavage activity. Moreover, in the presence of Mn2+ ions the CrRNA-independent DNase activity of FnCas12a is activated for both dsDNA and ssDNA.

References

1. Li, B., Yan, J., Zhang, Y., Li, W., Zeng, C., Zhao, W., Hou, X., Zhang, C., & Dong, Y. (2020). CRISPR-CAS12A possesses unconventional DNase activity that can be inactivated by synthetic oligonucleotides. Molecular Therapy - Nucleic Acids, 19, 1043–1052. https://doi.org/10.1016/j.omtn.2019.12.038

3D printable case for light standardization

For the purposes of our project, we created a 3D printable case that enables light standardization. The 3D cube model was developed in FreeCad, starting from a solid cube and extruding the needed shapes. The microfluidics channel was developed in Comsol, by filling a square channel with cylinders of varying sizes, aiming to break the fluid flow, creating vortexes, leading to the homogenization of the sample.

3D view of the printable photo-equalization cube

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