Tell us about your research project and the challenge that led you to seek CellEDIT's services.

Our research focuses on the Hedgehog signaling pathway, particularly why the SMO receptor initiates biased signaling. We introduce non-canonical amino acids into receptors, especially photosensitive amino acids, to establish structure-function relationships.

We needed functional assays with measurable signals in appropriate cellular contexts. SK-OV-3 ovarian cancer cells were ideal, but they contain endogenous SMO genes that we needed to remove for clean readouts in our light-sensitive amino acid incorporation studies. 

What factors influenced your decision to choose Cytosurge over other options?

After contacting several companies, we chose Cytosurge because it provided exceptional scientific expertise and even competitive quotations. When talking to the sales team, we speak with scientists who understand our language and can implement our needs effectively into experiments, providing essential guidance on the technical approach and strategy, which was invaluable given my limited experience with CRISPR-Cas9 editing in mammalian cells.

How would you describe your overall experience with the project process?

I'm extremely happy with the service. The company’s specialists demonstrated exceptional professionalism from the beginning. The precise timeline was easy to follow, and the post-delivery report explanation of each knockout clone was incredibly educational.

Can you tell us about the technical approach used for your project?

Cytosurge used a sophisticated double-guide CRISPR approach in SK-OV-3 cells: Guide 1 targeted exon 2 to disrupt gene function. Guide 2 targeted exon 6 at the receptor binding site.

What makes Cytosurge's approach unique is its single-cell gene editing technology, which allows for precise targeting and editing of individual cells before expansion into clonal populations. The SK-OV-3 cells showed high CRISPR efficiency, with both guides inducing cuts in all edited clones. This led to successful deletions of the region between exons 2 and 6, as confirmed by agarose gel electrophoresis and Sanger sequencing.

The report indicates you received both homozygous and heterozygous knockout clones. How valuable was this for your research?

Extremely valuable. We received two edited clones (heterozygous and homozygous) plus two wild-type controls, all with 94-98% viability. After knockout, SMO-elicited GLI transcription is completely absent. GLI transcription is a key readout of Hedgehog pathway activity, so this confirms that our knockout was successful. However, when we overexpress SMO in these cells, we see clear GLI responses. This confirms we have the clean background needed for our synthetic biology studies.

We're now expressing mutants with photosensitive amino acids and seeing encouraging differences compared to wild-type.

How critical were these engineered cell lines for your research progress?

Absolutely essential. We actually have two types of cell lines for comparison: the SK-OV-3 cancer cell line where we used Cytosurge's service to remove the background SMO gene, and another MEF cell line that had the SMO gene knocked out through traditional methods 10-20 years ago. Having both allows for very interesting comparative studies in our synthetic biology framework, and all this data is contributing to our manuscript submission.

How does this work fit into your broader synthetic biology research program?

This SMO knockout project is part of our larger effort to develop optical methods to regulate neuronal signaling processes with light. We've expanded the genetic code in mammalian cells and are engineering light-responsive neuronal receptors for optogenetic studies. The clean SK-OV-3 background allows a precise study of how unnatural amino acids affect receptor function without interference from endogenous proteins.

Based on your experience, would you recommend Cytosurge to colleagues?

Definitely. I would recommend Cytosurge to anyone wanting to engineer mammalian cells, cancer cells, or any cell types. The company is very reliable, professional, and the single-cell gene editing technology is remarkable.

Would you work with CellEDIT again?

Yes, absolutely. In fact, following the success of this initial collaboration, we already ordered and successfully received our next project with CellEDIT: a knock-in for point mutations. Their reliable results have made them our preferred choice for future gene editing projects as we continue to expand our synthetic biology toolkit.

Dr. Ye-Lehmann's story demonstrates how professional expertise and innovative single-cell technology provide researchers with precise tools for complex synthetic biology studies. The successful SMO knockout exemplifies the technical excellence enabling cutting-edge research in Hedgehog signaling and optogenetics.