Cell perturbation using the CRISPR technique
Earlier on www.flowcytometry-news.com, we featured an article on the use of the BD Rhapsody™ system to perform highly scalable and more sensitive CRISPR screens. In cell perturbation using the CRISPR technique, single guide RNAs (sgRNAs) are used to guide the endonuclease Cas9 to create a targeted double-strand break, the repair of which results in a frameshift mutation. If this double-strand break occurs in a coding region, it can result in the gene becoming inactivated. The high specificity of the mechanism enables accurate perturbation of thousands of genes, using what are called CRISPR screens. In industrial R&D, this technique is widely used to identify potential drug targets.
Combining CRISPR screening with single-cell RNA sequencing – The Aelian® CROP-seq workflow
In a typical CRISPR screen, Cas9 expressing cells have sets of genes perturbed by CRISPR editing, so that every single cell represents a knockout for a single gene. Single-cell RNA sequencing can then reveal the changes in global transcription, which can be mapped back to the individual sgRNAs in cells. The main challenge in this process has been to combine the two technologies of CRISPR screening and single-cell RNA sequencing. This challenge has now been addressed by the CROP-seq (CRISPR Droplet sequencing) workflow, developed by Aelian’s co-founder, Christoph Bock.
The advantages offered by the Aelian® CROP-seq are
- Combining CRISPR screening with single-cell RNA sequencing
- Seamless integration of CRISPR screening based on single-cell transcriptomics with the BD Rhapsody™ workflow
- The microwell-based capture system of the BD Rhapsody™ Single-cell Analysis System allows flexible cell loads with high recovery rates and minimisation of multiplets.
- The imaging system of the BD Rhapsody™ provides detailed information that allows for quality control
- The targeted sequencing minimises costs and increases sensitivity
- The design of both the sgRNA library and the targeted primer panel is fully customisable with a quick turnaround time
The webinar accompanying this article showcases the advantages of the Aelian® CROP-seq workflow and its use in a case study involving the perturbation of the signalling pathway elicited by interferon-β (IFN-β) as an example. Coupled with targeted sequencing, which lowers the cost and improves the sensitivity of the experiment, the Aelian® CROP-seq workflow enables the detection of more subtle phenotypes. This approach is expected to pave the way towards genome-scale single-cell CRISPR screens, a paradigm shift in functional genomics from simplistic viability screens to high-content screens.
Learn more about the Aelian® CROP-seq workflow by watching the webinar here.