How to Combine CRISPR with Single-Cell RNA Sequencing

What is CRISPR screening used for?

Single-cell multi-omics (scM) instruments can be used to perform highly scalable and sensitive clustered regularly interspaced short palindromic repeat (CRISPR) screens. In cell perturbation using CRISPR screening, single-guide ribonucleic acids (sgRNAs) are used to guide the endonuclease Cas9 to create a targeted double-strand break, the repair of which results in a frameshift mutation¹. If this double-strand break occurs in a coding region, it can result in the gene becoming inactivated¹. The high specificity of the mechanism enables accurate perturbation of thousands of genes². Unfortunately, they lack the complexity necessary to perform transcriptome profiling². In industrial R&D, this technique is widely used to identify potential drug targets.

What is the purpose of single-cell RNA sequencing?

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 gene³. Single-cell RNA sequencing can then reveal the changes in global transcription, which can be mapped back to the individual sgRNAs in cells³. The main challenge is in finding a way to perform targeted single-cell RNA sequencing at scale.

Why combine CRISPR and single-cell RNA sequencing?

Christoph Bock developed the CROP-seq (CRISPR Droplet sequencing) workflow, which combines these two technologies². The new technology was adapted to the BD Rhapsody™ Single-Cell Analysis System. The advantages of this combination include:

• The seamless integration of CRISPR screening based on single-cell transcriptomics with the BD Rhapsody™ Single-Cell Analysis System workflow
• The microwell-based capture system of the BD Rhapsody™ Single-Cell Analysis System allows for flexible cell loads with high recovery rates and minimises multiplets
• The imaging system of the BD Rhapsody™ Single-Cell Analysis System provides detailed information that allows for quality control
• The possibility of pooled costs and increased sensitivity with targeted sequencing
• The design of both the sgRNA library and the targeted primer panel is fully customisable with a quick turnaround time

Where can you learn more about CROP-seq?

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 complex single-cell transcriptome readouts at scale². 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.