Combining CRISPR Screening with Single-Cell RNA Sequencing

What is CRISPR screening used for?

Single-cell multiomics (scM) instruments can be used to perform highly scalable and sensitive clustered regularly interspaced short palindromic repeat (CRISPR) screens.¹ 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 mutation.² If this double-strand break occurs in a coding region, it can result in the gene becoming inactivated.² The high specificity of the mechanism enables accurate perturbation of thousands of genes.¹ Unfortunately, the CRISPR screening method lacks the complexity necessary to perform transcriptome profiling.¹ 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 each single cell represents a knockout for a single gene.³ Single-cell RNA sequencing can then reveal changes in global transcription, which can be mapped back to the individual sgRNAs in the cells.³ A challenge is in finding a way to perform targeted single-cell RNA sequencing at scale. 

CROP-Seq: combining CRISPR and single-cell RNA sequencing

The CROP-seq (CRISPR Droplet sequencing) workflow combines these two technologies.¹ This new technology has since been adapted for the BD Rhapsody™ Single-Cell Analysis System.

The advantages of this combination include:

- 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 that allows for flexible cell loading with high recovery rates and minimal multipletss

- The imaging system of the BD Rhapsody™ Single-Cell Analysis System, which provides detailed information that allows for quality control

- The design of both the sgRNA library and the targeted primer panel that is fully customizable with quick turnaround time.

The webinar accompanying this article highlights the case study of a CROP-seq workflow used in the perturbation of the signalling pathway elicited by interferon-β (IFN-β). Coupled with targeted sequencing, this CROP-seq workflow enables complex single-cell transcriptome readouts at scale.¹ This approach has the potential to simplify single-cell CRISPR screens, while enabling the comprehensive characterisation of large CRISPR libraries.¹.