Single-cell CRISPR screens and targeted sequencing
Introduction
The CRISPR-Cas9 method is a well-understood technique for the targeted disruption of genes across a wide range of cell types. Single-cell RNA sequencing (ScRNAseq) can be used to link genetic perturbations elicited by CRISPR-Cas9 to their transcriptomic phenotypes.
However, CRISPR technology and single-cell RNA sequencing can benefit from being combined and adapted to a more targeted approach. In fact, targeted single-cell RNA sequencing can lead to more sensitive and affordable CRISPR screens compared to the whole transcriptome approach (WTA).
Discover how the BD Rhapsody™ System can achieve targeted single-cell CRISPR screens for greater efficiency.
The limitations of conventional single-cell RNA sequencing
Conventional single-cell RNA sequencing experiments are associated with high costs due to single-cell library preparation and next-generation sequencing (NGS).
They also suffer from lower sensitivity, as relevant mRNAs are often sparsely represented and outshone by abundant mRNAs from, for example, housekeeping genes.
Lastly, conventional single-cell RNA sequencing platforms often lack process control. It can be difficult to monitor metrics like cell capture efficiency and multiplet rate that strongly influence the NGS data quality.
Targeted single-cell RNA sequencing
A single-cell RNA sequencing experiment using the whole transcriptome approach (WTA) would result in the amplification of all expressed genes in an unbiased manner.
However, most of the time only differentially expressed genes across cells are considered informative for the study of specific phenotypes.
Sequencing a complex WTA library consequently implies that many reads are spent on constitutively expressed mRNAs that are considered non-informative and thus cause unnecessary sequencing cost.
Therefore, it may be useful and economical to focus on a specific set of mRNAs chosen before the experiment, if feasible.
Learn more about targeted mRNA panels and other BD Biosciences tools for single-cell multi-omic analyses.
The emergence of single-cell CRISPR screens (Perturb-seq or CROP-seq)
The CRISPR-Cas9 method utilises a single guide RNA (sgRNA) strand to guide the Cas9 endonuclease and create a double-strand break at a site that is complementary to the sgRNA. The resulting frameshift mutation can lead to the disruption of the open reading frame.
The use of multiple sgRNAs or sgRNA libraries allows for unbiased functional genomic screens, also called CRISPR screens.
Initially, CRISPR screens had been confined to readouts such as cell proliferation or flow cytometry.
However, pooled CRISPR screens combined with single-cell RNA sequencing, known as “single-cell CRISPR screens,” “Perturb-seq” or “CROP-seq,” have recently allowed the tracking of phenotypic changes in global transcription of each cell to be mapped back onto the causative sgRNAs.
The resulting matrix of sgRNAs versus perturbed transcriptomes provides a strong dataset to address many complex biological questions.
Cost effectiveness and sensitivity in targeted single-cell CRISPR screens
Using the BD Rhapsody™ System, one model experiment combines CRISPR screens with targeted single-cell RNA sequencing, leveraging two transformative technologies to enable genetic screening for complex phenotypes.
The experiment, led by Aelian Biotechnology compares an unbiased WTA with a targeted approach using a panel of mRNAs nominated a priori.
The experiment shows that a targeted single-cell RNA sequencing approach in conjunction with CRISPR screens provides a greater dynamic range of detection of transcripts at a lower cost.
This approach holds promise in identifying novel drug targets or elucidating unknown mechanisms of actions of drugs.
Discover the full experiment in this commercial application note published in Nature Methods:
Scalable, more sensitive CRISPR screens*
Greater sensitivity with the targeted approach
The experiment showcased an efficient approach based on Aelian’s well established CROP-seq workflow in combination with the targeted single-cell RNA sequencing assay on the BD Rhapsody™ Single-Cell Analysis System, whereby targeted sequencing lowered the cost and at the same time enhanced the sensitivity of the experiment, enabling the detection of more subtle transcriptomic phenotypes.
Learn more about the BD Rhapsody™ Single-Cell Analysis System.
