High-Throughput Single-Cell Screening with CRISPR

Phenotypic analysis has been key to our understanding of gene function and cellular processes. CRISPR-based genetic screens have proved to be a powerful approach for unbiased functional genomic research to evaluate gene function at high-throughput. But by combining CRISPR screening with single-cell sequencing, researchers can now leverage the flexibility and efficiency of CRISPR with single-cell platforms to interrogate gene function at the cellular level with unprecedented resolution—achieving both high-content readouts and high-throughput genetic screening for transcriptional profiling, chromatin accessibility, epigenetics, and single-cell imaging. This article discusses single-cell CRISPR screening (scCRISPR) and its potential impact.

CRISPR screening: A powerful tool for functional screening

CRISPR pooled and arrayed screens have revolutionized and accelerated biological research, providing a potent tool to identify gene function, elucidate molecular pathways, and identify potential therapeutic targets for drug discovery. Pooled and arrayed CRISPR screens both rely on gene perturbations having a measured effect on phenotype—from simple proliferation and survival with pooled screens to more high-content readouts with flow cytometry or microscopy from arrayed-based methods. But these screens rely on bulk analysis and so can omit more complex molecular mechanisms such as transcriptomic profiling, as well as cell-type specific gene function. Readouts also rely on measurement of distinct phenotypes and so may miss more subtle changes—especially important if the cell population is heterogeneous. It is widely known that groups of cells, even in culture, display heterogeneity in gene expression and response, particularly observed in cancer cell lines showcasing different drug resistance or immune response, which may be missed with current screening methods.

CRISPR at single-cell resolution

Single cell sequencing, however, links data back to individual cells and has been used to measure the genome, transcriptome, or methylome of each individual cell within a population.¹ Single-cell sequencing relies on the physical isolation of a cell from the population by microfluidics, or even flow or mass cytometry, before the genetic material is extracted and sequenced. The ability to determine cell-specific variation has been shown to identify tumor heterogeneity in a cancer population, epigenetic changes during development, and provide insight into the impact of the expression of a gene on other genetic pathways.

One challenge with scCRISPR screening is correctly linking the sgRNA that has caused the perturbation with the single-cell profile. To do this, single-cell RNA sequencing platforms typically use polyadenylated indexes for screening—but issues with recombination of these indexes during delivery have resulted in them becoming uncoupled from assigned sgRNAs, limiting the use of the platforms to arrayed samples at a restricted scale. To address this, scientists have developed various approaches. CROP-seq uses a vector that duplicates the sequence of a single encoded sgRNA during lentiviral transduction, resulting in two expression cassettes on a single construct: one that expresses a polyadenylated transcript carrying the sgRNA sequence at the 3’ end, and the other expressing the functional sgRNA.² Direct capture Perturb-seq instead uses guide-specific primers during reverse transcription, and then combines this with hybrid capture to enable the expressed sgRNAs to be sequenced alongside single-cell transcriptomes.³

Transcriptomic profiling with scCRISPR-RNAseq

The most common form of single-cell profiling is single-cell RNA-seq (scRNA-seq), which allows transcriptomic profiling of individual cells to understand gene activities and regulatory networks and includes methods such as CROP-seq and Perturb-seq, as well as DROP-seq, CRISPRseq, and Mosaic-seq. Combining scRNA-seq with CRISPR screening (scCRISPR-RNA-seq) enables researchers to investigate the effect a particular guide has on the whole transcriptomic readout and so interrogate the phenotype of a given perturbation in detail to identify nuanced and cell-specific cellular responses to drugs. Cells can be isolated by flow cytometry or microfluidics—platforms such as 10x Chromium, Drop-seq, and Fluidigm C1 isolate individual cells within nanoliter droplets from which reverse transcriptase creates the cDNA pool for amplification and sequencing.

Since 2016, scCRISPR-RNAseq has evolved and developed rapidly, with improvements to accuracy and efficiency, as well as incorporating the expanding CRISPR toolkit including CRISPR-knockout, dCas9, Cas9 nickases and base editing, and performing screens in vivo. Hou et al. integrated both Perturb-seq and CROP-seq with both in vitro and in vivo immune screens and were able to show an interactive relationship between targeting tumor intrinsic factors and T-cell mediated antitumor immune response, which could not be assessed with bulk sequencing associated with RNAseq.⁴

Most scCRISPR-RNAseq screens focus on single-gene function and interactions, but Yang et al. developed a single-cell CRISPR gene tiling pipeline, or sc-Tiling, a method to examine functional gene domains at sub-gene resolution.⁵ Their work was able to provide a high-resolution transcriptomic profiling of methyltransferase DOT1L, a potential therapeutic candidate for mixed-lineage leukemia gene-rearranged (MLL-r) leukemia and link it to a three-dimensional structure to discover novel self-regulatory domains that modulate chromatin interaction, enzymatic activation and therapeutic sensitivity to MLL-r leukemia.

Chromatin accessibility screening with scCRISPR

Several scCRISPR methodologies have also combined chromatin-accessibility profiling with scCRISPR to investigate epigenomic regulatory mechanisms at large-scale. Rubin et al. developed Perturb-ATAC, a method for simultaneously detecting sgRNA with open chromatin sites by Assay of Transposase-Chromatin Accessibility sequencing (ATAC-seq) to dissect gene regulatory networks.⁶ By applying Perturb-ATAC, they were able to uncover regulators of chromatin accessibility in human B-cells and identify those transcription factors that control B-cell development. However, Perturb-ATAC did come with limited throughput and high background noise—Spear-ATAC instead uses bespoke sgRNA with pre-integrated adapters and biotin-tagged primers, which resulted in a substantial increase in throughput and reduced background.⁷

A deeper understanding of tumor biology with scCRISPRseq

By combining single-cell sequencing with CRISPR screening methodologies, researchers can perform high-content profiling of the genome, transcriptome, and epigenome at the level of the individual cell. High-content, large-scale functional studies at single-cell resolution look set to revolutionize cellular characterization, allowing us to dissect molecular networks, and further our understanding of tumor biology by detecting responses to treatment or stimulus in a heterogeneous cell population.

For example, Roth et al. discovered chimeric antigen receptors that improved T cell anti-tumor activity under immune suppression, and Jun et al. used it with vemurafenib selection to identify mutations in genes MAP2K1, KRAS, and NRAS that contribute to melanoma drug resistance.^8,9 scCRISPR has also provided insights into various processes such as epithelial-to-mesenchymal transition, interactions between oncogenic genes, T cell exhaustion, differentiation blockade, immune checkpoint regulation, and the function of druggable proteins. Methods to combine scCRISPR screening with proteome analysis have also been developed to provide protein-level phenotyping at single-cell resolution as well as screening of cellular dynamics and morphology with single-cell spatial imaging facilitating a multi-omic approach to cancer biology.1

Advancing the next breakthroughs

Single-cell CRISPR screening can provide critical phenotypic information on a heterogenous population to increase our understanding on subpopulation structure and differences in cellular responses—and how subpopulations of cancer cells respond to a particular treatment. The combination of single-cell sequencing and CRISPR screening is an exciting technology that links phenotype to the genetics of an individual cell, and in doing so truly links genotype with phenotype. Already, this technology is changing our understanding of biology and shattering established paradigms in cell development and disease development. As the technology evolves, making the approach more robust and more accessible to researchers, we can expect our understanding of the complexities of biology to grow still further.

High-throughput screening with CRISPR

• Pooled CRISPR screens involve hundreds of sgRNA that are delivered to a pool of cells at a low enough viral titer to ensure a single sgRNA, and so single gene perturbation, per cell
• A screening condition is then applied, for example drug treatment, and those sgRNA present or absent in the pool identified by next-generation sequencing
• Pooled library screening relies on simple cell fitness phenotypes, such as cell proliferation or survival whereas arrayed-based methods can be combined with more high-content measurements, such as flow cytometry or microscopy, to measure a broader range of phenotypic end points, such as cell surface receptor localization or cell morphology

References

1. Cheng, Junyun et al. “Massively Parallel CRISPR-Based Genetic Perturbation Screening at Single-Cell Resolution.” Advanced science (Weinheim, Baden-Wurttemberg, Germany) vol. 10,4 (2023): e2204484. 

2. Datlinger, Paul et al. “Pooled CRISPR screening with single-cell transcriptome readout.” Nature methodsvol. 14,3 (2017): 297-301. 

3. Replogle, Joseph M et al. “Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing.” Nature biotechnology vol. 38,8 (2020): 954-961. doi:10.1038/s41587-020-0470-y

4. Hou, Jiakai et al. “Single-cell CRISPR immune screens reveal immunological roles of tumor intrinsic factors.” NAR cancer vol. 4,4 zcac038. 9 Dec. 2022, doi:10.1093/narcan/zcac038

5. Yang, L., Chan, A.K.N., Miyashita, K. et al. High-resolution characterization of gene function using single-cell CRISPR tiling screen. Nat Commun 12, 4063 (2021). 

6. Rubin, Adam J et al. “Coupled Single-Cell CRISPR Screening and Epigenomic Profiling Reveals Causal Gene Regulatory Networks.” Cell vol. 176,1-2 (2019): 361-376.e17. 

7. Pierce, S.E., Granja, J.M. & Greenleaf, W.J. High-throughput single-cell chromatin accessibility CRISPR screens enable unbiased identification of regulatory networks in cancer. Nat Commun 12, 2969 (2021). 

8. Roth, Theodore L et al. “Pooled Knockin Targeting for Genome Engineering of Cellular Immunotherapies.” Cell vol. 181,3 (2020): 728-744.e21. 

9. Jun, Soyeong et al. “Single-cell analysis of a mutant library generated using CRISPR-guided deaminase in human melanoma cells.” Communications biology vol. 3,1 154. 2 Apr. 2020,