The Next Generation of CRISPR Screening

CRISPR-based genetic screens have revolutionized functional genomics, providing researchers with the means to perform unbiased phenotypic analysis, allowing interrogation of gene function on a genome-wide scale. Pooled library screening with CRISPR has now been widely adopted for high-throughput phenotypic studies, enabling analysis of thousands of genes in a single experiment to elucidate biological pathways, identify therapeutic targets for drug discovery, and genetically profile therapeutics for precision medicine.

The expanding CRISPR toolkit has now given rise to the next generation of CRISPR screening capability, including gene expression studies with CRISPRi and CRISPRa, combinatorial screening, high-content phenotypic analysis with single-cell and optical screening, as well as identification of genes involved in complex diseases such as diabetes. This article looks at the next generation of CRISPR screens and how they are being used to reveal cellular and disease mechanisms.

CRISPR screening for complex disease

CRISPR-Cas9 gene-editing technology is inherently simple, requiring only a single-guide RNA (sgRNA) to target the Cas9 endonuclease to the gene of interest, where it introduces a double-strand break (DSB). This activates the endogenous non-homologous end joining (NHEJ) DNA repair pathway that causes a frameshift mutation and subsequent gene knockout. In pooled library screening, multiple sgRNA are introduced to cells via lentiviral vector at sufficient concentration so that each cell receives only a single guide. As the lentiviral vector randomly integrates into the host genome, this provides each cell with a barcode, allowing sgRNA that have been enriched or depleted by the screen to be identified by next-generation sequencing, resulting in a list of genes that confer sensitivity or resistance to the biological challenge.

But identifying those genes and processes that play a causal role in complex diseases remains a significant challenge. In a 2022 paper, Rottner et al. performed a genome-wide pooled CRISPR screen in a human pancreatic beta cell line, assessing regulation of insulin content—they identified 580 genes that regulate beta cell function, along with 20 genes that play a causal role at the type 2 diabetes loci, including the autophagy receptor CALCOCO2. This work shows the utility of genome-wide CRISPR screening as a multi-omic approach to link gene expression, regulatory elements, and disease-relevant biology.¹

Genome-wide gene expression studies with CRISPR

CRISPR knockout screening is a powerful tool for perturbation studies but does come with limitations. CRISPR knockout produces permanent loss of function with a true null phenotype—but pharmacological inhibition of gene products with a drug is rarely absolute, and so rather than full knockout, a partial knockdown of gene expression would better mimic the action of a drug to reveal the impact on cellular pathways.

CRISPR interference, or CRISPRi, utilizes a catalytically inactive version of Cas9 (dCas9) tethered to a transcriptional repressor to upregulate gene expression. Alternatively, fusing dCas9 with transcriptional activators allows gain of function studies with CRISPR activation (CRISPRa)—as genes such as KRAS and BRAF are frequently activated during cancer. CRISPRa has enormous potential for studying molecular pathways, as well as drug-resistance mechanisms that are caused by gain of function events.

CRISPRi and CRISPRa can also be used to determine the function of regulatory elements. The human genome contains thousands of long non-coding RNAs (lncRNAs), which despite playing a crucial role in cellular processes, the function of the vast majority remains unknown. Haswell et al. used CRISPRi to perform an unbiased, genome-wide screen to target lncRNA loci that are expressed during endoderm differentiation, identifying FOXD3-AS1, an essential lncRNA for pluripotency and differentiation.² lncRNAs could provide a raft of potential therapeutic targets. Morelli et al. used a large-scale CRISPRi screen to interrogate cell-growth dependency of lncRNAs in multiple myeloma and identified an MIR17HG-derived lncRNA with novel oncogenic function and a potential therapeutic target.³

CRISPR screening for cancer research

Genome-wide association studies (GWAS) is an approach used to identify those variants that are associated with a particular trait or increased disease risk—and evidence that implicates a gene within a disease etiology is crucial for the identification of potential therapeutic targets. For example, ESR1 was discovered within the known breast cancer locus and is targeted by tamoxifen, one of the most commonly used drugs. In their recent paper, Tuano et al. performed 60 high-throughput CRISPR screens, including knockout, CRISPRi, and CRISPRa, and measured proliferation in 2D, 3D, and in immune-deficient mice, identifying 20 GWAS targets that are likely to promote cancer by increasing proliferation or inhibiting DNA repair mechanisms in breast cells. This work shows the effectiveness of CRISPR screens to identify and define gene targets and phenotypes within a risk locus.⁴

Combinatorial screening with CRISPR

One therapeutic approach to target cancer is to exploit synthetic lethality, where the inhibition or expression of one gene in the presence of an oncogenic mutation leads to cell death. CRISPR has been used to investigate synthetic lethality by using combinatorial CRISPR screening, where multiple sgRNA are introduced to a single cell for simultaneous knockdown of gene pairs. In their 2021 Nature paper, Thompson et al. screened 1191 gene pairs, identifying 105 gene combinations that resulted in loss of cellular fitness, including FAM50A/FAM50B. As silencing of FAM50B has been shown in a range of cancer types, this suggests it is a significant synthetic lethal interaction that could potentially be exploited for therapeutic benefit.⁵

High-content optical screening with CRISPR

The majority of pooled CRISPR screens rely on measuring simple phenotypes, such as simple proliferation and survival. Measurement of more complex phenotypes with flow cytometry or microscopy is possible with arrayed-based screens but comes with increased technical burden and cost. Both pooled and arrayed methods require enrichment of the cell population, meaning measurement is at the population-level only and limits the phenotypes assessed to a few parameters. High-throughput pooled CRISPR screening has been successfully used at a single-cell resolution, thereby achieving high-dimensional readouts—but as these methods involve cell destruction, they cannot be used to monitor dynamic processes over time, such as morphology and subcellular localization.

Optical screening is an approach that combines high-content imaging with in situ sequencing therefore allowing identification of genes that affect spatially and temporally defined phenotypes. For example, Feldman et al. performed an optical pooled CRISPR loss of function screen across 3 cell lines, screening 952 genes for their involvement in NF-κB signaling. By imaging p65 nuclear translocation in response to inflammatory signals, they were able to identify 15 known pathway components and showed a role of Mediator complex subunits in regulating nuclear retention of p65.⁶ In a recent paper, a pooled optical screen was successfully scaled to the genome level—80,000 perturbations from approximately 10 million cells were analyzed to uncover pathways that govern innate immune responses to viral infection.⁷

Next-generation models for CRISPR screening

When embarking on a CRISPR screen, it’s important to use a biologically relevant model, but this is not always technically possible with the large number of cells involved in a genome-wide study. For the most part, cancer cell lines are employed initially, and then subsequent follow up validation steps performed in a more biologically relevant model—but for some diseases such as non-alcoholic fatty liver disease (NAFLD), relevant biological models are scarce. However, a recent study has performed CRISPR screening in organoid models for NAFLD target drug discovery. Using human fetal hepatocyte organoids, the screen identified FADS2 (fatty acid desaturase 2) as playing a role in heptatic steatosis, the first stage of nonalcholic fatty liver disease.⁸

The next generation of biological breakthroughs with CRISPR screening

The flexibility, ease of use, efficacy, and lower off-target effects of CRISPR screening compared to other methodologies such as RNAi, have fueled its widespread adoption since its development over a decade ago. CRISPR screening has proven a powerful tool in functional genomics, enabling unbiased, systematic interrogation of biological systems on a genome-wide scale. The development of CRISPR gene-editing technology has been rapid, and efforts in developing the next generation of CRISPR screening capacity look set to continue.

Key Takeaways

• CRISPR gene-editing technology now allows researchers to perform unbiased interrogation of gene function on a genome-wide scale
• Pooled and arrayed library screening with CRISPR has overtaken existing methodologies such as RNAi due to its flexibility and ease of use
• As well as gene knockout, modulation of gene expression with CRISPRi and CRISPRa can be incorporated into screening workflows
• CRISPR screens usually involve measurement of simple phenotypes, such as cellular proliferation and survival—but more complex phenotypes can be assessed with flow cytometry and microscopy
• Screening can also be performed at the single-cell resolution—as well as optical screening for assessment of dynamic cellular processes

References

1. Rottner, A.K., Ye, Y., Navarro-Guerrero, E. et al. A genome-wide CRISPR screen identifies CALCOCO2 as a regulator of beta cell function influencing type 2 diabetes risk. Nat Genet 55, 54–65 (2023). 

2. Haswell, Jeffrey R et al. “Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency.” PloS one vol. 16,11 e0252848. 3 Nov. 2021, doi:10.1371/journal.pone.0252848

3. Morelli, Eugenio et al. “A MIR17HG-derived long noncoding RNA provides an essential chromatin scaffold for protein interaction and myeloma growth.” Blood vol. 141,4 (2023): 391-405. 

4. Tuano, N.K., Beesley, J., Manning, M. et al. CRISPR screens identify gene targets at breast cancer risk loci. Genome Biol 24, 59 (2023). 

5. Thompson, Nicola A et al. “Combinatorial CRISPR screen identifies fitness effects of gene paralogues.” Nature communications vol. 12,1 1302. 26 Feb. 2021

6. Feldman, David et al. “Optical Pooled Screens in Human Cells.” Cell vol. 179,3 (2019): 787-799.e17. 

7. Carlson, Rebecca J et al. “A genome-wide optical pooled screen reveals regulators of cellular antiviral responses.” Proceedings of the National Academy of Sciences of the United States of America vol. 120,16 (2023): e2210623120. 

8. Hendriks, Delilah et al. “Engineered human hepatocyte organoids enable CRISPR-based target discovery and drug screening for steatosis.” Nature biotechnology, 10.1038/s41587-023-01680-4. 23 Feb. 2023,