Why You Should Consider Arrayed Library Screening

Functional genomics aims to establish the relationship between phenotype and genotype, enabling the dissection of molecular pathways and elucidation of biological processes. High-throughput screening with the CRISPR-Cas9 gene-editing tool has revolutionized functional genomics and allowed identification of gene-function in a broad range of applications and species.¹

CRISPR screens can be performed in either a pooled or arrayed format, but pooled CRISPR screens have become a widely used and cost-effective tool in functional genomics due to their efficiency, scalability, efficacy, and ease of use. Pooled library screening with CRISPR only requires standard molecular biology techniques, and so can be performed within most laboratories to achieve genome-wide scale data. However, despite the popularity of pooled CRISPR screening, arrayed-based methods offer several advantages over a pooled approach. This article discusses the benefits of arrayed CRISPR library screening and why it should be considered for your CRISPR screening experiments.

Easier data interpretation

A typical high-throughput pooled CRISPR screen involves the delivery of a library of single-guide RNAs (sgRNA) to a pool of cells via lentiviral transduction, at a viral titre that results in a single perturbation per cell. The pool of perturbed cells is then challenged, for example treatment with a drug or toxin, as the lentivirus stably integrates into the genome, this provides each cell with a ‘barcode’ so any sgRNA that have been enriched or depleted can be easily identified from the pool by massively parallel sequencing.² In contrast, in an arrayed screen, cells are physically separated throughout all stages in a multi-well plate and sgRNA delivered by transduction or transfection.²

A pooled library screen therefore requires significant bioinformatic analyses of the sequencing data to identify those sgRNA that have been enriched or depleted. Pooled CRISPR screens must also deal with the inherent noise from the pool, which can be exacerbated by off-target activity or incomplete knockouts. With arrayed screening, as the cells are already separated the effect of the perturbation on individual cells can be observed more easily and so accurately link phenotype to genotype.³ Using an arrayed CRISPR screen means phenotypes can therefore be attributed to individual genes with higher confidence and specificity, and so make downstream analysis more accurate.

More biologically relevant data with reduced optimization

A pooled CRISPR screen is generally a labor-intensive and time-consuming process, largely due to needing to optimize every aspect of experimental design to ensure good data can be achieved. Key to the success of a pooled CRISPR screen is maintenance of library representation, as sgRNA library diversity can be lost at every stage in the pooled CRISPR screening workflow. This can lead to bias and misinterpretation of data, making downstream validation more difficult.⁴

As well as an optimized assay and a good sgRNA library, a successful CRISPR screen needs a biologically relevant model to ensure confidence in the resulting hits. Primary cells can be incompatible for pooled library screening with CRISPR, especially those from the human nervous system, those not amenable to transduction, or those that are unable to stably express the Cas9 protein. The usual practice is to perform an initial whole-genome pooled screen using a surrogate model system, and then follow up resulting hits with a smaller arrayed screen in a more relevant biological model. However, surrogate model systems are sometimes not an accurate model of the disease studied, which can compromise validation of hits obtained in the follow up screen.

An often-cited disadvantage of arrayed library screening is that it comes with increased cost due to the reliance on automation for liquid handling—but without the need for optimization, and the ability to perform initial whole-genome screens in appropriate biological models from the start, arrayed library screening could significantly save on time and reagents and generate more relevant hits. So even with the initial capital outlay, arrayed library screening could be a more cost-effective option overall.

High content readouts with arrayed screens

As pooled CRISPR screens rely on survival or proliferation, readouts typically result in a binary “Yes or No” answer as to whether particular genes are involved in a biological process. However, in arrayed screens, the physical separation of cells allows for the design of sophisticated multiparametric assays to increase the value of the results generated from the screen—and unlike pooled library screens, the gene-edited cells of arrayed screens are compatible with downstream analyses, for example confocal microscopy or flow cytometry, which further increases the value of the screen data.⁵

Save time and money in the long run

In recent years, CRISPR-Cas9 has emerged as a valuable tool for genetic manipulation and has transformed the field of functional genomics by enabling large-scale loss- and gain-of-function screens. Pooled library screening with CRISPR has gained popularity due to its scalability and ease of use, but it is limited to dividing cells and simple, binary assay readouts only, and comes with a heavy bioinformatic burden, requiring significant data deconvolution to produce the final hit list, which then requires further validation.

Arrayed CRISPR screening could therefore provide significant advantages with the potential to overcome these limitations—providing more precise and reliable data, better identification of gene-specific phenotypes, and increased confidence in subsequent validation. Arrayed CRISPR screening does come with higher initial costs due to the requirement for automation, but this higher upfront cost could be outweighed by the decreased time needed for optimization, better reproducibility, and greater confidence in hits, and so save time and money in the long run.

Key Takeaways

• Pooled library screening with CRISPR has revolutionized functional genomics due to its scalability and ease of use, but is limited to simple, binary assay readouts only, and requires significant data deconvolution to identify hits
• Arrayed CRISPR library screens maintain physical separation of gene-edited cells, omitting the extensive bioinformatic analysis associated with pooled screens and making data interpretation easier
• CRISPR screening utilizing arrayed-based methods are compatible with sophisticated multiparametric primary and downstream assays, increasing the value of the results obtained
• Arrayed CRISPR library screens do require automation, but the higher upfront capital investment could ultimately be outweighed by the decreased time needed for optimization, better reproducibility, and greater confidence in hits making for easier downstream target validation

References

1. Shalem O, Sanjana NE, Zhang F. High-throughput functional genomics using CRISPR-Cas9. Nat Rev Genet. 2015 May;16(5):299-311.

2. Bock C, Datlinger P, Chardon F, Coelho MA, Dong MB, Lawson KA, Lu T, Maroc L, Norman TM, Song B, Stanley G, Chen S, Garnett M, Li W, Moffat J, Qi LS, Shapiro RS, Shendure J, Weissman JS, Zhuang X. High-content CRISPR screening. Nat Rev Methods Primers. 2022;2(1):9.

3. Agrotis A, Ketteler R. A new age in functional genomics using CRISPR/Cas9 in arrayed library screening. Front Genet. 2015 Sep 24;6:300.

4. Doench JG. Am I ready for CRISPR? A user's guide to genetic screens. Nat Rev Genet. 2018 Feb;19(2):67-80.

5. Panepucci RA, de Souza Lima IM. Arrayed functional genetic screenings in pluripotency reprogramming and differentiation. Stem Cell Res Ther. 2019 Jan 11;10(1):24.