The first step to a successful CRISPR screen: Making the right choice between arrayed or pooled library


CRISPR screening empowers researchers to interrogate the biological activity of tens, hundreds, or thousands of genes to reveal what genes may be critical to cancer biology pathways, find drug targets, test probe functions, stratify patients, and many other important biological applications.

To realize the full potential of the screening assay, the first important step is to choose the screening format that is relevant to the types of biological question you are investigating. Here are a few questions our Technical Support team commonly ask to guide our customers in choosing a library format:

  • What is the biological question you are attempting to answer?
  • Can the phenotype be selected or separated from background in the cell population?
  • Is your facility equipped with lab automation to perform high-throughput screening?
  • Is handling of lentiviral particles allowed?
  • How much storage and cell culture space do you have in the facility?
  • What do you know about the transfectability and growth characteristics of the cells?

Depending on these aspects, one format may be more suitable compared to the other, we have listed some of the applications, advantages, and special considerations for each format to help you make the decision:

Lentiviral Pooled Screening Synthetic Arrayed Screening
Applications • Suitable for phenotypes that can be shifted in response to selective pressure, such as cell viability/proliferation assays or FACS analysis
• Identification of drug resistance genes
• Suitable for difficult-to-transfect cells
• Simpler genotype-phenotype correlation
• Allows for complex screening assays, such as morphological phenotypes by microscopy
• Suitable for high-content screening assays
Advantages • Relatively consistent set-up regardless of library size
• Requires minimal automation or specialized equipment
• Requires less experimental manipulation
• Custom-made libraries are available
• Suitable for longer time points
• Potential hits easily identifiable, one gene per well
• Can perform very complex readouts
• Multi-parametric readouts possible
• Custom-made libraries are available
• Applicable for 3D and co-culture studies
• Cells do not have to be actively dividing and growing
Special requirements • Require ability to select/separate cells with particular phenotype from the population
• Requires ability to start with a very large cell population and maintain large flasks throughout the screen
• Cells typically must be actively dividing and growing throughout the screen
• NGS and extensive bioinformatics required to deconvolute hits
• Viral handling required
• Cells must be amenable to transfection or electroporation
• Requires automated liquid handling equipment
• Higher reagent costs due to consumables such as plates, media, tips required by automation
• Potential plate-to-plate or well-to-well variability
• May require sufficient freezer space
• Shorter assay time points

No matter which format you choose, we offer a variety of predefined and custom libraries with deep and broad coverage of 5-10 guide RNAs per gene across the human and mouse genome. These guide RNAs were designed by a functionally validated proprietary algorithm that provide efficient knockout with unparalleled specificity to ensure your best chance at success.

Authors:Annie Zhang Bargsten, M.D., Ph.D. | Scientific Support Specialist 2 and Ivonne Rubio, Ph.D. | Scientific Support Specialist 3

Check out Horizon's CRISPR library options:
Interested in Horizon's screening services?
Learn more
  • A guide to CRISPR screening - blog post
  • Lentiviral sgRNA screening in primary immune cells - blog post
  • Benefits of cell panel and functional genomics screens in drug discovery - blog post

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  1. A. Cluse, I. Nikolic, et al. A Comprehensive Protocol Resource for Performing Pooled shRNA and CRISPR Screens. Methods Mol Biol. 1725, 201–227 (2018). doi: 10.1007/978-1-4939-7568-6_17
  2. S. J. Pettitt, D. B. Krastev, et al. Genome-wide and high-density CRISPRCas9 screens identify point mutations in PARP1 causing PARP inhibitor resistance. Nat Commun. 9, 1849 (2018). doi: 10.1038/s41467-018-03917-2
  3. Ž. Strezoska, M. Perkett, et al. High-content analysis screening for cell cycle regulators using arrayed synthetic crRNA libraries. J. Biotechnol. 251, 189-200 (2017). doi: 10.1016/j.jbiotec.2017.04.017
  4. J. Tan, S. E. Martin. Validation of Synthetic CRISPR Reagents as a Tool for Arrayed Functional Genomic Screening. PLoS One 11, e0168968 (2016). doi: 10.1371/journal.pone.0168968
  5. JR Costa, BE Bejcek et al.Genome Editing Using Engineered Nucleases and Their Use in Genomic Screening.Assay Guidance Manual. 2017.