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Related Experiment Video

Updated: Jan 10, 2026

Pooled CRISPR-Based Genetic Screens in Mammalian Cells
09:05

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

Published on: September 4, 2019

23.1K

Automation of high-throughput workflow for arrayed CRISPR activation library screening.

Chih-Cheng Yang, Aniruddha J Deshpande, Michael Jackson

    Biorxiv : the Preprint Server for Biology
    |November 26, 2025
    PubMed
    Summary
    This summary is machine-generated.

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    This study introduces an automated workflow for whole-genome CRISPR-mediated gene activation (CRISPRa) screening. The system efficiently screens gene activation libraries, identifying phenotypes like EPHA2 receptor promoter activation causing cell growth reduction.

    Area of Science:

    • Molecular Biology
    • Genetics
    • Biotechnology

    Background:

    • CRISPR-mediated gene activation (CRISPRa) effectively mimics endogenous gene expression.
    • Genome-wide CRISPRa screens utilize guide-RNA (gRNA) libraries in arrayed or pooled formats.
    • The T. gonfio library offers reduced complexity with four tandem gRNAs per target for arrayed screens.

    Purpose of the Study:

    • To develop a high-throughput automated workflow for streamlined genome-wide arrayed CRISPRa screening.
    • To establish and maintain transduced cell libraries for extended screening periods.
    • To enable the identification of phenotypes requiring longer development times, even in rapidly proliferating cells.

    Main Methods:

    • Development of a high-throughput automated workflow using the Biomek i7 Hybrid liquid-handling platform.

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  • Implementation of three pipelines: lentiviral library transduction, cell library passaging, and assay processing.
  • Utilized a T. gonfio mini-library targeting kinases and phosphatases in a pilot arrayed screen.

    • Main Results:

    • The automated workflow successfully established and maintained cell libraries for extended screening.
    • Activation of the EPHA2 receptor promoter induced a growth reduction phenotype in HEK293 cells.
    • The observed phenotype was validated in parallel pooled CRISPRa and co-culture assays.

    Conclusions:

    • The developed automated workflow significantly streamlines genome-wide arrayed CRISPRa screening.
    • This approach enhances the ability to detect phenotypes that manifest over extended periods.
    • The system is adaptable for various cell models, including rapidly proliferating ones, and validates findings across different screening formats.