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Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform

Author

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  • Nazanin Esmaeili Anvar

    (The University of Texas MD Anderson Cancer Center
    The University of Texas MD Anderson Cancer Center UTHealth)

  • Chenchu Lin

    (The University of Texas MD Anderson Cancer Center)

  • Xingdi Ma

    (The University of Texas MD Anderson Cancer Center
    The University of Texas MD Anderson Cancer Center UTHealth)

  • Lori L. Wilson

    (The University of Texas MD Anderson Cancer Center)

  • Ryan Steger

    (Broad Institute of MIT and Harvard)

  • Annabel K. Sangree

    (Broad Institute of MIT and Harvard)

  • Medina Colic

    (The University of Texas MD Anderson Cancer Center)

  • Sidney H. Wang

    (The University of Texas Health Science Center at Houston)

  • John G. Doench

    (Broad Institute of MIT and Harvard)

  • Traver Hart

    (The University of Texas MD Anderson Cancer Center
    The University of Texas MD Anderson Cancer Center)

Abstract

Genetic interactions mediate the emergence of phenotype from genotype, but technologies for combinatorial genetic perturbation in mammalian cells are challenging to scale. Here, we identify background-independent paralog synthetic lethals from previous CRISPR genetic interaction screens, and find that the Cas12a platform provides superior sensitivity and assay replicability. We develop the in4mer Cas12a platform that uses arrays of four independent guide RNAs targeting the same or different genes. We construct a genome-scale library, Inzolia, that is ~30% smaller than a typical CRISPR/Cas9 library while also targeting ~4000 paralog pairs. Screens in cancer cells demonstrate discrimination of core and context-dependent essential genes similar to that of CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes. Importantly, the in4mer platform offers a fivefold reduction in library size compared to other genetic interaction methods, substantially reducing the cost and effort required for these assays.

Suggested Citation

  • Nazanin Esmaeili Anvar & Chenchu Lin & Xingdi Ma & Lori L. Wilson & Ryan Steger & Annabel K. Sangree & Medina Colic & Sidney H. Wang & John G. Doench & Traver Hart, 2024. "Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-47795-3
    DOI: 10.1038/s41467-024-47795-3
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