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Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements

Author

Listed:
  • Florence M. Chardon

    (University of Washington
    Seattle Hub for Synthetic Biology)

  • Troy A. McDiarmid

    (University of Washington
    Seattle Hub for Synthetic Biology)

  • Nicholas F. Page

    (University of California, San Francisco
    University of California, San Francisco
    University of California, San Francisco)

  • Riza M. Daza

    (University of Washington
    Seattle Hub for Synthetic Biology)

  • Beth K. Martin

    (University of Washington
    Seattle Hub for Synthetic Biology)

  • Silvia Domcke

    (University of Washington)

  • Samuel G. Regalado

    (University of Washington)

  • Jean-Benoît Lalanne

    (University of Washington)

  • Diego Calderon

    (University of Washington)

  • Xiaoyi Li

    (University of Washington
    Seattle Hub for Synthetic Biology)

  • Lea M. Starita

    (University of Washington
    Brotman Baty Institute for Precision Medicine)

  • Stephan J. Sanders

    (University of California, San Francisco
    University of California, San Francisco
    University of Oxford)

  • Nadav Ahituv

    (University of California, San Francisco
    University of California, San Francisco)

  • Jay Shendure

    (University of Washington
    Seattle Hub for Synthetic Biology
    Brotman Baty Institute for Precision Medicine
    Howard Hughes Medical Institute)

Abstract

CRISPR-based gene activation (CRISPRa) is a strategy for upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to a library of 493 gRNAs targeting candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically upregulating intended target genes and no other neighboring genes within 1 Mb, including gRNAs yielding upregulation of six autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes in neurons. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate genes in a cell type-specific manner.

Suggested Citation

  • Florence M. Chardon & Troy A. McDiarmid & Nicholas F. Page & Riza M. Daza & Beth K. Martin & Silvia Domcke & Samuel G. Regalado & Jean-Benoît Lalanne & Diego Calderon & Xiaoyi Li & Lea M. Starita & St, 2024. "Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-52490-4
    DOI: 10.1038/s41467-024-52490-4
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    References listed on IDEAS

    as
    1. Jesse R. Dixon & Siddarth Selvaraj & Feng Yue & Audrey Kim & Yan Li & Yin Shen & Ming Hu & Jun S. Liu & Bing Ren, 2012. "Topological domains in mammalian genomes identified by analysis of chromatin interactions," Nature, Nature, vol. 485(7398), pages 376-380, May.
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