Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

  1. Joseph M Replogle
  2. Jessica L Bonnar
  3. Angela N Pogson
  4. Christina R Liem
  5. Nolan K Maier
  6. Yufang Ding
  7. Baylee J Russell
  8. Xingren Wang
  9. Kun Leng
  10. Alina Guna
  11. Thomas M Norman
  12. Ryan A Pak
  13. Daniel M Ramos
  14. Michael Emmerson Ward
  15. Luke A Gilbert
  16. Martin Kampmann
  17. Jonathan S Weissman  Is a corresponding author
  18. Marco Jost  Is a corresponding author
  1. University of California, San Francisco, United States
  2. Whitehead Institute for Biomedical Research, United States
  3. Harvard Medical School, United States
  4. National Institutes of Health, United States
  5. National Institute of Neurological Disorders and Stroke, United States

Abstract

CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions in cell biology, genetics, and biotechnology, but wider deployment of CRISPRi screening has been constrained by the large size of single guide RNA (sgRNA) libraries and challenges in generating cell models with consistent CRISPRi-mediated knockdown. Here, we present next-generation CRISPRi sgRNA libraries and effector expression constructs that enable strong and consistent knockdown across mammalian cell models. First, we combine empirical sgRNA selection with a dual-sgRNA library design to generate an ultra-compact (1-3 elements per gene), highly active CRISPRi sgRNA library. Next, we compare CRISPRi effectors to show that the recently published Zim3-dCas9 provides an excellent balance between strong on-target knockdown and minimal nonspecific effects on cell growth or the transcriptome. Finally, we engineer a suite of cell lines with stable expression of Zim3-dCas9 and robust on-target knockdown. Our results and publicly available reagents establish best practices for CRISPRi genetic screening.

Data availability

Sequencing data are available on NCBI GEO under accession number GSE205310 (Perturb-seq) and GSE205147 (bulk RNA-seq). sgRNA counts from CRISPRi screens are included as supplemental tables. All data generated or analyzed during this study are included in the manuscript and supporting files.

The following data sets were generated

Article and author information

Author details

  1. Joseph M Replogle

    Medical Scientist Training Program, University of California, San Francisco, San Francisco, United States
    Competing interests
    Joseph M Replogle, consults for Maze Therapeutics and Waypoint Bio.
  2. Jessica L Bonnar

    Whitehead Institute for Biomedical Research, Cambridge, United States
    Competing interests
    No competing interests declared.
  3. Angela N Pogson

    Whitehead Institute for Biomedical Research, Cambridge, United States
    Competing interests
    No competing interests declared.
  4. Christina R Liem

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  5. Nolan K Maier

    Department of Microbiology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6103-6726
  6. Yufang Ding

    Department of Microbiology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  7. Baylee J Russell

    Department of Microbiology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  8. Xingren Wang

    Department of Microbiology, Harvard Medical School, Boston, United States
    Competing interests
    No competing interests declared.
  9. Kun Leng

    Medical Scientist Training Program, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  10. Alina Guna

    Whitehead Institute for Biomedical Research, Cambridge, United States
    Competing interests
    No competing interests declared.
  11. Thomas M Norman

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    Thomas M Norman, consults for Maze Therapeutics. The Regents of the University of California with TMN, MJ, LAG, and JSW as inventors have filed patent applications related to CRISPRi/a screening and Perturb-seq..
  12. Ryan A Pak

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    No competing interests declared.
  13. Daniel M Ramos

    Center for Alzheimer's Disease and Related Dementias, National Institutes of Health, Bethesda, United States
    Competing interests
    No competing interests declared.
  14. Michael Emmerson Ward

    National Institute of Neurological Disorders and Stroke, Bethesda, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5296-8051
  15. Luke A Gilbert

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, United States
    Competing interests
    Luke A Gilbert, declares outside interest in Chroma Medicine. The Regents of the University of California with TMN, MJ, LAG, and JSW as inventors have filed patent applications related to CRISPRi/a screening and Perturb-seq. LAG, MK, and JSW are inventors on US Patent 11,254,933 related to CRISPRi/a screening..
  16. Martin Kampmann

    Institute for Neurodegenerative Diseases, University of California, San Francisco, San Francisco, United States
    Competing interests
    Martin Kampmann, serves on the Scientific Advisory Boards of Engine Biosciences, Casma Therapeutics, Cajal Neuroscience, and Alector, and is an advisor to Modulo Bio and Recursion Therapeutics. LAG, MK, and JSW are inventors on US Patent 11,254,933 related to CRISPRi/a screening..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3819-7019
  17. Jonathan S Weissman

    Whitehead Institute for Biomedical Research, Cambridge, United States
    For correspondence
    weissman@wi.mit.edu
    Competing interests
    Jonathan S Weissman, declares outside interest in 5 AM Venture, Amgen, Chroma Medicine, KSQ Therapeutics, Maze Therapeutics, Tenaya Therapeutics, Tessera Therapeutics, and Third Rock Ventures. The Regents of the University of California with TMN, MJ, LAG, and JSW as inventors have filed patent applications related to CRISPRi/a screening and Perturb-seq. LAG, MK, and JSW are inventors on US Patent 11,254,933 related to CRISPRi/a screening..
  18. Marco Jost

    Department of Microbiology, Harvard Medical School, Boston, United States
    For correspondence
    marco_jost@hms.harvard.edu
    Competing interests
    Marco Jost, consults for Maze Therapeutics and Gate Bioscience. The Regents of the University of California with TMN, MJ, LAG, and JSW as inventors have filed patent applications related to CRISPRi/a screening and Perturb-seq..
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1369-4908

Funding

National Institutes of Health (R00GM130964)

  • Marco Jost

National Institutes of Health (T32AI132120)

  • Baylee J Russell

Human Frontier Science Program (2019L/LT000858)

  • Alina Guna

Chan Zuckerberg Initiative (Ben Barres Early Career Acceleration Award)

  • Martin Kampmann

Howard Hughes Medical Institute (Investigator)

  • Jonathan S Weissman

National Institutes of Health (RM1HG009490-01)

  • Jonathan S Weissman

Springer Nature Global Grant for Gut Health (1772808)

  • Marco Jost

Charles H. Hood Foundation (Child Health Research Award)

  • Marco Jost

Defense Advanced Research Projects Agency (HR0011-19-2-0007)

  • Jonathan S Weissman

Ludwig Center for Molecular Oncology (NA)

  • Jonathan S Weissman

Chan Zuckerberg Initiative (NA)

  • Jonathan S Weissman

National Institutes of Health (F31NS115380)

  • Joseph M Replogle

National Institutes of Health (F30AG066418)

  • Kun Leng

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Jan E Carette, Stanford University School of Medicine, United States

Version history

  1. Preprint posted: July 13, 2022 (view preprint)
  2. Received: July 13, 2022
  3. Accepted: December 23, 2022
  4. Accepted Manuscript published: December 28, 2022 (version 1)
  5. Version of Record published: January 9, 2023 (version 2)

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

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  1. Joseph M Replogle
  2. Jessica L Bonnar
  3. Angela N Pogson
  4. Christina R Liem
  5. Nolan K Maier
  6. Yufang Ding
  7. Baylee J Russell
  8. Xingren Wang
  9. Kun Leng
  10. Alina Guna
  11. Thomas M Norman
  12. Ryan A Pak
  13. Daniel M Ramos
  14. Michael Emmerson Ward
  15. Luke A Gilbert
  16. Martin Kampmann
  17. Jonathan S Weissman
  18. Marco Jost
(2022)
Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors
eLife 11:e81856.
https://doi.org/10.7554/eLife.81856

Share this article

https://doi.org/10.7554/eLife.81856

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