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Cited by 1,043CRISPR and Genetic EngineeringChromatin Remodeling and CancerRNA Interference and Gene Delivery

Optimized libraries for CRISPR-Cas9 genetic screens with multiple modalities

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Sanson, Kendall R., Hanna, Ruth E., Hegde, Mudra, Donovan, Katherine F., Strand, Christine, Sullender, Meagan E., Vaimberg, Emma W., Goodale, Amy +3 more·2018·Nature Communications
DOI 10.1038/s41467-018-07901-8PMC PMC6303322

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Abstract

The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene function. Here, we show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we present genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese), and show in negative selection screens that Dolcetto, with fewer sgRNAs per gene, outperforms existing CRISPRi libraries and achieves comparable performance to CRISPRko in detecting essential genes. We also perform positive selection CRISPRa screens and demonstrate that Calabrese outperforms the SAM approach at identifying vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities.

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  • CRISPRa-based screens are inherently less reproducible than CRISPRi or knockout screens.

    “In addition, CRISPRa-based screens are inherently less reproducible than CRISPRi or knockout screens43.”

    From Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions (2025) · cites this paper as [43]

    The Results section (‘Optimized genome-wide CRISPRa library’; page unavailable) reports low replicate correlations in specific CRISPRa resistance screens, but says low correlation is generally expected in positive-selection screens. It does not establish that CRISPRa is inherently less reproducible than CRISPRi or knockout screens. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • The iCRISPRi screen showed high consistency between independent experiments, while the iCRISPRa screen exhibited lower replicate correlation.

    “Our data for each screen, which included two independent experiments as replicates, showed high consistency in the iCRISPRi screen, while the iCRISPRa screen exhibited lower replicate correlation, consistent with known technical challenges associated with CRISPRa-based perturbations43 (Fig. S2D).”

    From Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions (2025) · cites this paper as [43]

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Cited by

Sorted by citations

Authors
Sanson, K. R., Hanna, R. E., Hegde, M., Donovan, K. F., Strand, C., Sullender, M. E., Vaimberg, E. W., Goodale, A., Root, D. E., Piccioni, F., & Doench, J. G.
Affiliations
Broad Institute (US)
Corresponding author
Doench, John G.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-12-21
Volume
9
Issue
1
Pages
5416-5416
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-018-07901-8
PMC
PMC6303322
OpenAlex
W2904226822
Cited by
1,043
References
12
Citations by year
2026: 175 · 2025: 192 · 2024: 194 · 2023: 115 · 2022: 125 · 2021: 109 · 2020: 102 · 2019: 29 · 2018: 1
FWCI
21.51
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Broad Institute
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
Keywords
CRISPR interference, CRISPR activation, vemurafenib resistance, geckos
MeSH terms
CRISPR-Associated Protein 9, Streptococcus pyogenes, Genomic Library, CRISPR-Cas Systems

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