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Cited by 164CRISPR and Genetic EngineeringRNA Interference and Gene DeliveryGene Regulatory Network Analysis

Titrating gene expression using libraries of systematically attenuated CRISPR guide RNAs

Open access
Jost, Marco, Santos, Daniel A., Saunders, Reuben A., Horlbeck, Max A., Hawkins, John S., Scaria, Sonia M., Norman, Thomas M., Hussmann, Jeffrey A. +3 more·2020·Nature Biotechnology
DOI 10.1038/s41587-019-0387-5PMC PMC7065968

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Abstract

A lack of tools to precisely control gene expression has limited our ability to evaluate relationships between expression levels and phenotypes. Here, we describe an approach to titrate expression of human genes using CRISPR interference and series of single-guide RNAs (sgRNAs) with systematically modulated activities. We used large-scale measurements across multiple cell models to characterize activities of sgRNAs containing mismatches to their target sites and derived rules governing mismatched sgRNA activity using deep learning. These rules enabled us to synthesize a compact sgRNA library to titrate expression of ~2,400 genes essential for robust cell growth and to construct an in silico sgRNA library spanning the human genome. Staging cells along a continuum of gene expression levels combined with single-cell RNA-seq readout revealed sharp transitions in cellular behaviors at gene-specific expression thresholds. Our work provides a general tool to control gene expression, with applications ranging from tuning biochemical pathways to identifying suppressors for diseases of dysregulated gene expression.

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  • CRISPRi and CRISPRa offer advantages of precise, adjustable gene expression without causing genomic indels or nonspecific toxicity associated with Cas9-induced DNA double-strand breaks.

    “CRISPRi and CRISPRa offer the advantages of precise, adjustable gene expression without causing genomic indels or the nonspecific toxicity associated with Cas9-induced DNA double-strand breaks31.”

    From Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions (2025) · cites this paper as [31]

    The Abstract (page unavailable) describes programmable CRISPRi and CRISPRa effectors and a method for titrating gene expression with nuclease-dead Cas9. That supports adjustable expression and the absence of nuclease-mediated cutting in this approach, but the paper does not address genomic indels or nonspecific toxicity from Cas9-induced double-strand breaks as claimed. (LLM, conf=0.85)

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Cites

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Cited by

Sorted by citations

Authors
Jost, M., Santos, D. A., Saunders, R. A., Horlbeck, M. A., Hawkins, J. S., Scaria, S. M., Norman, T. M., Hussmann, J. A., Liem, C. R., Gross, C. A., & Weissman, J. S.
Affiliations
  • QB3 (US)
  • University of California, San Francisco (US)
Corresponding author
Weissman, Jonathan S.
ORCID
Venue
Nature Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2020-01-13
Volume
38
Issue
3
Pages
355-364
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
DOI
10.1038/s41587-019-0387-5
PMC
PMC7065968
OpenAlex
W3000149627
Cited by
164
References
14
Citations by year
2026: 19 · 2025: 22 · 2024: 34 · 2023: 26 · 2022: 17 · 2021: 22 · 2020: 24
FWCI
7.30
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Biotechnology (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
Funders
  • Howard Hughes Medical Institute
  • University of California, San Francisco
  • National Human Genome Research Institute (RM1HG009490)
  • National Cancer Institute (U01CA217882, U01CA168370)
  • National Institute of General Medical Sciences (K99GM130964, F32GM116331, T32GM007618, R35GM118061)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
Keywords
CRISPR interference, single guide RNA library, essential genes, single-cell RNA sequencing, gene expression regulation
MeSH terms
Gene Editing, Deep Learning, RNA, Guide, CRISPR-Cas Systems, HeLa Cells, Humans, Phenotype, Genomic Library, Gene Expression, RNA, Guide, Kinetoplastida, Sequence Analysis, RNA, Computational Biology, K562 Cells, Single-Cell Analysis, CRISPR-Cas Systems

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