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PreprintCited by 36CRISPR and Genetic EngineeringRNA Interference and Gene DeliveryPluripotent Stem Cells Research

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Multiplexed and Inducible Gene Modulation in Human Pluripotent Stem Cells by CRISPR Interference and Activation

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Hazelbaker, Dane Z., Beccard, Amanda, Mazzucato, Patrizia, Angelini, Gabriella, Messana, Angelica, Lam, Daisy, Eggan, Kevin, Barrett, Lindy E.·2019·Scientific Reports
DOI 10.1101/603951PMC PMC6971260

Preprint — published as 10.1038/s41598-020-57500-1

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Abstract

CRISPR-Cas9-mediated gene interference (CRISPRi) and activation (CRISPRa) approaches hold promise for functional genomic studies and genome-wide screens in human pluripotent stem cells (hPSCs). However, in contrast to CRISPR-Cas9 nuclease approaches, the efficiency of CRISPRi/a depends on continued expression of the dead Cas9 (dCas9) effector and guide RNA (gRNA), which can vary substantially depending on transgene design and delivery. Here, we design new fluorescently labeled piggyBac (PB) vectors to deliver robust and stable expression of multiplexed gRNAs. In addition, we generate hPSC lines harboring AAVS1-integrated, inducible and fluorescent dCas9-KRAB and dCas9-VPR transgenes to allow for accurate quantification and tracking of cells that express both the dCas9 effectors and gRNAs. We then employ these systems to target the TCF4 gene and conduct a rigorous assessment of expression levels of the dCas9 effectors, gRNAs and targeted gene. Collectively, these data provide proof-of-principle application of a stable, multiplexed PB gRNA delivery system that can be widely exploited to further enable genome engineering studies in hPSCs. Paired with diverse CRISPR tools including our dual fluorescence CRISPRi/a cell lines, this system would facilitate functional dissection of individual genes and pathways as well as larger-scale screens for studies of development and disease.

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Metadata

Authors
Hazelbaker, D. Z., Beccard, A., Mazzucato, P., Angelini, G., Messana, A., Lam, D., Eggan, K., & Barrett, L. E.
Affiliations
Broad Institute (US)
Corresponding author
Barrett, Lindy E.
ORCID
Venue
Scientific Reports
Publisher
openRxiv
Published
2019-04-09
Volume
10
Issue
1
Pages
635-635
ISSN
2045-2322 (ISSN-L)
DOI
10.1101/603951
PMC
PMC6971260
OpenAlex
W3000424462
Cited by
36
References
1
Citations by year
2026: 3 · 2025: 5 · 2024: 7 · 2023: 8 · 2022: 8 · 2021: 4 · 2020: 1
FWCI
1.70
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
  • Broad Institute
  • Tallinna Tehnikaülikool
  • Stanley Center for Psychiatric Research, 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 › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
Keywords
piggyBac transposon, CRISPR interference, CRISPR activation, dCas9, human pluripotent stem cells, dCas9-KRAB, TCF4, genome-wide screen, genome editing
MeSH terms
RNA, Guide, CRISPR-Cas Systems, Cell Line, DNA Transposable Elements, Genetic Vectors, Humans, Gene Transfer Techniques, Transgenes, Drosophila Proteins, Pluripotent Stem Cells, Basic Helix-Loop-Helix Proteins, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems

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