Cited by 57CRISPR and Genetic EngineeringGenomics and Chromatin DynamicsChromatin Remodeling and Cancer

Development of compact transcriptional effectors using high-throughput measurements in diverse contexts

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Tycko, Josh, Van, Mike V., Aradhana, DelRosso, Nicole, Ye, Hanrong, Yao, David, Valbuena, Raeline, Vaughan-Jackson, Alun +18 more·2025·Nature Biotechnology

Published version of a preprint

Abstract

Transcriptional effectors are protein domains known to activate or repress gene expression; however, a systematic understanding of which effector domains regulate transcription across genomic, cell type and DNA-binding domain (DBD) contexts is lacking. Here we develop dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous target genes and test effector function for a library containing 5,092 nuclear protein Pfam domains across varied contexts. We also map context dependencies of effectors drawn from unannotated protein regions using a larger library tiling chromatin regulators and transcription factors. We find that many effectors depend on target and DBD contexts, such as HLH domains that can act as either activators or repressors. To enable efficient perturbations, we select context-robust domains, including ZNF705 KRAB, that improve CRISPRi tools to silence promoters and enhancers. We engineer a compact human activator called NFZ, by combining NCOA3, FOXO3 and ZNF473 domains, which enables efficient CRISPRa with better viral delivery and inducible control of chimeric antigen receptor T cells.

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Authors
Tycko, J., Van, M. V., Aradhana, DelRosso, N., Ye, H., Yao, D., Valbuena, R., Vaughan-Jackson, A., Xu, X., Ludwig, C., Spees, K., Liu, K., Gu, M., Khare, V., Mukund, A. X., Suzuki, P. H., Arana, S., Zhang, C., Du, P. P., Ornstein, T. S., Hess, G. T., Kamber, R. A., Qi, L. S., Khalil, A. S., Bintu, L., & Bassik, M. C.
Affiliations
  • Harvard University (US)
  • Stanford University (US)
  • Boston University (US)
  • Chan Zuckerberg Biohub San Francisco (US)
  • University of Wisconsin–Madison (US)
Corresponding author
  • Bintu, Lacramioara
  • Bassik, Michael C.
Venue
Nature Biotechnology
Published
2024-11-01
Volume
43
Issue
9
Pages
1525-1538
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
OpenAlex
W4403984400
Cited by
57
References
134
Citations by year
2026: 18 · 2025: 20 · 2024: 17 · 2023: 2
FWCI
7.49
Type
journal article
Language
en
Open access
Locations
  • Nature Biotechnology (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
Funders
  • National Science Foundation (T32 GM007276, 1921677, DGE-165651 8, 1656518, 2234657)
  • U.S. Department of Defense (DGE-1656518, N000142012825)
  • Achievement Rewards for College Scientists Foundation
  • Department of Neurobiology, Harvard Medical School
  • National Institutes of Health (NIH T32, 1dp2hd084069-01, F99/K00, NIH-4K00DK126120-03, NIH-1F99DK126120-01)
  • Chemistry, Engineering and Medicine for Human Health, Stanford University
  • National Human Genome Research Institute (UM1HG009436, R01HG011866, T32HG000044)
  • National Institute of General Medical Sciences (T32GM007276, R35M128947)
  • National Institute of Diabetes and Digestive and Kidney Diseases (F99DK126120, K00DK126120)
  • National Institute of Biomedical Imaging and Bioengineering (R01EB029483)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
Keywords
dCas9, high-throughput screening, DNA-binding domain, chromatin regulators, transcription factors, CRISPR interference, CRISPR activation, NCOA3, FOXO3, chimeric antigen receptor T cells, viral gene delivery, enhancer silencing
MeSH terms
Protein Domains, Gene Expression Regulation, Humans, Transcription Factors, Transcription, Genetic, High-Throughput Screening Assays, HEK293 Cells, CRISPR-Cas Systems

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