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

Affiliations
  • 1Department of Genetics, Stanford University, Stanford, CA, USA.
  • 2Department of Neurobiology, Harvard Medical School, Boston, MA, USA.
  • 3Department of Biology, Stanford University, Stanford, CA, USA.
  • 4Biophysics Program, Stanford University, Stanford, CA, USA.
  • 5Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, MA, USA.
  • 6Chan Zuckerberg Biohub-San Francisco, San Francisco, CA, USA.
  • 7Department of Bioengineering, Stanford University, Stanford, CA, USA.
  • 8Department of Cancer Biology, Stanford University, Stanford, CA, USA.
  • 9Department of Biomolecular Chemistry and Center for Human Genomics and Precision Medicine, University of Wisconsin-Madison, Madison, WI, USA.
  • 10Sarafan ChEM-H, Stanford University, Stanford, CA, USA.
  • 11Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.
  • 12Department of Bioengineering, Stanford University, Stanford, CA, USA. lbintu@stanford.edu.
  • 13Department of Genetics, Stanford University, Stanford, CA, USA. bassik@stanford.edu.
  • 14Sarafan ChEM-H, Stanford University, Stanford, CA, USA. bassik@stanford.edu.

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November 2, 2024

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.