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

Josh Tycko; Mike V Van; Aradhana; Nicole DelRosso; Hanrong Ye; David Yao; Raeline Valbuena; Alun Vaughan-Jackson; Xiaoshu Xu; Connor Ludwig; Kaitlyn Spees; Katherine Liu; Mingxin Gu; Venya Khare; Adi Xiyal Mukund; Peter H Suzuki; Sophia Arana; Catherine Zhang; Peter P Du; Thea S Ornstein; Gaelen T Hess; Roarke A Kamber; Lei S Qi; Ahmad S Khalil; Lacramioara Bintu; Michael C Bassik

doi:10.1038/s41587-024-02442-6

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

Nat Biotechnol. 2024 Nov 1. doi: 10.1038/s41587-024-02442-6. Online ahead of print.

Authors

Josh Tycko^#^{1

2}, Mike V Van^#³, Aradhana¹, Nicole DelRosso⁴, Hanrong Ye⁵, David Yao¹, Raeline Valbuena¹, Alun Vaughan-Jackson^{1

6}, Xiaoshu Xu⁷, Connor Ludwig⁷, Kaitlyn Spees¹, Katherine Liu³, Mingxin Gu¹, Venya Khare⁵, Adi Xiyal Mukund⁴, Peter H Suzuki⁷, Sophia Arana¹, Catherine Zhang⁸, Peter P Du⁸, Thea S Ornstein⁵, Gaelen T Hess⁹, Roarke A Kamber¹, Lei S Qi^{6

7

10}, Ahmad S Khalil^{5

11}, Lacramioara Bintu¹², Michael C Bassik^{13

14}

Affiliations

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

^# Contributed equally.

PMID: 39487265
DOI: 10.1038/s41587-024-02442-6

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.

Grants and funding

R01 HG011866/HG/NHGRI NIH HHS/United States