Development of compact transcriptional effectors using high-throughput measurements in diverse contexts
Josh Tycko1,2, Mike V Van3, Aradhana1
1Department of Genetics, Stanford University, Stanford, CA, USA.
Nature Biotechnology
|November 2, 2024
Summary
Researchers systematically screened protein domains for gene regulation effects, discovering context-dependent functions. They developed improved CRISPR tools for gene silencing and activation, enhancing applications like CAR T cell therapy.
Area of Science:
- Molecular Biology
- Genomics
- Epigenetics
Background:
- Transcriptional effectors regulate gene expression but their functions across different genomic and cellular contexts are not fully understood.
- A systematic understanding is needed to leverage these domains for precise gene regulation.
Purpose of the Study:
- To systematically quantify the function of nuclear protein domains in regulating transcription across diverse genomic and cell type contexts.
- To identify context-robust effector domains for improving CRISPR-based gene editing tools.
- To engineer novel activators for enhanced CRISPR activation (CRISPRa) applications.
Main Methods:
- Developed a high-throughput recruitment (HT-recruit) screening method using dCas9 to quantify effector function at endogenous genes.
- Tested a library of 5,092 nuclear protein Pfam domains and a larger library of unannotated regions.
- Selected context-robust domains and engineered novel CRISPR activators.
Main Results:
- Many transcriptional effector domains exhibit context-dependent activity, functioning as activators or repressors based on target and DNA-binding domain (DBD) contexts.
- Identified context-robust domains, such as ZNF705 KRAB, for improved CRISPR interference (CRISPRi) gene silencing.
- Engineered a novel compact human activator (NFZ) by combining NCOA3, FOXO3, and ZNF473 domains for efficient CRISPRa.
Conclusions:
- Systematic screening reveals context-specific transcriptional effector functions, crucial for understanding gene regulation.
- Developed improved CRISPRi and CRISPRa tools with enhanced efficiency, viral delivery, and inducible control for therapeutic applications like CAR T cell engineering.
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